DNA Microarrays and Component-Level Sequencing for Nucleic Acid-Based Data Storage and Processing

Abstract

The technology includes systems, devices, and methods that use nucleic acid molecules (e.g., DNA) to write, store, read, and perform calculations on digital information. The technology includes, for example, devices that include one or more individually or block-addressable electrode microarrays or nanoarrays for writing, storing, retrieving, reading, and performing calculations / manipulations on digital information.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 316,812, entitled "DNA MICROARRAYS," filed March 4, 2022, U.S. Provisional Patent Application No. 63 / 326,598, entitled "COMPONENT LEVEL SEQUENCING," filed April 1, 2022, U.S. Provisional Patent Application No. 63 / 329,111, entitled "MULTISENSOR COMPONENT LEVEL SEQUENCING," filed April 8, 2022, and U.S. Provisional Patent Application No. 63 / 333,698, entitled "MULTISENSOR COMPONENT LEVEL SEQUENCING," filed April 22, 2022. The entire contents of each of the above-cited applications are incorporated herein by reference. [Background technology]

[0002] background

[0002] Nucleic acid digital data storage is a stable method for encoding and storing information over the long term, and data can be stored at a higher density than magnetic tape or hard drive storage systems. Furthermore, digital data stored in nucleic acid molecules stored under low-temperature, dry conditions can be retrieved for as long as 60,000 years or more. To access the digital data stored in nucleic acid molecules, the nucleic acid molecules can be sequenced. Therefore, nucleic acid digital data storage can be an ideal method for storing or archiving large amounts of information that is not accessed frequently and can be stored or archived over the long term. Summary of the Invention [Means for solving the problem]

[0003] overview Current DNA data storage technologies focus on one aspect of the problem, such as writing information to nucleic acid molecules (e.g., DNA) or reading information encoded in nucleic acid molecules. Described herein are integrated platforms for writing digital information to nucleic acid molecules, reading digital information encoded in nucleic acid molecules, storing nucleic acid molecules encoding digital information, and performing computational operations on nucleic acid molecules. The technologies described herein include fully integrated nucleic acid molecule (e.g., DNA) writer / reader / storage / computing devices, systems and methods for assembling nucleic acid molecules (e.g., DNA) under ideal buffer conditions, systems and methods for efficient high-speed quantification of ligation, systems and methods for high-speed purification of fully formed nucleic acid molecules (e.g., DNA), and systems and methods for writing large data sets to nucleic acid molecules (e.g., DNA) at high throughput. Further, the technology described herein includes a device for reading a nucleic acid sequence, the device including a nanochannel disposed on a substrate and configured to receive an input nucleic acid molecule comprising an input strand, and a sensor device disposed on or within the nanochannel, the sensor device including an electronic sensing device, the electronic sensing device having an electronic gate with a gate voltage, the gate voltage being modulated using a charge of a reading component during the translocation of the input nucleic acid molecule to vary a source-drain current at the gate.

[0004] Incorporation by Reference

[0004] All publications, patents, and patent applications mentioned in this specification are incorporated herein 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 that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, it is intended that the present specification take precedence and / or supersede any such conflicting material.

[0005] BRIEF DESCRIPTION OF THE DRAWINGS 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 referred to herein as "Figure" and "FIG."). [Brief explanation of the drawings]

[0006] [Figure 1]

[0006] FIG. 1 shows a schematic perspective view of an example channel containing multiple cells including conductive plates and counter electrodes. [Figure 2]

[0007] FIG. 1 shows a schematic top view of an example (micro)array of small metal plates that can be used in conjunction with the techniques described herein. [Figure 3]

[0008] FIG. 1 shows a schematic top view of an electrode microarray that can be used with the techniques described herein, where the array is subdivided into multiple blocks. [Figure 4A]

[0009] FIG. 1 shows a schematic top view of a 5×5 electrode microarray that can be used in conjunction with the techniques described herein. [Figure 4B] 1 is a schematic diagram of an example dynamic random access memory (DRAM) cell array having a 4×4 electrode array. [Figure 5]

[0010] 1 shows a cross-sectional view of an example cell of a microarray. [Figure 6]

[0011] 1 shows a schematic cross-sectional view of an example cell configuration. [Figure 7]

[0012] 1 shows a schematic diagram of an example system for translating digital information into nucleic acid sequences, as described herein. [Figure 8]

[0013] 1 shows a schematic diagram of an example system for translating digital information into nucleic acid sequences, as described herein. [Figure 9A]

[0014] 1 shows a schematic cross-sectional view of a nanopore reader module that can be used in conjunction with the techniques described herein. [Figure 9B]

[0014] FIG. 1 shows a schematic top view of an example nanopore reader module that can be used in conjunction with the techniques described herein. [Figure 10A]

[0015] 1 shows a schematic top view of a nanochannel reader module that can be used with the technology described herein. [Figure 10B]

[0015] Figure 1 shows a schematic cross-sectional view of a nanochannel reader module that can be used in conjunction with the techniques described herein. [Figure 11A]

[0016] 1 shows a schematic cross-sectional view of a zero mode waveguide module that can be used in conjunction with the technology described herein. [Figure 11B]

[0016] Figure 1 shows a schematic top view of an example zero mode waveguide module that can be used in conjunction with the technology described herein. [Figure 12]

[0017] 1 shows a schematic cross-sectional view of an example nanopore sequencing module that can be used in conjunction with the technology described herein. [Figure 13]

[0018] FIG. 1 shows a schematic perspective view of an example nanopore sequencing device that can be used in conjunction with the techniques described herein. [Figure 14]

[0019] FIG. 1 is a schematic top view of an example nanopore sequencing device that can be used in conjunction with the techniques described herein. [Figure 15]

[0020] 1 shows a schematic diagram of an example system for translating digital information into a nucleic acid sequence. [Figure 16]

[0021] 1 is a flow chart of an example DNA writing process described herein. [Figure 17]

[0022] 1 is a flow chart of an example DNA reading process using the nanopore reader described herein. [Figure 18]

[0023] 1 is a flow chart of an example DNA reading process using a zero-mode waveguide reader as described herein. [Figure 19]

[0024] 1 shows a schematic diagram of an example of a nucleic acid molecule in multiple component layers for use in the technology described herein. [Figure 20]

[0025] 1 shows a schematic diagram of a process for flowing nucleic acid molecules of a zeroth component layer (A0 0) into a channel having two cells in one example nucleic acid assembly process described herein. [Figure 21]

[0026] FIG. 1 shows a schematic diagram of the buffer rinsing process in one example nucleic acid assembly process described herein. [Figure 22]

[0027] 1 shows a schematic diagram of a process for flowing a zeroth component (A1 0) layer of nucleic acid molecules into a channel having two cells in one example nucleic acid assembly process described herein. [Figure 23]

[0028] FIG. 1 shows a schematic diagram of the buffer rinsing process in one example nucleic acid assembly process described herein. [Figure 24]

[0029] 1 shows a schematic diagram of a process for flowing a first component (B0 1) layer of nucleic acid molecules through a channel having two cells in one example nucleic acid assembly process described herein. [Figure 25]

[0030] 1 shows a schematic diagram of the process of flowing nucleic acid molecules of a second component (CO 2) layer into a channel having two cells in one example nucleic acid assembly process described herein. [Figure 26]

[0031] 1 shows a schematic diagram of the process of flowing a layer of nucleic acid molecules of a second component (C1 2) into a channel having two cells in one example nucleic acid assembly process described herein. [Figure 27]

[0032] 1 shows a schematic diagram of the final buffer rinse process through a channel with two cells in one example nucleic acid assembly process described herein. [Figure 28]

[0033] 1 shows a schematic diagram of a quality control step in a channel with two cells in one example nucleic acid assembly process described herein. [Figure 29]

[0034] FIG. 1 shows a schematic diagram of quality control in a channel with two cell steps to determine ligation efficiency in one example nucleic acid assembly process described herein. [Figure 30]

[0035] FIG. 1 shows a schematic diagram of a quality control step in a channel with two cells to identify the distribution of abortive products in one example nucleic acid assembly process described herein. [Figure 31]

[0036] 1 shows a schematic diagram of a binary result (data vs. no data) readout in a channel with two cells in one example nucleic acid assembly process described herein. [Figure 32]

[0037] 1 shows a schematic fluorescence map of an electrode array obtained from an example QC process. [Figure 33]

[0038] A schematic diagram of the removal of incomplete nucleic acid products in a channel with two cells is shown. [Figure 34]

[0039] 1 shows a schematic diagram of an example data extraction step in a channel having two cells as described herein. [Figure 35]

[0040] 1 shows a schematic diagram of an example calculation step in a channel having two cells as described herein. [Figure 36]

[0041] 1 shows a schematic cross-sectional view of an example nanochannel and MOSFET sensing device with an example nucleic acid molecule containing a readout component. [Figure 37]

[0042] 1 shows a schematic cross-sectional view of a nanochannel with a first read component transitioning through the gate of an example MOSFET producing a change in source-drain current. [Figure 38]

[0043] 1 shows a series of current changes measured as an example nucleic acid molecule (eg, DNA) translocates through a nanochannel sensor device, as described herein. [Figure 39]

[0044] As described herein, an example shows a series of current changes measured as a nucleic acid molecule (e.g., DNA) migrates through a nanochannel sensor device, with the molecule having initial and final readout components that induce large electrical signals indicative of nucleic acid sequence boundaries. [Figure 40]

[0045] As described herein, an example shows a series of current changes measured as a nucleic acid molecule (e.g., DNA) migrates through a nanochannel sensor device, with the molecule having initial and final readout components that induce a pattern of small electrical signals that indicate the boundaries of the nucleic acid sequence. [Figure 41]

[0046] An example read element having four regions is shown. [Figure 42]

[0047] 1 shows a series of current changes measured as an example nucleic acid molecule (e.g., DNA) translocates through a nanochannel sensor device as described herein, the molecule having a read component with four regions, e.g., as used in the DNA writing process described herein. [Figure 43]

[0048] As described herein, an example shows a series of current changes measured as a nucleic acid molecule (e.g., DNA) translocates through a nanochannel sensor device, the molecule having a readout component with a secondary readout component hybridized thereto. [Figure 44]

[0049] 1 shows a schematic cross-sectional view of an example nanochannel with an optical fluorescence measurement device with an example nucleic acid molecule that includes a readout component having a fluorophore. [Figure 45]

[0050] 1 shows a series of fluorescence intensity changes measured as an example nucleic acid molecule (e.g., DNA) travels through an optical fluorescence measurement device as described herein, the molecule having a readout component with a fluorophore, e.g., as used in the DNA writing process described herein. [Figure 46]

[0051] 1 shows a series of current changes measured as an example nucleic acid molecule (e.g., DNA) is translocated through a nanochannel sensor device as described herein, the molecule having a read component at the layer boundary, e.g., as used in the DNA writing process described herein. [Figure 47]

[0052] 1 shows a series of current changes measured as an example nucleic acid molecule (e.g., DNA) is translocated through a nanochannel sensor device as described herein, where the molecule has a reduced number of read components at the layer boundaries, e.g., as used in the DNA writing process described herein. [Figure 48]

[0053] 1 is a series of fluorescence intensity changes measured as an example nucleic acid molecule (e.g., DNA) travels through an optical fluorescence measurement device as described herein, the molecule having a readout component at the layer boundary with a fluorophore, e.g., as used in the DNA writing process described herein. [Figure 49]

[0054] 1 shows a series of current changes measured as an example nucleic acid molecule (e.g., DNA) is translocated through a nanochannel sensor device as described herein, the molecule having a readout component comprising an aptamer and a peptide, for example, as used in the DNA writing process described herein. [Figure 50]

[0055] 1 shows a series of current changes measured as an example nucleic acid molecule (e.g., DNA) is translocated through a nanochannel sensor device as described herein, the molecule having a readout component comprising a dendrimer, e.g., as used in the DNA writing process described herein. [Figure 51]

[0056] 1 shows a schematic diagram of an example nucleic acid molecule with multiple readout components translocating through an example nanochannel and sensor device, as described herein. [Figure 52]

[0057] 1 shows a schematic diagram of an example nucleic acid molecule with multiple readout components translocating through an example nanochannel and a "slow" sensor device. [Figure 53]

[0058] A schematic diagram of an example nucleic acid molecule with multiple reading components moving through an example nanochannel and multiple sensor devices is shown, where each sensor (e.g., sensor device (or electronic or optical sensing device)) reads at least one piece of information. [Figure 54]

[0059] 1 shows a schematic diagram of an example nucleic acid molecule with multiple reading components traveling through an example nanochannel and multiple sensor devices, where each sensor (e.g., sensor device (or electronic or optical sensing device)) may miss some of the information read from the nucleic acid. [Figure 55]

[0060] 1 shows a schematic diagram of an example nucleic acid molecule with multiple readout components translocating through an example nanochannel and n multiple sensor devices. [Figure 56]

[0061] 1 shows a schematic diagram of an example nucleic acid molecule with multiple readout components translocating through an example nanochannel with multiple sensor device clusters. [Figure 57]

[0062] 1 shows a schematic overview of the process of encoding, writing, accessing, querying, reading, and decoding digital information stored in nucleic acid sequences. [Figure 58A]

[0063] 1 illustrates a schematic diagram of an example method for encoding digital data, referred to as "data at an address," using objects or identifiers (e.g., nucleic acid molecules), and shows combining a rank object (or address object) with a byte value object (or data object) to create an identifier. [Figure 58B]

[0063] An example method for encoding digital data, referred to as "data-at-address," using objects or identifiers (e.g., nucleic acid molecules) is shown schematically, and an embodiment of the data-at-address method is shown in which rank objects and byte value objects are themselves combinatorial concatenations of other objects. [Figure 59A]

[0064] 1A-1C are schematic diagrams illustrating an example method for encoding digital information using objects or identifiers (e.g., nucleic acid sequences), illustrating the encoding of digital information using rank objects as identifiers. [Figure 59B]

[0064] An example method for encoding digital information using objects or identifiers (eg, nucleic acid sequences) is shown schematically, illustrating an embodiment of the encoding method in which the address object is itself a combinatorial concatenation of other objects. [Figure 60]

[0065] Shows a contour plot in logarithmic space of the combinatorial space of possible identifiers (C, x-axis) versus the average number of identifiers (k, y-axis) that can be constructed to store information of a given size (contour). [Figure 61]

[0066] 1 shows a schematic overview of how information can be written into a nucleic acid sequence (e.g., deoxyribonucleic acid). [Figure 62A]

[0067] An example method, termed the "product approach," for constructing an identifier (e.g., a nucleic acid molecule) by combinatorially assembling distinguishable components (e.g., nucleic acid sequences) is shown, and the architecture of an identifier constructed using the product approach is shown. [Figure 62B]

[0067] An example method, called the "product method," for constructing an identifier (e.g., a nucleic acid molecule) by combinatorially assembling distinguishable components (e.g., nucleic acid sequences) is provided, along with an example of the combinatorial space of identifiers that can be constructed using the product method. [Figure 63]

[0068] 1 illustrates a schematic representation of the use of overlap extension polymerase chain reaction to construct an identifier (e.g., a nucleic acid molecule) from components (e.g., nucleic acid sequences). [Figure 64]

[0069] 1 illustrates a schematic representation of the use of cohesive end ligation to construct an identifier (e.g., a nucleic acid molecule) from components (e.g., nucleic acid sequences). [Figure 65]

[0070] 1A-1C illustrate a schematic representation of the use of recombinase assembly to construct an identifier (e.g., a nucleic acid molecule) from components (e.g., nucleic acid sequences). [Figure 66A]

[0071] 1 shows template-directed ligation and schematically illustrates the use of template-directed ligation to construct an identifier (e.g., a nucleic acid molecule) from components (e.g., nucleic acid sequences). [Figure 66B]

[0071] Template-directed ligation is shown, showing a histogram of copy numbers (abundances) of 256 distinct nucleic acid sequences each assembled combinatorially from six nucleic acid sequences (e.g., components) in one pooled template-directed ligation reaction. [Figure 67A]

[0072] 1 shows a schematic representation of an example method, referred to as a "sorting scheme," for constructing identifiers (e.g., nucleic acid molecules) with sorted components (e.g., nucleic acid sequences), and illustrates the architecture of an identifier constructed using the sorting scheme. [Figure 67B]

[0072] An example of a method, referred to as a "sorting scheme," for constructing identifiers (e.g., nucleic acid molecules) having permuted components (e.g., nucleic acid sequences) is shown schematically, along with an example of a combinatorial space of identifiers that can be constructed using the sorting scheme. [Figure 67C]

[0072] An example of a method, referred to as a "sorting method," for constructing an identifier (e.g., a nucleic acid molecule) having sorted components (e.g., nucleic acid sequences) is shown schematically, and an example of an implementation of the sorting method using template-directed ligation is shown. [Figure 67D]

[0072] Figure 67C schematically illustrates an example of a method, referred to as a "reordering scheme," for constructing an identifier (e.g., a nucleic acid molecule) having permuted components (e.g., a nucleic acid sequence), and shows an example of how the embodiment from Figure 67C can be modified to construct an identifier having permuted and repeated components. [Figure 67E]

[0072] An example of a method, termed the "sorting method," for constructing an identifier (e.g., a nucleic acid molecule) having sorted components (e.g., a nucleic acid sequence) is shown schematically, and the example embodiment from Figure 67D shows how undesirable by-products can be derived that can be removed using nucleic acid size selection. [Figure 67F]

[0072] An example of a method, referred to as a "reordering method," for constructing identifiers (e.g., nucleic acid molecules) with permuted components (e.g., nucleic acid sequences) is shown schematically, and another example of how template-directed ligation and size selection are used to construct identifiers with permuted and repeated components is shown. [Figure 67G]

[0072] A schematic diagram of an example of a method, referred to as a "sorting scheme," for constructing an identifier (e.g., a nucleic acid molecule) with sorted components (e.g., a nucleic acid sequence) illustrates an example of when size selection may fail to separate a particular identifier from undesired by-products. [Figure 68A]

[0073] 1 shows a schematic diagram of an example of a method, referred to as the "MchooseK" method, for constructing an identifier (e.g., a nucleic acid molecule) using any number, K, of assembled components (e.g., nucleic acid sequences) out of a larger number, M, of possible components, and illustrates the architecture of an identifier constructed using the MchooseK method. [Figure 68B]

[0073] A schematic diagram of an example of a method, referred to as the "MchooseK" method, for constructing an identifier (e.g., a nucleic acid molecule) using any number, K, of assembled components (e.g., nucleic acid sequences) out of a larger number, M, of possible components is shown, along with an example of the combinatorial space of identifiers that can be constructed using the MchooseK method. [Figure 68C]

[0073] Schematic diagram of an example method, referred to as the "MchooseK" method, for constructing an identifier (e.g., a nucleic acid molecule) using any number, K, of assembled components (e.g., nucleic acid sequences) out of a larger number, M, of possible components, and an example implementation of the MchooseK method using template-directed ligation is shown. [Figure 68D]

[0073] An example of a method, referred to as the "MchooseK" method, for constructing an identifier (e.g., a nucleic acid molecule) using any number, K, of assembled components (e.g., nucleic acid sequences) out of a larger number, M, of possible components is shown schematically, and the example embodiment from Figure 68C shows how undesirable by-products can be removed using nucleic acid size selection. [Figure 69A]

[0074] 1 illustrates a schematic diagram of an example of a method, referred to as a "compartmentalization scheme," for constructing identifiers with compartmentalized components, and shows an example of the combinatorial space of identifiers that can be constructed using the compartmentalization scheme. [Figure 69B]

[0074] An example of a method called the "compartmentalization method" for constructing an identifier having compartmentalized components is shown schematically, and an example of an implementation of the compartmentalization method using template-directed ligation is shown. [Figure 70A]

[0075] 1 shows a schematic diagram of an example of a method called the "unconstrained string" (or USS) method for constructing an identifier composed of a string of any component derived from several possible components, and an example of the combinatorial space of identifiers that can be constructed using the USS method. [Figure 70B]

[0075] A schematic diagram of an example of a method called the "unconstrained string" (or USS) approach for constructing an identifier composed of a string of any component derived from several possible components is shown, along with an example implementation of the USS approach using template-guided ligation. [Figure 71A]

[0076] 1 illustrates a schematic example of a method called "component elimination" for constructing an identifier by removing components from a parent identifier, and shows an example of a combinatorial space of identifiers that can be constructed using the component elimination method. [Figure 72B]

[0076] A schematic illustration of an example of a method called "component deletion" for constructing an identifier by removing components from a parent identifier is shown, and an example of an implementation of the component deletion method using double-stranded targeted cleavage and repair is shown. [Figure 72]

[0077] 1 shows a schematic representation of a parent identifier with a recombinase recognition site from which further identifiers can be constructed by applying a recombinase to the parent identifier. [Figure 73A]

[0078] 10A-10C are schematic diagrams outlining examples of methods for accessing portions of information stored in nucleic acid sequences by accessing a few specific identifiers from a larger number of identifiers, and show examples of methods for accessing identifiers containing specified components using polymerase chain reaction, affinity tagged probes, and degradation targeted probes. [Figure 73B]

[0078] A schematic overview of an example method for accessing some of the information stored in a nucleic acid sequence by accessing several specific identifiers from a larger number of identifiers is shown, and an example of a method for using the polymerase chain reaction to perform an "OR" or "AND" operation to access identifiers containing multiple specified components is shown. [Figure 73C]

[0078] A schematic overview of an example method for accessing some of the information stored in a nucleic acid sequence by accessing several specific identifiers from a larger number of identifiers is shown, and an example of a method for using affinity tags to perform an "OR" or "AND" operation to access identifiers containing multiple specified components is shown. [Figure 74A]

[0079] 1 shows an example of encoding, writing, and reading data encoded in a nucleic acid molecule, and an example of encoding, writing, and reading 5,856 bits of data. [Figure 74B]

[0079] An example of encoding, writing, and reading data encoded in a nucleic acid molecule is shown, and an example of encoding, writing, and reading 62,824 bits of data is shown. [Figure 75]

[0080] 1 illustrates a computer system programmed or otherwise configured to implement the methods presented herein. [Figure 76]

[0081] 1 shows an example of a scheme for the assembly of any two selected double-stranded components from a single parental set of double-stranded components. [Figure 77]

[0082] A possible cohesive end component structure composed of two oligos, X and Y, is shown. [Figure 78]

[0083] 1 shows an example of constructing an identifier from components with multiple functionalities. [Figure 79A]

[0084] An example of the effect of identifier rank on PCR-based random access is shown. [Figure 79B]

[0084] An example of the effect of identifier rank on PCR-based random access is shown. [Figure 80A]

[0085] 1 shows an example of the effect of an identifier architecture with a non-uniform component distribution on PCR-based random access. [Figure 80B]

[0085] An example of the effect of an identifier architecture with non-uniform component distribution on PCR-based random access is shown. [Figure 81]

[0086] 1 shows an example of the effect of increasing layers in an identifier architecture on PCR-based random access. [Figure 82]

[0087] An example of a multi-bin positional coding scheme across an alphabet of nine symbols is shown. [Figure 83]

[0088] An example of a multi-bin identifier distribution coding scheme is shown, with an identifier library of two identifiers and a bin set of three bins, that allows encoding any of nine possible messages in a four-bit string. [Figure 84]

[0089] We present an example of a multi-bin identifier distribution coding scheme with identifier reuse having a library of two identifiers and a bin set of three bins that allows encoding any of 64 possible messages in a 6-bit string. [Figure 85]

[0090] Here is an example of encoding information into DNA using integer division. [Figure 86]

[0091] 1 illustrates an example of an encoding pipeline that includes algorithmic modules that prepare and convert a source bitstream into a building program specification that is interpreted by a writer. [Figure 87]

[0092] 1 illustrates an example of one embodiment of a data structure for representing an identifier library in serialized form. [Figure 88]

[0093] 1 shows an example of two source bitstreams and a generic identifier library prepared for computation using operations defined in the identifier pool. [Figure 89]

[0094] 1 shows the inputs and results of three example logical operations performed on the identifier pool, illustrating how the identifier library can be used as a platform for in vitro computation. [Figure 90A]

[0095] An example of storing an image file and reading the image file at multiple resolutions is shown. [Figure 90B]

[0095] An example of storing an image file and reading the image file at multiple resolutions is shown. [Figure 90C]

[0095] An example of storing an image file and reading the image file at multiple resolutions is shown. [Figure 90D]

[0095] An example of storing an image file and reading the image file at multiple resolutions is shown. [Figure 90E]

[0095] An example of storing an image file and reading the image file at multiple resolutions is shown. [Figure 90F]

[0095] An example of storing an image file and reading the image file at multiple resolutions is shown. [Figure 90G]

[0095] An example of storing an image file and reading the image file at multiple resolutions is shown. [Figure 91]

[0096] An example method for generating entropy that can be used to create a random bit string is shown. [Figure 92A]

[0097] An example method for generating and storing entropy (random bit strings) is shown. [Figure 92B]

[0097] An example method for generating and storing entropy (random bit strings) is shown. [Figure 92C]

[0097] An example method for generating and storing entropy (random bit strings) is shown. [Figure 93A]

[0098] 1 illustrates an example method for organizing and accessing random bit strings using an input. [Figure 93B]

[0098] An example method for organizing and accessing random bit strings using input is shown. [Figure 94]

[0099] An example method for securing and authenticating access to artifacts using physical DNA keys is presented. DETAILED DESCRIPTION OF THE INVENTION

[0007] explanation

[0100] While various embodiments of the present invention have been shown and described herein, it will be apparent 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 is understood that various alternatives to the embodiments of the invention described herein may be employed.

[0008]

[0101] The term "symbol," as used herein, generally refers to the representation of a unit of digital information. Digital information can be divided or translated into a string of symbols. In one example, a symbol can be a bit, and can have a value of "0" or "1."

[0009]

[0102] The terms "distinguishable" or "unique," as used herein, generally refer to an object that can be distinguished from other objects in a group. For example, a distinguishable or unique nucleic acid sequence can be a nucleic acid sequence that does not have the same sequence as any other nucleic acid sequence. A distinguishable or unique nucleic acid molecule may not have the same sequence as any other nucleic acid molecule. A distinguishable or unique nucleic acid sequence or molecule may also share regions of similarity with another nucleic acid sequence or molecule.

[0010]

[0103] The term "component," as used herein, generally refers to a nucleic acid sequence. A component can be a distinct nucleic acid sequence. A component can be linked or assembled with one or more other components to generate other nucleic acid sequences or molecules.

[0011]

[0104] The term "stratum," as used herein, generally refers to a group or pool of components. Each stratum may contain a set of distinguishable components such that the components in one stratum differ from the components in another stratum. Components from one or more stratums may be assembled to generate one or more identifiers.

[0012]

[0105] The term "identifier," as used herein, generally refers to a nucleic acid molecule or sequence that represents the position and value of a bit string within a larger bit string. More generally, an identifier may refer to any object that represents or corresponds to a symbol in a symbol string. In some embodiments, an identifier may include one or more concatenated components.

[0013]

[0106] The term "combinatorial space," as used herein, generally refers to the set of all possible distinct identifiers that can be generated from a starting set of objects, such as components, and an allowable set of rules for how to modify those objects to form identifiers. The size of the combinatorial space of identifiers created by assembling or linking components can depend on the number of layers of components, the number of components in each layer, and the particular assembly method used to generate the identifiers.

[0014]

[0107] The term "identifier rank," as used herein, generally refers to a relationship that defines the ordering of identifiers in a set.

[0015]

[0108] The term "identifier library," as used herein, generally refers to a collection of identifiers that correspond to symbols in a string representing digital information. In some embodiments, the absence of a given identifier in an identifier library can indicate a symbol value at a particular location. One or more identifier libraries can be combined into a pool, group, or set of identifiers. Each identifier library can include a unique barcode that identifies the identifier library.

[0016]

[0109] The term "nucleic acid," as used herein, generally refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or variants thereof. Nucleic acids can include one or more subunits selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. Nucleotides can include A, C, G, T, or U, or variants thereof. Nucleotides can include any subunit that can be incorporated into a growing nucleic acid chain. Such subunits can be A, C, G, T, or U, or any other subunit that can be specific to one of more complementary A, C, G, T, or U, or that can be complementary to a purine (i.e., A or G, or variants thereof) or a pyrimidine (i.e., C, T, or U, or variants thereof). In some instances, nucleic acids can be single-stranded or double-stranded, and in some cases, nucleic acid molecules are circular.

[0017]

[0110] The terms "nucleic acid molecule" or "nucleic acid sequence," as used herein, generally refer to a polymeric form of nucleotides or polynucleotides, which may be of various lengths, either deoxyribonucleotides (DNA) or ribonucleotides (RNA), or analogs thereof. The term "nucleic acid sequence" can refer to the alphabetical representation of a polynucleotide; alternatively, the term can apply to the physical polynucleotide itself. This alphabetical representation can be entered into a database in a computer having a central processing unit and used to encode digital information, for mapping nucleic acid sequences or nucleic acid molecules to symbols or bits. A nucleic acid sequence or oligonucleotide may contain one or more non-standard nucleotides, nucleotide analogs, and / or modified nucleotides.

[0018]

[0111] "Oligonucleotide," as used herein, generally refers to a single-stranded nucleic acid sequence, typically composed of a specific sequence of the four nucleotide bases adenine (A), cytosine (C), guanine (G), and thymine (T), or, if the polynucleotide is RNA, adenine (A), cytosine (C), guanine (G), and uracil (U).

[0019]

[0112] Examples of modified nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylguanine, 5-methylaminomethyl-2-thiouridine ... methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, and the like. Nucleic acid molecules may have modified base moieties (e.g., at one or more atoms normally available to form hydrogen bonds with a complementary nucleotide and / or at one or more atoms normally unavailable to form hydrogen bonds with a complementary nucleotide), modified sugar moieties, or modified phosphate backbones. Nucleic acid molecules may also contain amine-modifying groups such as aminoallyl-dUTP (aa-dUTP) and aminohexhylacrylamide-dCTP (aha-dCTP) to allow covalent attachment of amine-reactive moieties such as N-hydroxysuccinimide ester (NHS).

[0020]

[0113] The term "primer," as used herein, generally refers to a nucleic acid strand that serves as the starting point for nucleic acid synthesis, such as in polymerase chain reaction (PCR). In one example, during replication of a DNA sample, an enzyme that catalyzes replication initiates replication at the 3' end of a primer bound to the DNA sample and copies the opposite strand. For more information regarding PCR, including details about primer design, see Chemical Methods Section D.

[0021]

[0114] The terms "polymerase" or "polymerase enzyme," as used herein, generally refer to any enzyme capable of catalyzing a polymerase reaction. Examples of polymerases include, but are not limited to, nucleic acid polymerases. Polymerases can be naturally occurring or synthetic. An example of a polymerase is Φ29 polymerase or a derivative thereof. In some cases, a transcriptase or a ligase (i.e., an enzyme that catalyzes the formation of a bond) is used in conjunction with or as a substitute for a polymerase to construct new nucleic acid sequences. Examples of polymerases include DNA polymerases, RNA polymerases, thermostable polymerases, wild-type polymerases, modified polymerases, E. coli DNA polymerase I, T7 DNA polymerase, bacteriophage T4 DNA polymerase Φ29 (phi29) DNA polymerase, Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, Pwo polymerase, VENT polymerase, DEEPVENT polymerase, Ex-Taq polymerase, LA-Taw polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tca polymerase, Tih polymerase, Tfi polymerase, and Platinum Examples include Taq polymerase, Tbr polymerase, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment polymerase with 3' to 5' exonuclease activity, and variants, modifications, and derivatives thereof. For additional polymerases that can be used with PCR, and for more information on how polymerase properties can affect PCR, see Chemical Methods section D.

[0022]

[0115] The term "species," as used herein, generally refers to one or more DNA molecules of the same sequence. When "species" is used in the plural sense, it can be assumed that every species within the plural has a distinguishable sequence, which is sometimes clarified by writing "distinguishable species" instead of "species."

[0023]

[0116] Digital information, such as computer data, in the form of binary code may include a sequence or string of symbols. Binary code may, for example, encode or represent text or computer processor instructions using a binary system with two binary symbols called bits, typically 0 and 1. Digital information may be represented in the form of a non-binary code, which may include a sequence of non-binary symbols. Each encoded symbol may be reassigned to a unique bit string (or "byte"), and the unique bit strings or bytes may be arranged into a byte string or byte stream. The bit value for a given bit may be one of two symbols (e.g., 0 or 1). A byte may contain a string of N bits, for a total of 2 N For example, a byte containing 8 bits can have a total of 2 8 Or 256 possible unique byte values ​​can be produced, where each of the 256 bytes can correspond to one of the 256 possible distinct symbols, characters, or instructions that can be encoded in the byte. Raw data (e.g., text files and computer instructions) can be represented as a sequence of bytes or a byte stream. Zip files, or compressed data files that contain raw data, can also be stored as a byte stream; these files can be stored in compressed form as a byte stream and then restored to raw data before being read by a computer.

[0024]

[0117] The disclosed methods and systems can be used to encode computer data or information with multiple identifiers, each of which can represent one or more bits of primary information. In some examples, the disclosed methods and systems encode data or information using identifiers, each of which can represent two bits of primary information.

[0025]

[0118] Previous methods for encoding digital information into nucleic acids have relied on the base-by-base synthesis of nucleic acids, which can be expensive and time-consuming. Alternative methods can improve the efficiency and commercial viability of digital information storage by reducing the reliance on base-by-base nucleic acid synthesis to encode digital information, and can eliminate the need for de novo synthesis of distinct nucleic acid sequences for every new information storage requirement.

[0026]

[0119] Rather than relying on base-by-base or de novo nucleic acid synthesis (e.g., phosphoramidite synthesis), the new methods can encode digital information (e.g., binary code) into multiple identifiers or nucleic acid sequences comprising combinatorial sequences of components. Thus, the new strategies can generate a first set of distinct nucleic acid sequences (or components) for a first request for information storage, and then reuse the same nucleic acid sequences (or components) for subsequent information storage requests. These approaches can significantly reduce the cost of DNA-based information storage by reducing the role of de novo synthesis of nucleic acid sequences in the process of encoding and writing information to DNA. Furthermore, unlike embodiments of base-by-base synthesis (e.g., phosphoramidite chemistry-based or template-free polymerase-based nucleic acid extension), which may use cyclic delivery of each base to each extended nucleic acid, the new methods of writing information to DNA using identifier construction from components are highly parallelizable processes that do not necessarily use cyclic nucleic acid extension. Thus, the new methods can increase the speed at which digital information is written to DNA compared to traditional methods.

[0027]

[0120] Described herein are technologies including systems, devices, and methods for writing, storing, reading, and performing computations on digital information using nucleic acid molecules (e.g., DNA). The technologies include, for example, devices that include one or more individually or block-addressable electrode microarrays or nanoarrays for writing, storing, retrieving, reading, and performing computations / manipulations on digital information.

[0028]

[0121] The technology includes methods for combinatorial assembly of oligonucleotide fragments that use local magnetic fields to synthesize unique identifiers to encode data or to directly represent data using other encoding schemes. The technology includes methods for performing quality control of the assembly process (e.g., by calculating ligation efficiency). The technology includes methods for performing post-processing of synthesized nucleic acid molecules, e.g., DNA, using electric fields or heat (e.g., filtering out shorter abortive products). The technology includes methods for retrieving data encoded in nucleic acid molecules from addressable locations. The technology includes methods for reading digital information stored in or retrieved from nucleic acid molecules, e.g., DNA. The technology includes methods for performing computations using nucleic acid molecules, e.g., DNA, retrieved from one or more stored locations.

[0029]

[0122] The techniques involve the use of local electric fields to manipulate nucleic acid molecules, e.g., DNA, in applications involving DNA storage and / or computation. The techniques described herein involve arrays, e.g., microarrays or other miniature arrays, either micro- or nanoscale. Each element of a microarray (e.g., a plate, as described below) has a size of 0.1 micrometers. 2 ~1mm 2 ;1 micrometer 2 ~10,000 micrometers 2 ;10 micrometers 2 ~1,000 micrometers 2 ; or 100 micrometers 2 ~500 micrometers 2Each element of the nanoarray (e.g., a plate, as described below) can have an area of ​​0.1 nanometers. 2 ~1 micrometer 2 ; nanometers 2 ~10,000 nanometers 2 ;10 nanometers 2 ~1,000 nanometers 2 ; or 100 nanometers 2 ~500 nanometers 2 The technology described herein involves an electric field induced when a voltage is applied to a pair of charged structures placed in close proximity to one another. Nucleic acids, such as DNA, are negatively charged molecules. When nucleic acids are placed in an electric field, a force is induced on the molecules that depends on the charge of the molecules and the strength of the electric field.

[0030]

[0123] In some embodiments, the techniques described herein utilize electric field-dependent forces to attract or repel nucleic acids, e.g., DNA, at specific microarray locations. Such local electric fields can be generated by charging (small) conductive plates, e.g., metal plates, and placing them parallel to a counter electrode. Figure 1 shows a schematic perspective view of an example channel 100 containing multiple conductive plates 110 and counter electrodes 130. A fluid containing multiple nucleic acid molecules can flow parallel to the plates 110 and counter electrodes 130, for example, to transport nucleic acids from a reservoir to the array. The multiple conductive plates 110 and counter electrodes 130 are arranged opposite each other along the (first) dimension of the channel. Each plate 110 and a portion of the counter bridge 130 form a cell 150. The conductive plates of a cell can also be referred to as a microspot. The plates 110 and cells 130 can be arranged in several arrays and patterns, for example, a square array, a rectangular array, or a circular array. Figure 2 shows a schematic top view of an example 5x5 (micro)array 200 of a small metal plate 110. Figure 3 shows a schematic top view of an example electrode microarray scaled up and subdivided into blocks (9 blocks of 25 cells each in this illustrative case). Each one of the microarray cells can be addressed individually or together with one or more other cells, for example, together with all the cells in a block.

[0031]

[0124] Each cell in the array is configured to selectively attract or repel one or more nucleic acid molecules, which can be used to store encoded digital information, for example, using a scheme for encoding digital information onto a plurality of identifier molecules as described herein. To manipulate nucleic acids (e.g., to perform information read, write, and / or computational operations as described later in this specification and shown in Figures 57-94), each one of the microarray cells can be independently addressed and charged using an addressing scheme such as that shown in Figures 4A and 4B. An example 5x5 array 202 including 25 plates 110 is shown in Figure 4A. Figure 4B is a schematic diagram of an example dynamic random access memory (DRAM) cell array 203 having 16 plates 110a. When row and column lines are addressed using row decoder 222 and column decoder 224, the corresponding cell 150 / plate 111 is turned on, e.g., an electric field is induced in that cell. The cell can function as a capacitor. Depending on the voltage level of the cell, the capacitor within the cell charges or discharges. The counter electrode 130 (top plate) of the parallel plate capacitor within each cell can be used to attract or repel nucleic acid molecules (e.g., DNA) in solution.

[0032]

[0125] FIG. 5 shows a cross section of a single plate 110 of a cell 150 described herein. The example plate 110 includes, for example, a) an adapter molecule 112, e.g., a solid support for an adapter complementary to a layer 0 component as described herein, and / or b) a conductive plate, e.g., a metal portion 114, that provides one electrode of an electrode pair (e.g., together with a counter electrode 130) to generate an electric field. The plate 110 of the cell 150 also includes a base layer 118 and a dielectric layer 116 disposed between the base layer 118 and the conductive metal portion 114. The base layer 1180 may be or include a conductor, a semiconductor, or both. FIG. 6 shows an example cell 150 illustrating how the conductive layer (e.g., metal layer 114) and the counter electrode 130 of the plate 110 can be connected to generate an electric field, and therefore a force. The counter electrode can include, for example, a dielectric layer 132 disposed facing the conductive plate.

[0033]

[0126] The systems described herein can include one, two, or three (or four or more) layers of dielectric, e.g., 1) a dielectric layer disposed on a base layer 118, e.g., dielectric layer 116; 2) a dielectric (not shown) disposed on a conductive layer (conductive plate); and / or 3) a dielectric layer disposed on a counter electrode 130, e.g., dielectric layer 132. In some embodiments, channel 100 is or includes an electrolyte-filled reaction chamber formed between two parallel electrodes (e.g., between conductive plate 110 and counter electrode 130). When a direct current (DC) voltage is applied between these two electrodes, an electric field is generated between the electrodes, and current flows between the anode and cathode. Conductive plate 110 can be the cathode, and counter electrode 130 can be the anode, or vice versa. Unlike current caused by the flow of electrons, the current in this case is due to the flow of ions, which creates an ionic current. Ionic current refers to the flow of charge that can be observed in a conductive material or fluid, such as an electrolyte, wire, or plasma. In some embodiments, when a dielectric layer is disposed on one or both electrodes (e.g., dielectric layers 116 and / or 132), the reaction chamber can act as a parallel-plate capacitor, and no DC current flows between the electrodes. This configuration creates a constant electric field between the parallel plates. This electric field induces a force on any charged particle within the field. Nucleic acids, such as DNA, which are negatively charged molecules suspended in an electrolyte, can experience a force when placed in an electric field. This mechanism can be used to attract or repel nucleic acid molecules, such as DNA, in the presence of an electric field. In some embodiments, the cell 150 and its dielectric layers are configured to generate an electric field strong enough to denature double-stranded nucleotides attached to the cell.

[0034]

[0127] The quality and thickness of the dielectric can play a role in modulating or blocking current flow between parallel plates, such as the conductive plate 110 and the counter electrode 130. For example, during fabrication, e.g., during dielectric (oxide) deposition, charge traps can form due to the presence of metal ions, which can contribute to the degradation of oxide integrity and promote a current flow called Poole-Frenkel flow. These trap-based currents can occur even with thicker (100 nm) dielectric layers. Some methods, including the deposition of dry chlorinated thermal oxides, can eliminate these metal ions, thereby blocking current flow. Even with these high-integrity oxides, thin oxides can leak some current through tunneling. Oxide layers less than 2 nm thick can contribute to direct tunneling, while oxide layers between 5 and 20 nm have been shown to contribute to Fowler-Nordheim tunneling (wave-mechanical tunneling of electrons through an exact or approximate triangular barrier). In some embodiments, the devices described herein can include one or more dielectric layers (e.g., layers 116 and / or 132) having a thickness of at least 100 nm (e.g., 100-200 nm, 200-400 nm, or 400-1000 nm). In some embodiments, the devices described herein can include one or more dielectric layers (e.g., layers 116 and / or 132) including an oxide (e.g., a high-integrity oxide), such as: 1) a dielectric layer disposed on base layer 118, e.g., dielectric layer 116; 2) a dielectric (not shown) disposed on a conductive layer (conductive plate); and / or 3) a dielectric disposed on counter electrode 130, e.g., dielectric layer 132, having a thickness of at least 100 nm to prevent any current flow between, e.g., parallel conductive plates.

[0035]

[0128] An example system for translating digital information into and / or from nucleic acid sequences includes an array, such as microarray 200, 201, 202, or 203, as described above, integrated into a fluidic system for manipulating nucleic acids, e.g., DNA. The example system shown in FIG. 7 includes a source reservoir 300 configured to hold a fluid containing a pool of nucleic acid molecules. The system includes a main channel 101 containing a plurality of cells 150, each including a conductive plate and a counter electrode, as described above, the plurality of conductive plates and counter electrodes being disposed opposite each other along a first dimension of the main channel 101. The system includes a destination reservoir 400 that holds a fluid containing target nucleic acids or discarded nucleic acids. The system includes an input channel 102 in fluid communication with the source reservoir 300 and the main channel 101, the input channel 102 configured to dispense a first volume of fluid containing a (first) plurality of nucleic acid molecules from the source reservoir 300 to the main channel 101. The system includes an output channel 103 in fluid communication with a main channel 101 and a destination reservoir 400, the output channel 103 configured to dispense a second volume of fluid from the main channel 101 to the destination reservoir 400. The system can be used for information reading, writing, and / or computational operations as described later in this specification and shown in Figures 57-94.

[0036]

[0129] An example embodiment of a system described herein is shown in FIG. 8. The example system includes a source reservoir 300 that can include one or more zones: i) one or more zones for a fluid containing nucleic acid (e.g., DNA) components (e.g., components A-D) of one or more identifier molecules for encoding digital information (such fluids are sometimes referred to as DNA inks); ii) one or more zones for a fluid containing nucleic acid (e.g., DNA) molecules of a quality control (QC) layer; and iii) one or more zones for a buffer solution (e.g., a wash buffer). The example system includes one or more valves 105 for controlling the selective flow of the buffer and one or more fluid pumps 106 capable of inducing pressure-driven flow. The main channel 101 is configured as one or more reaction chambers (e.g., in the form of a "chip") with an inlet (input channel 102) and an outlet (output channel 103) surrounding a microarray of cells 150. The example system includes one or more destination (e.g., waste) reservoirs 400. In one example embodiment, fluid flows from DNA ink / buffer reservoir 300 to waste reservoir 400 based on pressure-driven flow induced by pump 106 and controlled by valve 105. For example, if layer 0 (A) of one example of component nucleotide 0 is to flow to the reaction chamber, pump 106 induces pressure-driven flow and valve 105 should be closed on all DNA ink reservoirs except the reservoir containing the DNA ink of layer 0 (A). This system can be used for information reading, writing, and / or computational operations as described later in this specification and shown in Figures 57-94.

[0037]

[0130] In some embodiments, the systems described herein include one or more nucleic acid reading devices, such as one or more reader modules, which may be or include a nanochannel, a nanopore, a zero-mode waveguide, or a combination thereof.

[0038]

[0131] In some embodiments, a system for writing digital information to nucleic acids and / or reading digital information encoded in nucleic acids as described herein can include one or more nanopore readers. The nanopore reader monitors changes in electrical current as the nucleic acid passes through a protein nanopore. The resulting electrical signal is decoded to provide a specific nucleic acid (e.g., DNA or RNA) sequence. An example of a nanopore reader module 500 that can be used in conjunction with the technology described herein is shown in Figures 9A and 9B. The nanopore reader 500 (e.g., nanopore reader module) includes a plate 110 and a plurality of cells 150 having one or more nanopores 501. The nanopores 501 can be located, for example, within a block of cells 150, e.g., in the center of the block as shown in Figure 9B. The schematic cross-sectional view in Figure 9A shows the location of the nanopores 501 relative to an example substrate including a base layer 118 and a dielectric (oxide) 116. The reader includes, for example, a cavity 502 disposed against a layer (e.g., dielectric 116) that separates the main channel 101 and the cavity 502. An electrolyte fills the main channel 101 and the cavity 502. The main channel 101 serves as a reaction chamber for chemical read, write, and / or computation operations as described herein.

[0039]

[0132] For example, to perform a read operation, data stored in nucleic acids 10, e.g., dsDNA, is extracted from one or more cells 150 by applying an electric field between the conductive plate 110 (metal layer 114) and the counter (top) electrode 130, thereby releasing the nucleic acids 10 (e.g., ssDNA). A voltage is then applied (e.g., immediately thereafter) between the counter electrode 130 and the base electrode 131 within the cavity. This voltage forces the ssDNA molecules from the cells 150 to translocate through the nanopore 501. In the absence of any DNA in solution, the observed current will be primarily due to ions flowing through the nanochannel. In the presence of ssDNA in solution and translocating through the nanopore 501, the resistance at the pore temporarily increases, lowering the current as the base passes through the nanopore 501. The magnitude of the current varies based on the type of base passing through the nanopore 501. The measured current is reported, e.g., by an electrical detection circuit to a computer for further analysis and sequence calling.

[0040]

[0133] In some embodiments, a system for writing digital information to nucleic acids and / or reading digital information encoded in nucleic acids as described herein can include one or more nanochannel readers. Nanochannels operate similarly to nanopores. An example nanochannel reader module 1000 that can be used with the technology described herein is shown in FIGS. 10A and 10B. The nanochannel reader 1000 is disposed in a block of cells 150 containing plates 110 and includes a central electrode 1030. One or more nanochannels 1010 are formed between a substrate 1005 (e.g., a base layer and a dielectric (oxide)) and a ceiling 1040 formed by a thin layer of material supported by the underlying walls except in the nanochannel region. Each nanochannel also includes a dedicated sensor 1020 that electrically detects the translocation of DNA molecules through the nanochannel 1010. The block electrode 1050, which defines the boundary of the block, applies an electric field between itself and the central electrode 1030 to force the DNA molecules to translocate through the nanochannel 1010, e.g., toward the outlet and reservoir 400. In some embodiments, the central electrode 1030 can include one or more fluid channels that are part of a fluid system configured to translocate the DNA molecules toward the outlet and reservoir 400.

[0041]

[0134] In some embodiments, cells 150 contain dsDNA molecules immobilized on the surface of metal layer 114 of plate 110, with one strand tethered to the surface of metal layer 114. For example, to perform a read operation, a voltage is applied between counter electrode 130 and conductive metal layer 114 of each cell 150. A voltage is then (e.g., immediately thereafter) applied between block electrode 1050 and center electrode 1030. This voltage forces the ssDNA molecules to translocate through the nanochannel 1010. Sensors 1020 within each nanochannel 1010 detect current changes as the DNA translocates through the nanochannel. These current changes are then read by electrical detection circuitry. This data is then transmitted, for example, to a computer for further analysis and sequence calling.

[0042]

[0135] In some embodiments, systems for writing digital information to nucleic acids and / or reading digital information encoded in nucleic acids as described herein can include one or more zero-mode waveguide (ZMW) readers. A zero-mode waveguide refers to an optical waveguide that guides optical energy into a volume that is small in all dimensions compared to the wavelength of the light. A zero-mode waveguide can include optical nanostructures fabricated from thin metal films that can constrain the excitation volume to the attoliter range. This small volume confinement allows single-molecule fluorescence experiments to be performed with physiologically relevant concentrations of fluorescently labeled biomolecules.

[0043]

[0136] An example zero-mode waveguide reader module 600 that can be used in conjunction with the technology described herein is shown in FIGS. 11A and 11B. The zero-mode waveguide reader 600 (e.g., a zero-mode waveguide reader module) includes a plurality of cells 150 having a plate 110 and one or more nanopore zero-mode waveguides 601. The zero-mode waveguides can be located within a block of cells 150, for example, in the center of the block as shown in FIG. 11B. The schematic cross-section of FIG. 11A shows the location of the waveguide 601 relative to an example substrate, which can include a base layer 118 and a dielectric (oxide) 116. The waveguide 601 is transparent and can be fabricated on or within the dielectric layer 116 located above the base layer 118. When an ssDNA molecule is trapped within the waveguide 601, a fluorescent signal is emitted at each base incorporated as the new strand is synthesized. This signal can be detected by an optical excitation and detection system 603 through a cavity 602 in the base layer 118 .

[0044]

[0137] To read the data from cell 150, the ssDNA is released from the cell by applying an electric field between the conductive plate 110 (e.g., metal layer 114) and the counter electrode 130. The released ssDNA molecule 10 can then diffuse into the zero-mode waveguide 601 or be forced into the zero-mode waveguide 601 by applying an electric field between the cell 150 and the zero-mode waveguide electrode. A polymerase is immobilized inside the zero-mode waveguide. Once the ssDNA reaches the zero-mode waveguide, the polymerase can synthesize a complementary strand in the presence of primers and fluorescently labeled nucleotides. The incorporation of each nucleotide generates a fluorescent signal that can be detected by the optical detection system 603. The digitized information can be sent to a computer (CPU / GPU) for further analysis and sequence calling.

[0045]

[0138] The technology described herein includes a sensor device, e.g., as described above. In some embodiments, the sensor device is disposed at the end, e.g., the tip (downstream end), of the nanochannel. One example of such a sensor device is or can include an electric / electronic sensing device, e.g., an electrolyte oxide field effect transistor (EOSFET).

[0046]

[0139] As mentioned above, charge-based DNA sequencing can be performed using solid-state or organic nanopores by measuring the ionic current as DNA molecules translocate through the nanopore. The sensitivity and scalability of ionic current measurement techniques can be limited. Described herein is a technology involving a device comprising a planar electrolyte oxide field effect transistor (EOSFET) in combination with a nanopore to improve the sensitivity and scalability of nanopore-based nucleic acid sequencing.

[0047]

[0140] Nanopore FET devices are typically designed to meet the requirements of base-by-base sequencing. A single DNA base is approximately 0.7 nm long when extended and approximately 0.35 nm long in a helical configuration. To perform base-by-base sequencing, the height of the FET channel (e.g., n-channel) must be less than 0.7 nm to be sensitive enough to detect charge fluctuations as a nucleic acid (e.g., DNA) molecule translocates through the pore. Fabrication of such FET structures can be tedious. For example, annealing to dope (modulate the conductivity of a semiconductor material by chemically combining it with other elements) the source and / or drain of the FET is performed at high temperatures, which can lead to ionization of nucleic acids into the channel and reduce the channel's sensitivity.

[0048]

[0141] Described herein is a technology that includes a method for sequencing nucleic acid (e.g., DNA) molecules at the component level. An example nucleic acid (e.g., DNA) component (identifier component) within an identifier can be approximately 30 bases long. This implies that a readout component with a compact secondary structure is approximately 30 bases long, or approximately 21 nm long, in its extended form. This implies that a nanopore FET with a channel height (or length) of approximately 10-20 nm is sufficient to perform highly sensitive measurements of charge within the secondary structure of the readout component. This increase in channel dimensions improves the robustness and / or sensitivity of nanopore-based sequencing because the fabrication process is simpler than for smaller channels using conventional ion implantation and annealing methods.

[0049]

[0142] An example nanopore-field effect transistor (FET) combination reader module 700 that can be used in conjunction with the technology described herein is shown in Figure 12. The nanopore-FET reader 700 (e.g., nanopore-FET reader module) includes a plurality of cells 150 having plates 110 and one or more nanopores 701. The nanopores 701 can be located, for example, within a block of cells 150, e.g., in the center of the block as shown for nanopore reader 500 in Figure 9B. The schematic cross-sectional view in Figure 12 shows the location of the nanopores 701 relative to an example substrate including a base layer 118 and a dielectric (oxide) 116. The reader can include, for example, a cavity 702 disposed inversely to a layer (e.g., dielectric 116) that separates the main channel 101 and the cavity 702. An electrolyte containing nucleic acid molecules 10 fills the main channel 101 and the cavity 702. The main channel 101 serves as a reaction chamber for chemical read, write, and / or computation operations as described herein. Devices such as the nanopore reader 500 or nanopore FET reader 700 can be fabricated, for example, from a silicon wafer with a cavity 502, 702 constructed by chemical wet etching. The main channel 101 can be created, for example, by bonding another silicon wafer with a cavity or a polymer structure with a well-type structure. The chambers are separated by a dielectric layer or membrane 116, for example, a silicon dioxide or silicon nitride membrane. An exemplary dielectric 116 contains nanopores with a diameter of less than 10 nm. In some embodiments, the nanopores have a diameter between 5 nm and 10 nm. In some embodiments, the nanopores have a diameter of less than 20 nm. A field effect transistor (FET) 703 is attached to the membrane, for example, as shown in Figures 12-14.

[0050]

[0143] The structure of an example nanopore FET 703 is shown in Figures 13 and 14. One example FET is an n-channel depleted electrolyte oxide field-effect semiconductor (EOSFET). Unlike conventional MOSFETs, the gate (metal layer) is replaced by an electrolyte solution. The FET includes a source region 711 of heavily doped n-type silicon (silicon combined with other elements so that electrons have a negative charge), a drain region 712 of heavily doped n-type silicon, a substrate region 713 of lightly doped or undoped silicon of the opposite polarity (p-type), and a narrow n-channel 714 formed by the lightly doped n-type silicon. This configuration forms an n-channel 714 between the source 711 and drain 712 when the gate voltage is zero, allowing current to flow between the source and drain with a drain-source voltage applied. When a negative voltage is applied to the gate, the n-channel width decreases, depleting the majority carriers (electrons) and thus reducing the source-drain current. The n-channel can be depleted by placing negative charges near the oxide layer (dielectric 116), thereby inducing an electric field between the gate (electrolyte) and the channel. This introduction of negative charges is equivalent to applying an external negative voltage.

[0051]

[0144] The EOSFET technology described above can be used in conjunction with sequencing techniques, such as component-level sequencing, as described herein. In some embodiments, negative charges arise from the phosphate backbone of the nucleic acid (e.g., DNA) identifier component and / or secondary structures in the readout component hybridized to the single-stranded identifier molecule, as described below. When the identifier or identifier-readout component complex migrates through the nanopore FET, the source-drain current decreases depending on the charge present on the molecule. In example regions without secondary structure, the resulting current decrease can be smaller, while in regions with higher charge containing secondary structure, the resulting current decrease can be larger. Depending on the channel doping concentration, the decrease in source-drain current can be proportional to the magnitude of the charge in the identifier and / or secondary structure. Thus, if each component in the input sequence is tagged with a different amount of negative charge, nanopore FETs can be used to detect the sequence of nucleic acid (e.g., DNA) molecules at the component level.

[0052]

[0145] Figure 14 shows the dimensions of the components of an example nanopore FET described herein. The source 711 and drain 712 are heavily doped n-type silicon separated by a relatively large area of ​​substrate 713 made of lightly doped or undoped p-type silicon. This separation minimizes electron tunneling effects between the source and drain when a source-drain voltage is applied. The source and drain regions near the channel 714 are designed to be 5-10 nm wide. In some embodiments, the drain region near the channel can be 1-50 nm wide. The substrate region near the channel of the example nanopore FET can be the same length as the nanopore 701 and the same width as the source and drain regions. A small n-type channel of 3-5 nm can be formed by lightly doping the substrate near the nanopore between the source and drain regions. The silicon layers (10-20 nm thick) of the source 711, drain 712, and substrate 713 may be deposited to form an amorphous silicon FET or obtained by thinning the silicon layer of a silicon-on-insulator (SOI) wafer. The channel thickness (height) can have a greater effect on sensor sensitivity than the channel length (distance between the source and drain). In some embodiments, the channel length is equal to or less than the diameter of the nanopore 701. A thinner channel produces a more sensitive sensor. For SOI wafers, a doping method using ion implantation and annealing can be performed. For amorphous silicon FETs, different concentrations of doped silicon can be deposited to avoid a separate doping step. A 5-10 nm thick dielectric layer (SiO2 or Si3N4) can cover the source, drain, substrate, and channel regions. The dielectric layer can passivate molecules to prevent nonspecific absorption of molecules into the electrolyte.

[0053]

[0146] As described herein, the EOSFET sensing device, e.g., nanopore FET 703, can be one of an array of sensing devices, e.g., devices including 10, 20, 30, 40, 50, 100, 1,000, or more EOSFET sensing devices.

[0054]

[0147] A diagram of the architecture of an example system described herein is shown in FIG. 15. The example system includes a microarray chip described herein, e.g., a chip including a main channel 101 configured as one of one or more reaction chambers. The chip includes microarray electrodes or cells (e.g., cells 150) grouped into blocks, e.g., arranged in units or blocks, as shown in FIG. 3. Nucleic acid (e.g., DNA) molecules can be assembled and / or disposed on a conductive plate, e.g., plate 110 (also called a microspot). The example system includes a reader module. The reader module can be or include a nanopore (e.g., nanopore reader 500), a nanochannel (e.g., nanochannel array 1000), a zero-mode waveguide (e.g., zero-mode waveguide reader 600), or a nanopore FET reader 700, which can be, e.g., disposed within or between each block, as described herein. In some embodiments, one or more reader modules can be included within or fluidically connected to each block. In some embodiments, one or more reader modules can be connected to one or more cells or all cells on the entire chip. In some embodiments, the reader module can be on a separate portion of the chip or on a separate reader chip with fluidic and electrical connections to the microarray chip. The microarray chip can include one or more heater elements, such as resistive heaters or Peltier heaters (e.g., solid-state active heat pumps that transfer heat from one side of the device to the other), along with a heater controller to maintain a specific temperature. The heater can be configured to heat one or more conductive plates, such as plate 110. In some embodiments, each cell includes or is connected to a separate heater element so that each cell can be heated independently. In some embodiments, two or more cells 150 share a heater element.

[0055]

[0148] The microarray chips described herein can be part of a main channel (e.g., main channel 101) that can be connected to a set of fluid lines or channels (e.g., input channel 102 and / or output channel 103) to connect the chip to one or more fluid reservoirs (e.g., source reservoir 300 and / or destination reservoir 400). The systems described herein can include a fluid pumping system that includes one or more pumps (e.g., pump 106) and valves (e.g., valve 105) to control the flow of fluid through the system. The reservoirs and corresponding fluid lines can be designated as inputs or outputs depending on the direction of flow. The valves and pumps in the fluid pumping system can be used to control the flow of liquid between the reservoirs and the chip. The operation of the fluid pumping system can be controlled by a computing system that includes a memory and a CPU. A controller or driver circuit is connected between the computer and the chip via electrical wires to carry signals (e.g., analog signals). The controller can receive instructions from a computer via a communication bus (e.g., I2C, SPI) and convert them into electrical signals to the microfluidic chip, for example, to induce an electric field or activate a heater on or over a set of cells. The controller can also read the voltage at a given cell and report it to the computer. The controller circuitry can be a combination of analog and digital components.

[0056]

[0149] The system described herein may include an electrical detection circuit that reads signals from the reader module and / or the cell and transmits the signals to a computer's CPU or GPU (graphics processing unit) for further analysis or storage. This detection circuit may be connected to the chip via electrical wires and to the computer via a communication bus. When a nanopore or nanochannel is used as the readout technology, the circuit may, for example, measure analog currents as nucleic acid (e.g., DNA) molecules translocate through the nanopore or nanochannel, convert them to digital values, and report them to the CPU or GPU. The system may include an optical detection system including optical components (e.g., lenses, optical fibers, polarizers, etc.) and photodetectors (e.g., cameras, photon counters, etc.) to measure optical signals from either the cell or the readout module and report the digitized values ​​to the computer. For example, when a zero-mode waveguide is used as the readout technology, a camera in the optical system may detect the fluorescence intensity from the waveguide and convert the fluorescence signal into a digital signal.

[0057]

[0150] A system as described herein may include a computer system, for example, as shown in Figure 15. The computer system may include a CPU, a GPU, memory, storage, peripherals, and / or associated software. The computer may be used to control the system and all of its components. The GPU in the system may be used to perform analytics, including using machine learning, from data generated by the chip.

[0058]

[0151] Systems including microarray chips as described herein can be used in various configurations to write data, read DNA-encoded data, store, calculate, and / or perform QC. Example workflows utilizing one or more components of the system are described herein. All operations can be controlled by a computer (including a processor and memory in which the instructions are stored) via instructions sent to subsystems or components. For example, when a workflow statement states that "a controller issues ...," it is understood that the computer sends instructions / commands to the controller, and the controller processes the instructions and sends signals to or receives instructions from the chip or other components.

[0059]

[0152] An example workflow for operating a system as described herein can include the following processes (DNA is used as an example nucleic acid molecule):

[0060]

[0153] In some embodiments, the technology described herein includes systems and methods for writing data using DNA. An example workflow is shown in Figure 16. One or more source reservoirs (e.g., source reservoir 300) contain pre-synthesized oligonucleotides (components, e.g., referred to as DNA 10), e.g., about 30 bases in length. The temperature of a chip with multiple cells 150 is raised using a heater to 5°C below the melting temperature (Tm) of the components and the sticky ends of adapter molecules 112 disposed on a conductive plate 110. A controller applies voltage to specific cells. A pumping system (e.g., pump 106) flows the contents of one reservoir (a fluid containing nucleic acid components) into a main channel 101 configured as a reaction chamber. The solution is retained in the reaction chamber for a finite time to allow hybridization of the components to the adapters on the chip. To flush the reaction chamber, the pumping system then flows the contents of the reaction chamber (including any unhybridized components) into a waste reservoir (e.g., destination reservoir 400). The pumping system then forces the wash buffer from the source reservoir into the reaction chamber and out to the waste reservoir. The steps of applying a voltage to a particular cell, flowing a solution containing the components, holding the solution to hybridize the components, and flushing the chamber are repeated for all of the input reservoirs containing the components used in the data set. The pumping system (e.g., pump 106) flows the solution containing the ligase from the source reservoir into the reaction chamber. The solution is held in the reaction chamber for a finite time to allow the ligation reaction to complete. The pumping system forces the wash buffer into the reaction chamber and out to the waste reservoir. The cell now contains the data to be written to the DNA. This data may optionally be extracted for storage elsewhere, for example, as described herein.

[0061]

[0154] In some embodiments, the technology described herein includes systems and methods for extracting data for off-chip storage or processing. This use case assumes that the data has already been written to nucleotides on the chip and stored on the chip, for example, as described above. A pumping system (e.g., pump 106) flows buffer solution from the source reservoir 300. Data can be extracted by one or more of the following techniques: (a) The temperature of the chip is raised above the Tm of the full-length molecule. This can melt the single-stranded DNA containing the data. The remaining DNA on the chip still contains the encoded data. (b) A controller applies a voltage to one or more cells 150, melting one strand of DNA from the surface while the other strand containing the data remains on the chip plate 110. (c) An enzyme for restriction digestion at the base of the adapter 112 can be flowed into the chip, releasing the dsDNA containing the data. The pumping system flows buffer solution from the reaction chamber to a collection reservoir (e.g., destination reservoir 400).

[0062]

[0155] In some embodiments, the technology described herein includes systems and methods for reading data stored on a chip using a nanopore module, e.g., nanopore reader module 500. An example workflow is shown in Figure 17. A pumping system (e.g., pump 106) flows reader buffer from source reservoir 300 into main channel 101, configured as the reaction chamber of the chip. A controller applies a voltage to cell 150, from which data is read and ssDNA is extracted. The controller applies a voltage between counter electrode 130 and base electrode 131 of the chip. Molecules translocate through nanopore 501, causing a current fluctuation in the nanopore. An electrical detection circuit measures the current and reports the current value to a computer. A CPU / GPU performs analysis of the current value and converts the current reading into a DNA sequence (e.g., base calling). The CPU / GPU analyzes all sequences and decodes the data.

[0063]

[0156] In some embodiments, the technology described herein includes systems and methods for reading data stored on a chip using a zero-mode waveguide reader module, such as waveguide reader module 600. An example workflow is shown in FIG. 18. A pumping system (e.g., pump 106) flows reader buffer from source reservoir 300 into main channel 101, configured as the reaction chamber of the chip. A controller applies a voltage to cell 150, from which data is read and ssDNA is extracted. Molecules diffuse into waveguide 601 or a voltage is applied between the waveguide and counter electrode 130. Molecules are analyzed by waveguide 601 by generating light pulses as described above. Light detection system 603 measures the light intensity, converts the light signal to a digital signal, and reports the data to a computer. A CPU / GPU performs analysis of the digital values ​​of the light intensity and converts these values ​​to DNA sequences (e.g., by applying base-calling techniques). The CPU / GPU analyzes the sequence and decodes the data.

[0064]

[0157] In some embodiments, the techniques described herein include systems and methods for calculating information stored on a chip. A pumping system (e.g., pump 106) flows reader buffer from a source reservoir 300 into a main channel 101 configured as a reaction chamber of the chip. A controller / CPU applies a voltage to one or more cells 150 (sources) from which data is read, thereby releasing a DNA molecule 10 from the source cell. The controller / CPU applies a different voltage to one or more different cells 150 (destinations) to force the molecule into the destination cell. The pumping system flows enzymes and fluorophores from the source reservoir 300 to act on the molecule on the destination cell. An optical detection system detects optical signals in the source and destination cells and reports the signals to a computer. The source molecules are operands, the enzymes and fluorophores are operators, and the molecule in the destination cell is the result of the operation.

[0065]

[0158] In some embodiments, the technology described herein includes systems and methods for clearing incomplete product molecules after a writing step as described herein. A pumping system (e.g., pump 106) flows buffer solution from an input reservoir into the main channel 101, configured as the reaction chamber of the chip. A controller applies a specific voltage to all cells 150 to melt only the incomplete products (e.g., apply an electric field strong enough to denature the incomplete products but too weak to denature the complete products). Incomplete products can also be thermally denatured at a temperature lower than the melting temperature of the fully formed product. The pumping system flows buffer solution from the reaction chamber into a destination reservoir 400, e.g., a waste output reservoir. The dsDNA remaining on the chip are full-length molecules that can be extracted for reading or storage based on the use cases described herein.

[0066]

[0159] In some embodiments, the technology described herein includes systems and methods for measuring write efficiency. A pumping system (e.g., pump 106) flows quality control (QC) buffer containing tagged DNA molecules from a source reservoir 300 into a main channel 101 configured as a reaction chamber of the chip. An optical system measures fluorescence from each cell 150, quantifies the fluorescence values, and sends the digitized information to a computer. A CPU / GPU performs analysis of the cells 150 and calculates the write efficiency (the percentage of full-length molecules over the total possible molecules in the cell 150). A controller applies a voltage to all cells 150 to remove the QC molecules. The pumping system flushes the buffer through the reaction chamber and into a destination reservoir 400, washing the reaction chamber.

[0067]

[0160] In some embodiments, the techniques described herein include systems and methods for measuring the length distribution of written molecules. A pumping system (e.g., pump 106) flows a QC buffer containing a mixture of DNA molecules and different fluorophores corresponding to different layers into the main channel 101, configured as the reaction chamber of the chip. The molecules are captured by one or more cells 150 (optional). An optical system measures the fluorescence value from each cell for each color, quantifies the fluorescence, and sends the digitized information to a computer. A CPU / GPU performs the analysis of the cells 150 and calculates the molecular length distribution.

[0068] example

[0161] Described herein is a technology for using nucleic acids (e.g., DNA) to write, store, read, and / or compute digital information based on assembling smaller fragments of nucleic acid molecules (e.g., DNA) using ligation to encode digital information. Described herein are systems and methods for encoding digital information into nucleic acids. Exemplary components that can be ligated to encode digital information into so-called "identifiers" are shown in Figure 19. Components can be grouped into "layers," where a central double-stranded region contains a unique sequence and overhangs in one layer are complementary to components in an adjacent layer. In some embodiments, edge layers (layer 0 and layer n (e.g., 3)) can have blunt ends. In some embodiments, edge layers can be adapted to have sticky ends. The technology includes an optional QC layer that is complementary to layer n and contains single-stranded nucleic acids (e.g., DNA) with segments containing fluorophores.

[0069]

[0162] One application of the systems and methods described herein is the combinatorial assembly of shorter DNA fragments via ligation to construct longer (identifier) ​​molecules. To construct molecules, a solution containing nucleic acids (e.g., a so-called "DNA ink") is sequentially flowed through a main channel 101 configured as a reaction chamber of a chip as described herein, while a local electric field is applied to one or more cells 150 to attract or repel the DNA molecules to different cells or locations (e.g., simultaneously). In the example presented in Figures 20-27, two unique combinations of DNA molecules are assembled. First,

number

number

number

number

[0070]

[0163] The next steps in this example are:

number

number

[0071]

[0164] In some embodiments, the technique includes one or more quality control (QC) measures that can be performed on the assembled nucleic acid (e.g., nucleic acid) strands. For example, quantification of nucleic acids (e.g., DNA) can be performed at any location (cell 150), for example, by hybridizing a QC layer with a fluorophore and quantifying the emitted photons (Figure 27). One problem with ligation-based assembly is that the reaction can be inefficient. The number of fully formed nucleic acid (e.g., DNA) products may be only a fraction of the total amount of component nucleic acids and / or their products. Smaller fragments that did not complete the reaction may be attached to the cell, as shown, for example, in Figure 29. The ratio of fully formed products to the remaining products, indicating ligation efficiency, can be quantified. The left cell in Figure 29 shows a lower amount of fluorescence compared to the right cell, where all identifiers have a fluorophore, due to the fewer full-length identifiers present in this cell. Traditional methods for quantifying ligation efficiency require several time-consuming tasks, such as Monarch cleanup, gel extraction, and / or qPCR, with losses at every step. In one example of the method described herein, these steps are replaced with a single fluorescence-based readout, eliminating the need for any post-processing steps. This process can help save hours, if not days, of post-processing time.

[0072]

[0165] In some embodiments, the distribution of incompletely formed products can be assessed. This metric can indicate ligation efficiency for nucleic acid (e.g., DNA) strands of different lengths. Existing methods may require DNA purification / cleanup and (automated) electrophoresis runs, with associated losses at every step and low electrophoretic sensitivity. In some embodiments of the technology described herein, a QC readout distribution can be automatically obtained by attaching different fluorophores to nucleic acids (e.g., DNA) of different lengths and performing quantification of emitted photons (Figure 30). The left cell in Figure 30 exhibits fluorescence of different wavelengths due to the presence of different fluorophores hybridized to different layers above this cell compared to the right cell, where all identifiers have the same fluorophore. If this device is used as a memory chip, one desired rapid readout can be to identify locations containing or lacking data, as shown, for example, in the first and second locations in Figure 31, respectively. The fluorescence of the QC layer can be used to obtain a fluorescence map of an example array, as shown in Figure 32.

[0073]

[0166] In some embodiments, post-processing steps toward reading information written to nucleic acids (e.g., DNA) via the methods described herein can include removing any abortive ligation products. The abortive products can result in increased noise during sequencing and reading. Existing methods reduce noise through gel extraction, a manual and unreliable method with high variability. In some embodiments of the technology described herein, the method includes heat denaturing the abortive products below the melting temperature of the fully formed product, thereby separating and removing the abortive products. Figure 33 shows the removal of the abortive products from the left cell. In some embodiments, the abortive products can be denatured by applying a force, for example, using an electric field of lower strength than that required for denaturing the complete product.

[0074]

[0167] In some embodiments, once the fully formed product is purified, data can be stored in nucleic acid (e.g., DNA) at each location (cell), or retrieved and stored separately. To extract the data for subsequent processing, storage, or computation, data from any given location can be individually extracted, for example, by applying a local electric field. Figure 34 shows the retrieval of a full-length identifier from the left cell. Once the data is retrieved, it can be sequenced, for example, by using (integrated) nanopore-, nanochannel-, and / or zero-mode waveguide-based technologies within the same chip, for example, as described herein. Additionally, data retrieved from any given location can be used for on-chip computation, for example, as shown in Figure 35. In this example, an "add" operator is illustrated using a fluorescence-based readout of the ligation of two molecules retrieved from different locations. This operation is similar to retrieving electronic data from different locations on a memory chip and performing the addition in memory, except that this computation step is accomplished using nucleic acid (e.g., DNA). The computation step, e.g., ligation, can be performed in solution or in one or more cells.

[0075]

[0168] In some embodiments, data may be encoded in the nucleotide sequence or the length of the nucleic acid. The nucleic acid can be hybridized to a surface (e.g., a cell), and the electrical properties of the nucleic acid (and thus the information encoded therein) can be measured. Computation may be performed by placing nucleic acid strands from different cells in the same location, called a "result cell," thereby performing the computation (e.g., an addition step—the presence of identifiers from two different input cells indicates a successful addition). The computational processes described herein can be reversible (e.g., through selective denaturation), allowing for the erasure and rewriting of information. In some embodiments, the computation or operation can include bridging two bound nucleic acid molecules. Changes in electrical or fluorescent signals can be detected, from which information can be extracted. Examples of data storage, retrieval, and / or computational techniques that can be used in conjunction with the array technologies described above are described later in this specification.

[0076]

[0169] Described herein are technologies, including systems, devices, and methods, for writing, storing, reading, and performing computations on digital information using nucleic acid molecules (e.g., DNA). The technologies include, for example, devices and methods for detecting one or more components of a translocating nucleic acid strand to read a nucleic acid sequence using nanopores, nanochannels, and / or sensors. The reading devices and methods described herein may be or include standalone devices, or may be integrated into a device that includes one or more individually or block-addressable electrode microarrays or nanoarrays (e.g., arrays 200, 201, 202, 203) for writing, storing, retrieving, reading, and / or performing computations / manipulations on digital information.

[0077]

[0170] Current DNA sequencing technologies read one base at a time, leading to slower read rates. Examples of such technologies include sequencing-by-synthesis, Illumina®-type sequencing, or Oxford Nanopore®-type sequencing. For example, in certain applications where the sequence contains repeats or some known pattern, it may not be necessary to sequence nucleic acids (e.g., DNA) one base at a time. Instead, as described herein, approaches that read nucleic acid sequences (e.g., DNA) at a higher level, e.g., at sets of bases (e.g., identifier "components"), can be highly efficient and fast. Existing approaches can be cumbersome because they require prior modification of nucleic acid sequences (e.g., DNA) using cleavage enzymes and RecA proteins before the DNA can be sequenced. The techniques described herein can provide a simpler approach, e.g., because they can involve only a single hybridization step.

[0078]

[0171] The techniques described herein can provide a simple approach to rapidly sequence and / or extract information from nucleic acid molecules (e.g., DNA). The techniques described herein can use current-based sensing techniques, which can lead to cheaper sequencing compared to techniques using optical methods. The techniques described herein can use nanochannels, which can improve manufacturing scalability compared to other techniques that use biological molecules (e.g., proteins), such as protein nanopores. Nanochannels made from solid-state materials can have a longer lifespan than biological nanopore technologies.

[0079]

[0172] Described herein are devices and methods for reading nucleic acid (e.g., DNA) sequences in sets of bases (e.g., "components" of an identifier) ​​that encode digital information, as described herein. Traditional sequencing methods read one base at a time, detecting the base using optical or electrical methods and performing base calling on each base. Described herein is technology that accelerates the reading process by reading one or more sets of bases (e.g., "components") at a time instead of one base at a time.

[0080]

[0173] The technology described herein includes (A) a single-stranded nucleic acid (e.g., DNA) having a defined number of bases, e.g., at its 3' and 5' ends, complementary to a component to be sequenced (e.g., an identifier component that is a component of a nucleic acid identifier that encodes digital information), and (B) a reading component nucleic acid (e.g., DNA) organized into a compact structure and including a sequence of nucleic acid (e.g., DNA) between (e.g., at its 3' and 5' ends) that provides a charge. The charge of the reading component can vary and can be higher than the charge of the complementary strand of DNA.

[0081]

[0174] The technology described herein involves a set of readout components complementary to individual components (eg, identifier components) of the DNA to be sequenced, each readout component having a unique charge.

[0082]

[0175] The technology described herein includes a sensor device comprising an electronic device, e.g., a metal-oxide-semiconductor field-effect transistor (MOSFET), where the gate voltage can be modulated through the charge of a moving readout component to alter the source-drain current of the transistor. The sensor device can be disposed at the distal end (downstream end) of a nanochannel or nanopore. In some embodiments, the sensor device can be disposed at the proximal end (upstream end) of a nanochannel or nanopore.

[0083]

[0176] The technology described herein includes methods for sequencing nucleic acid molecules (e.g., DNA) by hybridizing a reading component to the nucleic acid molecule to be sequenced, translocating the DNA through a nanochannel or nanopore, and reading the sequence using a sensor.

[0084]

[0177] The technology described herein includes devices and methods for sequencing nucleic acid molecules (e.g., DNA) by sets of bases (e.g., components, e.g., identifier components) rather than individually. The nucleic acid molecules (e.g., DNA) to be sequenced can be in double-stranded or single-stranded form. In some embodiments, the nucleic acid molecules (e.g., DNA) to be sequenced are in single-stranded form. Single-stranded nucleic acid molecules can be easily obtained, for example, by using heat to melt the double-stranded nucleic acid molecules (e.g., DNA) and separate the single strands, e.g., by heating the plate 110 of the cell 150. The technology described herein includes a "reading component," which is or includes a "secondary structure," e.g., a single-stranded nucleic acid molecule (e.g., DNA) sequence having a nucleic acid sequence that forms a compact structure and remains single-stranded at the 3' and 5' ends. The identifier component can be a unique sequence of about 500 bases in length. In some embodiments, the identifier component can be a unique sequence between 10 and 5,000 bases, between 100 and 1,000 bases, or between 200 and 800 bases in length. A reading component, as described herein, is a 12-base complementary strand that is complementary to a specific site on the nucleic acid molecule (e.g., DNA) to be sequenced, e.g., the identifier component, at the 3' and 5' ends. In some embodiments, the complementary strand at the 3' and / or 5' ends can have a length of 1 base (b or bp) to 50 b, 2 b to 40 b, 3 b to 30 b, or 4 b to 20 b. In some embodiments, the secondary structure can have a length of 10 b to 50 b, 50 b to 100 b, 100 b to 200 b, 200 b to 300 b, or greater than 300 b. In some embodiments, such secondary structures can have the form of, for example, a loop, wave, braid, twist, coil, fold, knot, ball, or any combination thereof. In some embodiments, the reading component has a single secondary structure. In some embodiments, the reading component has two or more secondary structures, for example, 2, 3, 4, 5, or more. In some embodiments, such secondary structures can be located between the 3' and 5' ends of the reading component, at the 3' end of the reading component, at the 5' end of the reading component, or a combination thereof.The 3' and 5' ends of the readout component bind to specific sites on the nucleic acid molecule (e.g., DNA) to be sequenced. These sites represent known sequences or patterns, e.g., sites encoding digital information (e.g., identifier components), e.g., as described herein. Compact secondary structures, such as nanoparticles, provide a large charge in a small volume. Large amounts of digital information can be stored in a small volume, e.g., in libraries of any size, from 64 kilobytes to 1 megabyte or more. The single-stranded nucleic acid molecule (e.g., DNA) to be sequenced is then hybridized with the readout component to generate a structure, e.g., as shown in Figure 36. When the molecule translocates through a nanochannel (or nanopore) by application of an electric field, the nucleic acid molecule (e.g., DNA) can be elongated, as shown in Figure 36, allowing the readout components to pass or flow through the gate of the sensing device, one readout component at a time.

[0085]

[0178] The technology described herein includes a sensor device. In some embodiments, the sensor device is disposed at the end of a nanochannel (or nanopore), e.g., at the tip (downstream end). One example of such a sensor device is or includes an electric / electronic sensing device, e.g., a MOSFET. A MOSFET includes a source, a drain, and a gate, which may include a gate electrode. When a gate voltage is applied to the MOSFET, the MOSFET turns on, thereby allowing current to flow between the source and the drain. When the gate voltage is changed, the source-drain current changes accordingly. The gate voltage can be perturbed by charge introduction above and / or near the gate electrode. When a nucleic acid molecule (e.g., DNA) to be sequenced having a hybridized readout component (e.g., an "input strand" having an "input sequence") moves above and / or near the gate of the MOSFET, a change in the source-drain current can be detected. This current change can depend on the charge amount of secondary structures within the readout component. By limiting the stacking of secondary structures and packing large charges into small stacks, abrupt changes in current can be generated, as shown, for example, in Figure 37. Different secondary structures, e.g., nucleic acids of different lengths, can carry different charges. Therefore, by measuring the current, the corresponding charges, and therefore the readout components, can be identified.

[0086]

[0179] In some embodiments, the sensor device is or includes one or more electronic signal processing devices. In some embodiments, the sensor device includes or is electrically connected to one or more processors of a computing system, as described herein. The computing system can be configured to process signals received from the electronic or optical sensing devices, e.g., perform one or more steps of performing base calling, and convert the electrical or optical signals into sequence information, e.g., sequence information of an input strand.

[0087]

[0180] In some embodiments, as an entire nucleic acid molecule (e.g., DNA, e.g., identifier component) translocates through a nanochannel or nanopore, the measured current varies according to the charge amount of the various readout components hybridized to the DNA being sequenced (see Figure 38). For example, the longer the sequence of the readout component, the greater the charge and therefore the more pronounced the signal. Analysis of the series of current fluctuations can directly indicate variations in the nucleic acid molecule (e.g., DNA) sequence of the input strand.

[0088]

[0181] Nucleic acid molecules can be suspended in a liquid solution, for example, in the main channel 101, as described above. The liquid solution used in conjunction with the techniques described herein can include unincorporated readout components and nucleic acid molecule (e.g., DNA) strands to which the readout components are hybridized. Described herein are techniques for distinguishing signals arising from individual readout components (e.g., suspended) from nucleic acid molecules (e.g., DNA, e.g., identifier molecules) to be sequenced. For example, nucleic acid molecules (e.g., DNA) to be sequenced can be linked and / or hybridized at one or both ends to known start and / or stop sequences, e.g., sequences with large charges in the readout components. Large charges in the start / stop sequences lead to large changes in current (Figure 39). By analyzing a series of current changes and identifying the start / stop current levels, current changes of nucleic acid molecule (e.g., DNA, e.g., identifier molecule) sequences can be distinguished from unincorporated individual readout components or abortive products. In some embodiments, to minimize noise from unincorporated readout components, unincorporated readout components can be filtered out using one or more molecular purification methods.

[0089]

[0182] In some embodiments, the boundaries (start / stop) of a sequence (e.g., an input sequence of, for example, an identifier molecule) can be identified using start and / or stop sequences that have multiple small secondary structures that produce a characteristic pattern of current, as shown, for example, in FIG.

[0090]

[0183] In some embodiments, reading components having secondary structure can be applied as part of a nucleic acid "writing" process, e.g., when an input strand is assembled from, e.g., multiple identifier components, e.g., as described herein. Exemplary identifier components that can be ligated to encode digital information into so-called "identifiers," e.g., as described herein. Components can be grouped into "layers," e.g., as described above.

[0091]

[0184] In one embodiment, as shown in Figure 41 (Design A), one strand of the identifier contains multiple identifier components (e.g., components A and B). An example complementary strand (reading component) contains four distinct regions: (1) a region complementary to the previous (layer n) component, (2) a region complementary to the current (layer n+1) component, (3) a non-complementary component (e.g., a flap), and (4) a secondary structure, e.g., a compact secondary structure with a high charge density (e.g., a charge density higher than that of a DNA molecule without secondary structure). Regions 1 and 2 allow identifier components A and B from different layers to hybridize, allowing a ligase to ligate the nick between the identifier components. Region 3 is not complementary to any sequence within A or B, allowing a flap to be created. Region 4 can be or include a secondary structure as described herein, and the secondary structure can include a (long) sequence capable of self-hybridizing to form a compact secondary structure. The compact structure can exhibit a large charge density per unit volume (e.g., a charge density higher than that of a DNA molecule without secondary structure). In some embodiments, the secondary structure can have a length of 10-50 bases or base pairs (bp), 50-100 bp, 100-200 bp, 200-300 bp, or greater than 300 bp.

[0092]

[0185] The read component shown in Figure 41 (Design A) can constitute one strand of an identifier molecule assembled during a write process as described herein, for example, as shown in Figure 42. Different secondary structures induce different electrical signals in a reader, e.g., a device comprising a nanochannel (or nanopore) and a MOSFET sensor device as described herein. For example, the longer the secondary structure, the greater the current across the MOSFET. An advantage of this technology is that once an identifier molecule is created with the read component during the write process, no hybridization step is required during the subsequent read step. Molecules in a library can be directly fed into a nanochannel-based (or nanopore-based) reader device and readout can be performed without any post-processing. This technology can add significant value to technologies for reading, writing, computing, and / or storing digital information in nucleic acids (e.g., DNA), for example, because it eliminates some or all post-processing steps between the write and read steps of a nucleic acid (e.g., DNA) storage and retrieval pipeline.

[0093]

[0186] In some embodiments, the input strand is assembled from identifier components as described herein. The complementary strand includes components (e.g., "primary read components") that include only regions 1-3 of the components in Design A described above, as shown in Figure 43 (Design B). In some embodiments, this input strand is configured to allow hybridization (e.g., of the primary read component) and ligation of the identifier component during the writing process. To read the sequence, a secondary read component having a secondary structure as described above can hybridize to the flap (3) of the primary read component. The nucleic acid molecule (e.g., DNA) can now be read as described above.

[0094]

[0187] In some embodiments, the techniques described herein can read nucleic acids (e.g., identifiers) using optical signals instead of electrical signals. For example, light-emitting tags, e.g., fluorophores, can be used in place of or in combination with secondary nucleic acid structures as described above, e.g., in Designs A and B. In the example embodiment shown in Figure 44 (Design C), the readout component can be structured to include four regions, e.g., as described above for Design A, except that Region 4 is a fluorophore instead of a nucleic acid (e.g., DNA) with secondary structure. In some embodiments, the nanochannel (or nanopore) of a readout device as described herein can be configured to detect optical signals. In some embodiments, the nanochannel (or nanopore) can be configured to include a window for optical interrogation. Detection can be performed using, for example, a fluorescence measurement system including one or more lenses, optics, cameras, photon counters, or other optical detection or image processing implementations. As nucleic acid molecules translocate through a nanochannel (or nanopore) upon application of an electric field, the nucleic acid molecules (e.g., DNA) can be stretched, as shown, for example, in Figure 44, thereby allowing readout components to pass through windows in the nanochannel (nanopore) one at a time. In some embodiments, the fluorescence of each readout component can be measured as it passes through the window. Different colors or different light intensities (or both) can be used to provide each readout component with a distinguishable optical signature. The observed series of optical signals can be translated into an identifier sequence, as shown, for example, in Figure 45.

[0095]

[0188] In one embodiment, the read component can be a boundary read component that is designed similarly to the read component as shown in Figure 41 (Design A), but with region 2 truncated to not include the main part ("payload portion") of identifier component B. Thus, the (boundary) read component does not correspond to a single identifier component within each layer, but rather to a combination of identifier components from adjacent layers, as shown, for example, in Figure 46 (Design D). The advantage of this approach is that it reduces the material required for the read component and can accelerate the read process, for example by a factor of two.

[0096]

[0189] In some embodiments, the number of read components required for a given identifier component can be half that required in the previous design described above, as shown, for example, in FIG. 47. For example, each read component identifies both layers to which it is hybridized (at the boundary between the two layers). This implies that the read operation can be performed, for example, twice as fast as in the previous design. For example, if there is an odd number of layers in the identifier component, one additional read component is required. In some embodiments, this approach may require more unique read component sequences corresponding to combinations of components from adjacent layers.

[0097]

[0190] In some embodiments, boundary readout components (e.g., design D) as described above can use optical signals instead of electrical signals, as shown, for example, in Figure 48 (design E). For example, luminescent tags, such as fluorophores, can be used instead of charged nucleic acid (e.g., DNA) secondary structures. The nanochannel (or nanopore) design can be as shown in design C. Similarly, in some embodiments, the number of readout components required can be reduced (e.g., to half of that implemented in design C), as shown in Figures 46 and 47.

[0098]

[0191] In some embodiments, as shown in Figure 49 (Design F), the readout component can comprise a small nucleic acid (e.g., DNA) molecule, such as an aptamer, capable of binding to other molecules, e.g., peptides and / or proteins. In some embodiments, the technology described herein includes a library of aptamers capable of binding molecules with a high net (mobile) charge (relative to unmodified nucleic acid molecules) that can be detected by a nanochannel (or nanopore) sensor, as described above. An advantage of this design is that, in some embodiments, the size of the nucleic acid (e.g., DNA) probe can be 120 bases or less. However, the net charge can be controlled by charging the specific aptamer used. For example, a peptide with a sequence having several negative amino acids can be implemented, and secondary structures with this peptide can be generated. The sequence can be read using different net charges for each sensing readout component. In some embodiments, different peptide sequences can be used to achieve different net charges for each readout component, as shown above for Design A.

[0099]

[0192] In some embodiments, to increase the net signal change in a readout component (e.g., Design A) as described above, branched structures, e.g., dendrimers, can be used, as shown, for example, in Figure 50 (Design G). In some embodiments, the ends of the branches of such branched structures can be modified, for example, by attaching one or more other molecules as probes, such as streptavidin, biotin, or similar binding partners. These molecules can increase the size of the probe and allow for the attachment of other molecules that can increase or decrease the net charge of the readout component for detecting and reading nucleic acid molecule (e.g., DNA) sequences, as described above.

[0100]

[0193] In some embodiments, the technology described herein can be configured or arranged as a multi-sensor nucleic acid (eg, DNA) reader.

[0101]

[0194] Described herein are devices and methods for reading nucleic acid (e.g., DNA) sequences at speeds several orders of magnitude higher than current technologies, such as current nanopore sequencing technologies. As discussed above, current sequencing technologies infer nucleic acid (e.g., DNA) sequences by detecting electrical or optical signals one base at a time. Electrical methods involve measuring ionic current as nucleic acid molecules (e.g., DNA) translocate through a nanopore or measuring the charge on molecules as they translocate through a nanochannel with nanoelectrodes. Electronic circuits process the current fluctuations to derive the DNA sequence. These circuits currently cannot keep pace with DNA translocation speeds (e.g., 1 million bases per second). Therefore, nucleic acid molecules (e.g., DNA) are typically slowed down as they translocate through a nanopore to match the speed of the detection circuit. Described herein are technologies, including devices and methods, that utilize machine learning algorithms to decode nucleic acid (e.g., DNA) sequences based on information from multiple "slow" sensors operating in concert or parallel.

[0102]

[0195] In some embodiments, multiple sensor devices (e.g., "slow" sensor devices) are arranged in series along a migration path (e.g., of a nanopore or nanochannel), thereby accelerating the sequencing process by several orders of magnitude. In some embodiments, multiple electronic sensing devices, multiple optical sensing devices, or a combination thereof are arranged in series along a migration path (e.g., of a nanopore or nanochannel). While a single sensor device (or electronic or optical sensing device) (denoted "sensor" in the figures) operating at a speed slower than the migration rate of a nucleic acid molecule may not be able to detect every base in the molecule, an array of appropriately positioned sensor devices (or electronic or optical sensing devices) may collectively be able to collect enough sample to reconstruct all bases (or readout components, as described herein above) in a molecule, as shown, for example, in Figures 51-53. When sampling a signal, a sensor device (or electronic or optical sensing device) may completely fail to detect a single base (or readout component), or potentially only detect a single base (or readout component), as shown, for example, in Figure 51. Another situation in which a "slow" sensor (e.g., sensor device (or electronic or optical sensing device)) may be unable to resolve information from a nucleic acid molecule is shown in Figure 52. In this case, when the sensor's sampling time is much longer than the migration time of a single base (or readout component), the sensor may not have the resolution to distinguish individual bases (or readout components). By increasing the number of sensors (e.g., sensor devices (or electronic or optical sensing devices)) along the migration path, partial information from different sensors can be assembled, e.g., using machine learning, and more accurate information can be inferred.

[0103]

[0196] In some embodiments, nucleic acid (e.g., DNA) molecules that can be read using the techniques described herein can be single-stranded nucleic acid molecules (e.g., DNA), double-stranded nucleic acid molecules (e.g., DNA), or hybrid nucleic acid molecules having a complementary strand containing a secondary structure or readout moiety, e.g., as described above and shown, e.g., in Figures 36 and 37. In some embodiments, the sensor or sensor device can be or include an electrical / electronic sensing device along (on or within) the nanochannel, e.g., one or more MOSFET sensors and / or one or more resistive sensors, or one or more ionic current sensors on or within the nanopore. A series of sensors (e.g., sensor devices (or electronic or optical sensing devices)) can be positioned at defined intervals along the nucleic acid (e.g., DNA) migration path. Example intervals can be 1-10 nm, 10-100 nm, 100-300 nm, 200-500 nm, or 500-1000 nm. The intervals can be constant or variable. For example, a series of MOSFET sensors can be spaced at regular intervals of 500 nm. The width of the migration path (e.g., of the nanochannel) can be chosen so that only one molecule can migrate along the channel at any given time. Figure 53 shows an example device in which the total sensing capacity (number of sensors x speed of each sensor) is equal to the migration speed (e.g., sensor speed: one read component per second; migration speed: five read components per second). In this case, the signals from each sensor (e.g., sensor device (or electronic or optical sensing device)) can be collated, for example, using sensor fusion and machine learning algorithms to derive the sequence of the nucleic acid (e.g., DNA) being sequenced. The example shown in Figure 53 shows an ideal case in which each sensor (e.g., sensor device (or electronic or optical sensing device)) reads at least one piece of information (e.g., a base or read component) from the migrating DNA.

[0104]

[0197] FIG. 54 shows an example where the total sensing capacity of the sensor array is equal to the translation speed, but each sensor (e.g., sensor device (or electronic or optical sensing device)) may miss some of the information to be read from the nucleic acid. In this case, the collated information from each sensor may not reproduce the desired sequence. In some embodiments, multiple sensors (e.g., sensor device (or electronic or optical sensing device)) can be used along the translation path, as shown in FIG. 55, for example. In doing so, partial information from multiple sensors can increase the accuracy of the assembled information, for example, by redundant scanning and / or by gap-filling any missing information. In some embodiments, a readout component can include information about its relative position along the length of the nucleic acid molecule. In some embodiments, a set of readout components, read alone or together with information about the relative positions of the sensors reading the components relative to each other, can simultaneously provide information about the value and position of the readout component sequence (e.g., readout component B and readout component D read by sensor 2 and sensor 4, respectively, can indicate that components B and D are two sensor spaces apart). Scanning each component multiple times can increase the amount of information available for constructing the readout component and / or base sequence. In some embodiments, the more sensors, the greater the accuracy.

[0105]

[0198] In some embodiments, sensors (e.g., sensor devices (or electronic or optical sensing devices)) can be increased, thereby improving accuracy. For example, if sensors are placed very close to each other (e.g., 3-5 nm apart), a set of sensors ("clusters") can be used to detect more than one piece of information (e.g., bases or readout components) from each molecule per cluster, as shown, for example, in FIG. 56. In some embodiments, a set of readout components, read alone or together with information on the relative positions of the individual sensors and / or clusters reading the components, can simultaneously provide information on the value and position of the readout component sequence (e.g., readout component B and readout component D read by sensor 2 and sensor 4, respectively, can indicate that components B and D are two sensor spaces apart). Scanning each component or set of components multiple times can increase the amount of information available for constructing the readout component and / or base sequence. Again, increasing the number of such sensors increases the accuracy of the readout information.

[0106]

[0199] In some embodiments, machine learning algorithms can be used for base calling or other operations to convert signals received from one or more sensors (e.g., sensor devices (or electronic or optical sensing devices)) into nucleic acid sequence information.

[0107]

[0200] In some embodiments, the technology provided herein can provide for the reading of nucleic acid (e.g., DNA) sequences using machine learning at a speed equivalent to the natural translation rate (1 million bases per second).Currently, commercially available DNA sequencing speeds are 420 bases per second.The technology described herein can read nucleic acid sequences at a speed at least three orders of magnitude greater.

[0108]

[0201] The present specification provides systems and methods for storing digital information in nucleic acid molecules in various ways to improve the efficiency of retrieval and access of that digital information. For example, component nucleic acid molecules (e.g., components) are selected and linked together to form identifier nucleic acid molecules (e.g., identifiers), each corresponding to a particular symbol (e.g., a bit or series of bits) or the position (e.g., rank or address) of that symbol in a string (e.g., a bit stream). The components can be organized structurally to provide an efficient way to represent digital data. For example, the component structure can allow multiple component molecules to self-assemble or otherwise sort themselves into a predetermined order after being deposited or dispensed in the same area.

[0109]

[0202] Provided herein is a method for storing digital information in a nucleic acid molecule, the method comprising: (a) receiving the digital information as a symbol string, each symbol in the symbol string having a symbol value and a symbol position within the symbol string; and (b) forming a first identifier nucleic acid molecule, the method comprising: (1) selecting one component nucleic acid molecule from each of M layers from a set of distinguishable component nucleic acid molecules separated into M distinct layers; (2) depositing the M selected component nucleic acid molecules in an area; and (3) physically assembling the M selected component nucleic acid molecules in (2) to form a first identifier nucleic acid molecule having first and second terminal molecules and a third molecule located between the first and second terminal molecules, such that the component nucleic acid molecules from the first and second layers correspond to the first and second terminal molecules of the identifier nucleic acid molecule, and the component nucleic acid molecule in the third layer corresponds to the third molecule of the identifier nucleic acid molecule. (c) forming a first identifier nucleic acid molecule by forming an identifier nucleic acid molecule to define the physical order of the M layers in the first identifier nucleic acid molecule; (d) forming a plurality of additional identifier nucleic acid molecules, each of the plurality of additional identifier nucleic acid molecules having (1) a first and a second terminal molecule and a third molecule located between the first terminal molecule and the second terminal molecule, and (2) corresponding to each symbol position, wherein at least one of the first terminal molecule, the second terminal molecule, and the third molecule of the at least one additional identifier nucleic acid molecule is identical to a target molecule of the first identifier nucleic acid molecule in (b), and wherein the probe is capable of selecting at least two identifier nucleic acid molecules corresponding to each symbol having consecutive symbol positions in the symbol string; and (d) collecting the identifier nucleic acid molecules in (b) and (c) into a pool having a powder, liquid, or solid form.

[0110]

[0203] In some embodiments, a population of identifier nucleic acid molecules share the same target molecule, while other identifier nucleic acid molecules in the same pool may share different target molecules. At least one of the first and second terminal molecules of at least one additional identifier nucleic acid molecule may be identical to the target molecule of the first identifier nucleic acid molecule in (b). In some embodiments, physically assembling the M selected component nucleic acid molecules comprises ligating the component nucleic acid molecules.

[0111]

[0204] In some embodiments, the component nucleic acid molecules from each layer include at least one cohesive end that is complementary to at least one cohesive end of a component nucleic acid molecule from another layer, thereby enabling cohesive end ligation to form the identifier nucleic acid molecules in (b) and (c). For example, all components in each layer (e.g., A, B, C) can have the same cohesive ends as each other, with one cohesive end of every component in layer A complementary to one cohesive end of every component in layer B. Furthermore, the other cohesive end of every component in layer B can be complementary to one cohesive end of every component in layer C, and so on. In some embodiments, the first molecule of the at least one additional identifier nucleic acid molecule in (c) is identical to the first terminal molecule of the identifier nucleic acid molecule in (b), and the second terminal molecule of the at least one additional identifier nucleic acid molecule in (c) is identical to the second terminal molecule of the identifier nucleic acid molecule in (b).

[0112]

[0205] In some embodiments, the method further comprises using a probe to hybridize to target molecules of at least some of the identifier nucleic acid molecules in the first identifier nucleic acid molecule and the plurality of additional identifier nucleic acid molecules to select identifier nucleic acid molecules corresponding to each symbol having consecutive symbol positions. Symbols having consecutive symbol positions may be adjacent to each other and share similar properties due to being in similar neighborhoods. Therefore, it may be desirable to use the same probe to select identifier nucleic acid molecules located near each other. In some embodiments, the method further comprises applying a single PCR reaction to amplify at least two identifier nucleic acid molecules corresponding to each symbol having consecutive symbol positions. In some embodiments, the at least two identifier nucleic acid molecules corresponding to each symbol having consecutive symbol positions can be further amplified by another PCR reaction targeting specific component nucleic acid molecules in a third of the identifier nucleic acid molecules.

[0113]

[0206] In some embodiments, the component nucleic acid molecules in each layer are structured with first and second regions, such that the first terminal region of each authentic nucleic acid molecule from one of the M layers binds to the second terminal region of any component nucleic acid molecule from another of the M layers. In some embodiments, M is 3 or greater. In some embodiments, each symbol position in the symbol string has a corresponding different identifier nucleic acid molecule. In some embodiments, the identifier nucleic acid molecules in (b) and (c) represent a subset of the combinatorial space of possible identifier nucleic acid molecules, each comprising one component nucleic acid molecule from each of the M layers.

[0114]

[0207] In some embodiments, the presence or absence of an identifier nucleic acid molecule in the pool in (d) represents a symbol value at each corresponding symbol position in the symbol string. For example, the presence of an identifier can represent a symbol value of 1 at the corresponding symbol position, while its absence represents a symbol value of 0, or vice versa. In some embodiments, symbols having consecutive symbol positions encode similar digital information. In some embodiments, the distribution of the number of component nucleic acid molecules in each of the M layers is non-uniform. For example, one layer can have more component nucleic acid molecules than another layer, thereby adjusting the number and / or variety of possible permutations for creating identifier nucleic acid molecules.

[0115]

[0208] In some embodiments, if the third tier contains more component nucleic acid molecules than either the first tier or the second tier, the PCR query used to access the pool in (d) will access a larger pool of identifier nucleic acid molecules than if the third tier contained fewer component nucleic acid molecules than either the first tier or the second tier.

[0116]

[0209] In some embodiments, if the third tier contains fewer component nucleic acid molecules than either the first tier or the second tier, the PCR query used to access the pool in (d) will access a smaller pool of identifier nucleic acid molecules than if the third tier contained more component nucleic acid molecules than either the first tier or the second tier, and the smaller pool of identifier nucleic acid molecules accessed corresponds to a higher resolution of accessing symbols in the symbol string.

[0117]

[0210] In some embodiments, the first tier has the highest priority, the second tier has the next highest priority, and the remaining M-2 tiers have corresponding component nucleic acid molecules between the first and second end molecules. In some embodiments, the pool in (d) can be used to access all identifier nucleic acid molecules in the pool that have a particular component nucleic acid molecule in the first and second end molecules in a single PCR reaction.

[0118]

[0211] In one aspect, the disclosure provides a method for storing digital information in a nucleic acid molecule, the method comprising: (a) receiving the digital information as a symbol string, each symbol in the symbol string having a symbol value and a symbol position within the symbol string, the digital information including image data represented by a collection of vectors; and (b) forming a first identifier nucleic acid molecule by: (1) selecting one component nucleic acid molecule from a set of distinguishable component nucleic acid molecules separated into M different layers, from each of the M layers; and (2) depositing the M selected component nucleic acid molecules in an area. and (3) physically assembling the M selected component nucleic acid molecules in (2) to form a first identifier nucleic acid molecule having first and second terminal molecules and a third molecule located between the first and second terminal molecules, such that the component nucleic acid molecules from the first and second tiers correspond to the first and second terminal molecules of the identifier nucleic acid molecule, and the component nucleic acid molecule in the third tier corresponds to the third molecule of the identifier nucleic acid molecule, thereby defining a physical order of the M tiers in the first identifier nucleic acid molecule.

[0119]

[0212] In some embodiments, the method comprises steps (a) above; (b) forming a first identifier nucleic acid molecule by depositing M selected component nucleic acid molecules in an area and physically assembling the M selected component nucleic acid molecules, wherein the M selected component nucleic acid molecules are selected from a set of M distinct layered component nucleic acid molecules; (c) forming a plurality of identifier nucleic acid molecules, each of the plurality of identifier nucleic acid molecules corresponding to a respective symbol position; and (d) collecting the identifier nucleic acid molecules in (b) and (c) into a pool having a powder, liquid, or solid form.

[0120]

[0213] In some embodiments, at least some of the M layers correspond to different features of the image data. In some embodiments, the different features include an x-coordinate, a y-coordinate, and an intensity value or a range of intensity values. Storing the image data in nucleic acid molecules allows any neighborhood of a pixel to be queried for a color value using a random access method, such as any of the access methods described herein. In some embodiments, storing the image data in nucleic acid molecules allows the image data to be decoded at a fraction of the original resolution of the image data.

[0121]

[0214] In one aspect, the disclosure provides a method for storing digital information in a nucleic acid molecule, the method comprising: (a) receiving the digital information as a symbol string, each symbol in the symbol string having a symbol value and a symbol position within the symbol string, the digital information including image data represented by a collection of vectors; (b) forming a first identifier nucleic acid molecule by depositing M selected component nucleic acid molecules in an area, the M selected component nucleic acid molecules being selected from a set of M distinct component nucleic acid molecules separated into different layers; and (c) forming a plurality of identifier nucleic acid molecules, the plurality (b) each of the identifier nucleic acid molecules has a first and a second terminal molecule and a third molecule located between the first and second terminal molecules, corresponding to each symbol position, and at least one of the first terminal molecule, the second terminal molecule, and the third molecule of the at least one additional identifier nucleic acid molecule is identical to the target molecule of the first identifier nucleic acid molecule in (b), allowing a single probe to select at least two identifier nucleic acid molecules corresponding to each symbol having an associated symbol position in the symbol string; and (d) collecting the identifier nucleic acid molecules in (b) and (c) into a pool having a powder, liquid, or solid form. Storing image data in the nucleic acid molecules allows any neighborhood of a pixel to be queried for a color value using a random access method.

[0122]

[0215] In some embodiments, storing the image data in nucleic acid molecules allows the image data to be decoded at a fraction of the image data's original resolution, and the decoding of the image data at a fraction is used to search for particular visual features in a surveillance image archive or video archive to identify frames of interest.

[0123]

[0216] In one aspect, the disclosure provides a method for storing digital information in a nucleic acid molecule, the method comprising: (a) receiving the digital information as a symbol string, wherein each symbol in the symbol string has a symbol value and a symbol position within the symbol string; (b) forming a first identifier nucleic acid molecule by depositing M selected component nucleic acid molecules in an area to physically assemble the M selected component nucleic acid molecules, wherein the M selected component nucleic acid molecules are selected from a set of M distinct component nucleic acid molecules separated into different layers; and (c) forming a plurality of identifier nucleic acid molecules, each of the plurality of identifier nucleic acid molecules comprising a first and second terminal molecule and a first terminal molecule. and a third molecule located between the first and second terminal molecules, corresponding to each symbol position, wherein at least one of the first terminal molecule, the second terminal molecule, and the third molecule of the at least one additional identifier nucleic acid molecule is identical to the target molecule of the first identifier nucleic acid molecule in (b), allowing a single probe to select at least two identifier nucleic acid molecules corresponding to each symbol having an associated symbol position in the symbol string, and physically assembling the M selected component nucleic acid molecules to form the identifier nucleic acid molecule in (b) includes using click chemistry; and (d) collecting the identifier nucleic acid molecules in (b) and (c) into a pool having a powder, liquid, or solid form. Step (c) of the method for storing digital information may generally include forming a plurality of identifier nucleic acid molecules each corresponding to each symbol position, without performing the step of forming a molecule having first and second terminal molecules and a third molecule as described above.

[0124]

[0217] In one aspect, the disclosure provides a method for storing digital information in a nucleic acid molecule, the method including: (a) receiving the digital information as a symbol string, wherein each symbol in the symbol string has a symbol value and a symbol position within the symbol string; (b) forming a first identifier nucleic acid molecule by depositing M selected component nucleic acid molecules in an area and physically assembling the M selected component nucleic acid molecules using click chemistry, wherein the M selected component nucleic acid molecules are selected from a set of M distinct component nucleic acid molecules separated into different layers; (c) forming a plurality of identifier nucleic acid molecules, each of the plurality of identifier nucleic acid molecules corresponding to a respective symbol position; (d) collecting the identifier nucleic acid molecules in (b) and (c) in a pool having a powder, liquid, or solid form; and (e) deleting the data collected in the pool. In some embodiments, step (c) comprises physically assembling a plurality of identifier nucleic acid molecules, each having a first and second terminal molecule and a third molecule located between the first and second terminal molecules, corresponding to each symbol position, wherein at least one of the first terminal molecule, the second terminal molecule, and the third molecule of the at least one additional identifier nucleic acid molecule is identical to the target molecule of the first identifier nucleic acid molecule in (b), such that a single probe can select at least two nucleic acid molecules corresponding to each symbol having an associated symbol position in the symbol string, and wherein in (b), physically assembling the M selected component nucleic acid molecules to form the identifier nucleic acid molecules comprises using click chemistry.

[0125]

[0218] In some embodiments, the method further comprises pull-down selecting identifier nucleic acid molecules from the pool in (d) using sequence-specific probes to selectively remove data. In some embodiments, the selected identifier nucleic acid molecules are selectively removed using CRISPR-based methods. In some embodiments, the method further comprises non-selectively removing data by obfuscating the identifier nucleic acid molecules in the pool in (d) by making them inaccessible or difficult or impossible to read. In some embodiments, the method further comprises degrading the identifier nucleic acid molecules from the pool in (d) using sonication, autoclaving, bleaching, treatment with base, acid, ethidium bromide, other DNA modifying agents, irradiation, burning, and non-specific nuclease digestion to non-selectively remove data.

[0126]

[0219] In one aspect, the disclosure provides a method for storing digital information in a nucleic acid molecule, the method comprising: (a) receiving the digital information as a symbol string, each symbol in the symbol string having a symbol value and a symbol position within the symbol string; (b) dividing the symbol string into one or more blocks of a size equal to or less than a certain length; (c) forming a first identifier nucleic acid molecule by depositing M selected component nucleic acid molecules in an area to physically assemble the M selected component nucleic acid molecules, the M selected component nucleic acid molecules being selected from a set of M distinct component nucleic acid molecules separated into different layers; (d) forming a plurality of identifier nucleic acid molecules, each corresponding to a respective symbol position; and (e) collecting the identifier nucleic acid molecules in (b) and (c) into a pool having a powder, liquid, or solid form.

[0127]

[0220] In some embodiments, the plurality of identifier nucleic acid molecules in step (d) above each have a first and a second terminal molecule and a third molecule located between the first and second terminal molecules, corresponding to each symbol position, and at least one of the first terminal molecule, the second terminal molecule, and the third molecule of the at least one additional identifier nucleic acid molecule is identical to the target molecule of the first identifier nucleic acid molecule in (b), allowing a single probe to select at least two identifier nucleic acid molecules corresponding to each symbol having an associated symbol position in the symbol string.

[0128]

[0221] In some embodiments, the method further includes determining a size for each block based on the symbol string, processing requirements, or intended use of the digital information. In some embodiments, the method further includes calculating a hash of each block. In some embodiments, the method further includes applying one or more error detection and correction to each block and calculating one or more error protection bytes. In some embodiments, the method further includes mapping one or more blocks to a set of code words that optimize chemical conditions during encoding or decoding. In some embodiments, the set of code words has a fixed length such that a fixed number of identifier nucleic acid molecules are assembled in each reaction zone of the writing system at approximately equal concentrations within and across each reaction zone.

[0129]

[0222] In one aspect, the present disclosure provides a method for performing a calculation on digital information stored in nucleic acid molecules. Importantly, the calculation can be performed without having to read or decode the actual digital information from a pool of molecules. The calculation can include any combination of Boolean logic gates, such as AND, OR, NOT, or NAND operations. In particular, the present disclosure provides a method for storing digital information in nucleic acid molecules, the method comprising: (a) receiving the digital information as a symbol string, wherein each symbol in the symbol string has a symbol value and a symbol position within the symbol string; (b) forming a first identifier nucleic acid molecule by depositing M selected component nucleic acid molecules in an area to physically assemble the M selected component nucleic acid molecules, wherein the M selected component nucleic acid molecules are selected from a set of M distinct layered component nucleic acid molecules; (c) forming a plurality of identifier nucleic acid molecules, each of the plurality of identifier nucleic acid molecules corresponding to a respective symbol position; (d) collecting the identifier nucleic acid molecules in (b) and (c) into a pool having a powder, liquid, or solid form; and (e) performing a computation involving a Boolean logic operation, including AND, OR, NOT, or NAND, on the symbol string using the identifier nucleic acid molecules in (d), thereby generating a new pool of nucleic acid molecules. The new pool of nucleic acid molecules may represent the result or output of the computation.

[0130]

[0223] In some embodiments, the identifier nucleic acid molecules in (c) above each have a first and a second terminal molecule and a third molecule located between the first and second terminal molecules, corresponding to each symbol position, and at least one of the first terminal molecule, the second terminal molecule, and the third molecule of the at least one additional identifier nucleic acid molecule is identical to the target molecule of the first identifier nucleic acid molecule in (b), allowing a single probe to select at least two identifier nucleic acid molecules corresponding to each symbol having an associated symbol position in the symbol string.

[0131]

[0224] In some embodiments, the computation is performed on the pool of identifier nucleic acid molecules in (d) without decoding any of the identifier nucleic acid molecules to obtain any symbols in the string. In some embodiments, performing the computation comprises a series of chemical operations including hybridization and cleavage.

[0132]

[0225] In some embodiments, the string in (a) is denoted as alpha and comprises sub-bitstream s, and the plurality of identifier nucleic acid molecules in (d) are double-stranded and denoted as dsA, and the method further includes obtaining another pool of another plurality of identifier nucleic acid molecules, denoted as dsB, representing another string, denoted as b, comprising sub-bitstream t, and the calculation is performed on sub-bitstreams s and t by performing a series of steps on dsA and dsB. In some embodiments, the series of steps on dsA and dsB includes performing an initialization step, where the initialization step converts double-stranded identifier nucleic acid molecules in dsA to a plus single-stranded form, denoted as A, and converts double-stranded identifier nucleic acid molecules in dsA to a plus single-stranded form, denoted as A, and * and converting the nucleotide sequence to a minus single-stranded form shown as A * is the reverse complement of A, and converting the double-stranded identifier nucleic acid molecule in dsB into a plus single-stranded form designated as B, and converting the double-stranded identifier nucleic acid molecule in dsB into a plus single-stranded form designated as B. * and converting the nucleotide sequence to a minus single-stranded form shown as B * is the reverse complement of B, and selecting dsP as the identifier nucleic acid molecule in dsA corresponding to s, selecting P as the identifier nucleic acid molecule in A corresponding to s, selecting dsQ as the identifier nucleic acid molecule in dsB corresponding to t, and selecting B corresponding to t. * Q as an identifier nucleic acid molecule in * and selecting:

[0133]

[0226] In some embodiments, the calculation is an AND operation, and the sequence of steps for dsA and dsB is A and B. *and performing an AND operation between a and b by combining p and Q, hybridizing the complementary nucleic acid molecules, and selecting the perfectly complementary double-stranded nucleic acid molecules as a new pool of nucleic acid molecules. In some embodiments, the calculation is an OR operation, and the sequence of steps for dsA and dsB is * performing an AND operation between s and t by combining s with t, hybridizing the complementary nucleic acid molecules, and selecting the perfectly complementary double-stranded nucleic acid molecules as a new pool of nucleic acid molecules.

[0134]

[0227] In some embodiments, selecting a perfectly complementary nucleic acid molecule comprises chromatography, gel electrophoresis, single-strand-specific endonuclease, single-strand-specific exonuclease, or a combination thereof.

[0135]

[0228] In some embodiments, the calculation is an OR operation, and the sequence of steps for dsA and dsB includes performing an OR operation between a and b by combining dsA and dsB to generate a new pool of nucleic acid molecules. In some embodiments, the calculation is an OR operation, and the sequence of steps for dsA and dsB includes performing an OR operation between s and t by combining dsP and dsQ to generate a new pool of nucleic acid molecules.

[0136]

[0229] In some embodiments, the method further comprises updating A or dsA to include the new pool of nucleic acid molecules, thereby allowing A or dsA to represent the output of the operation.

[0137]

[0230] In one aspect, the disclosure provides a method for storing digital information in a nucleic acid molecule, the method comprising: (a) receiving the digital information as a symbol string, wherein each symbol in the symbol string has a symbol value and a symbol position within the symbol string; (b) forming a first identifier nucleic acid molecule by depositing M selected component nucleic acid molecules in an area to physically assemble the M selected component nucleic acid molecules, wherein the M selected component nucleic acid molecules are selected from a set of M distinct component nucleic acid molecules separated into different layers; (c) forming a plurality of identifier nucleic acid molecules; and (c) compartmentalizing the identifier nucleic acid molecules in (b) and (c) into separate bins, wherein each bin corresponds to a different symbol value.

[0138]

[0231] In some embodiments, forming the first identifier nucleic acid molecule in (b) comprises (1) selecting one component nucleic acid molecule from each of M layers from a set of distinguishable component nucleic acid molecules separated into M distinct layers, (2) depositing the M selected component nucleic acid molecules in an area, and (3) physically assembling the M selected component nucleic acid molecules in (2) to form a first identifier nucleic acid molecule having first and second terminal molecules and a third molecule located between the first and second terminal molecules, such that the component nucleic acid molecules from the first and second layers correspond to the first and second terminal molecules of the identifier nucleic acid molecule and the component nucleic acid molecule in the third layer corresponds to the third molecule of the identifier nucleic acid molecule, defining a physical order of the M layers in the first identifier nucleic acid molecule. In some embodiments, the symbol position of each symbol having a particular symbol value is recorded in a bin reserved for that value, and the bin is an area within (2).

[0139]

[0232] In one aspect, the disclosure provides a method for storing digital information in a nucleic acid molecule, the method comprising: (a) receiving the digital information as a symbol string, wherein each symbol in the symbol string has a symbol value and a symbol position within the symbol string; (b) forming a first identifier nucleic acid molecule by depositing M selected component nucleic acid molecules in an area to physically assemble the M selected component nucleic acid molecules, wherein the M selected component nucleic acid molecules are selected from a set of M distinct component nucleic acid molecules separated into different layers; (c) forming a plurality of identifier nucleic acid molecules, each corresponding to a respective symbol position; and (d) collecting the identifier nucleic acid molecules in (b) and (c) in a pool having a powder, liquid, or solid form.

[0140]

[0233] In some embodiments, step (c) above comprises forming a plurality of identifier nucleic acid molecules, each identifier nucleic acid molecule having a first and a second terminal molecule and a third molecule located between the first terminal molecule and the second terminal molecule, and at least one of the first terminal molecule, the second terminal molecule, and the third molecule of the at least one additional identifier nucleic acid molecule is identical to the target molecule of the first identifier nucleic acid molecule in (b), such that a single probe selects at least two identifier nucleic acid molecules corresponding to each symbol having an associated symbol position in the symbol string.

[0141]

[0234] In some embodiments, each of the M selected components comprises a plurality of moieties, each comprising a nucleic acid molecule, and each moiety is linked to the same identifier by one or more chemical methods. In some embodiments, the plurality of moieties each serve a distinct functional purpose for a different data storage operation. In some embodiments, the functional purpose includes ease of sequencing and accessibility by nucleic acid hybridization. In some embodiments, forming the first identifier nucleic acid molecule comprises programmatically mutating one or more bases in the parent identifier by applying a base editor, such as dCas9-deaminase.

[0142]

[0235] In one aspect, the present disclosure provides a method for storing digital information in a nucleic acid molecule, the method including: (a) receiving the digital information as a symbol string, each symbol in the symbol string having a symbol value and a symbol position within the symbol string; (b) applying a base editor to programmatically mutate one or more bases in a parent identifier to form a first identifier nucleic acid molecule; (c) forming a plurality of identifier nucleic acid molecules, each corresponding to a respective symbol position; and (d) collecting the identifier nucleic acid molecules in (b) and (c) in a pool having a powder, liquid, or solid form. In one example, one of the base editors applied in (b) is dCas9-deaminase.

[0143]

[0236] In one aspect, the disclosure provides a method for storing digital information generated from one or more random processes in a nucleic acid molecule, the method comprising: (a) receiving the digital information as a symbol string, wherein each symbol in the symbol string has a symbol value and a symbol position within the symbol string; (b) forming a first identifier nucleic acid molecule by depositing M selected component nucleic acid molecules in an area to physically assemble the M selected component nucleic acid molecules, wherein the M selected component nucleic acid molecules are selected from a set of M distinct component nucleic acid molecules separated into different layers; (c) forming a plurality of identifier nucleic acid molecules, each corresponding to a respective symbol position; and (d) collecting the identifier nucleic acid molecules in (b) and (c) in a pool having a powder, liquid, or solid form.

[0144]

[0237] In some embodiments, the present disclosure provides applications of the above method or any of the above methods, including encrypting information, authenticating entities, or using them as a source of entropy in applications involving randomization. In some embodiments, identifiers from one or more disjoint identifier libraries are used to uniquely identify entities or physical locations.

[0145]

[0238] In one aspect, the present disclosure provides a method for encoding digital information into sections of several random DNA species.

[0146]

[0239] In one aspect, the present disclosure provides a method for generating random data by randomly sampling and sequencing DNA species from a large combinatorial pool of possible DNA species.

[0147]

[0240] In one aspect, the present disclosure provides a method for generating and storing random data by randomly sampling and sequencing a subset of DNA species from a large combinatorial pool of possible DNA species.

[0148]

[0241] In some embodiments, the subset of DNA species is amplified to create multiple copies of the species. In some embodiments, error checking and correction nucleic acid molecules are added to the subset of DNA species to enable robust future readout. In some embodiments, the subset of DNA species are barcoded with unique molecules and combined into a pool of barcoded subsets of DNA species. In some embodiments, a specific subset of DNA species in the pool of barcoded subsets of DNA species is accessible using input nucleic acid probes for PCR or nucleic acid capture.

[0149]

[0242] In one aspect, the present disclosure provides a method for securing and authenticating an artifact using a system that includes (1) a DNA key composed of a subset of DNA species from a defined set, and (2) a DNA reader that accepts the key and searches for a matching key to either unlock the artifact locally or return a hashed token to access the artifact elsewhere. In some embodiments, the method further includes combinatorially assembling DNA fragments for biological applications.

[0150]

[0243] In one aspect, the disclosure provides a method for storing digital information in a nucleic acid molecule, the method comprising: (a) receiving the digital information as a symbol string, each symbol in the symbol string having a symbol value and a symbol position within the symbol string; and (b) (1) selecting one component nucleic acid molecule from each of M layers from a set of distinguishable component nucleic acid molecules separated into M distinct layers; (2) depositing the M selected component nucleic acid molecules in an area; and (3) physically assembling the M selected component nucleic acid molecules in (2) to form a first identifier nucleic acid molecule comprising designated components, wherein the designated components comprise at least one target molecule. (c) physically assembling a plurality of additional identifier nucleic acid molecules, each having a designated component, wherein the designated component comprises at least one target molecule of the first identifier nucleic acid molecule in (b), thereby enabling the probe to select at least two identifier nucleic acid molecules corresponding to each symbol having consecutive symbol positions within the symbol string; and (d) collecting the identifier nucleic acid molecules in (b) and (c) into a pool having a powder, liquid, or solid form.

[0151]

[0244] In one aspect, the present disclosure provides a method for encoding information into a nucleic acid sequence. The method for encoding information into a nucleic acid sequence may include (a) converting the information into a symbol string, (b) mapping the symbol string to a plurality of identifiers, and (c) constructing an identifier library including at least a subset of the plurality of identifiers. Each identifier of the plurality of identifiers may include one or more components. Each component of the one or more components may include a nucleic acid sequence. Each symbol at each position in the symbol string may correspond to a distinct identifier. Each identifier may correspond to each symbol at each position in the symbol string. Furthermore, one symbol at each position in the symbol string may correspond to the absence of an identifier. For example, each occurrence of a "0" in a string of binary symbols (e.g., bits) of "0" and "1" may correspond to the absence of an identifier.

[0152]

[0245] In another aspect, the present disclosure provides a method for nucleic acid-based computer data storage. The method for nucleic acid-based computer data storage may include (a) receiving computer data, (b) synthesizing nucleic acid molecules including a nucleic acid sequence that encodes the computer data, and (c) storing the nucleic acid molecules having the nucleic acid sequence. The computer data may be encoded in at least a subset of the synthesized nucleic acid molecules, rather than the sequence of each of the nucleic acid molecules.

[0153]

[0246] In another aspect, the present disclosure provides a method for writing and storing information in a nucleic acid sequence. The method may include (a) receiving or encoding a virtual identifier library representing the information, (b) physically constructing the identifier library, and (c) storing one or more physical copies of the identifier library in one or more separate locations. Each identifier in the identifier library may include one or more components. Each of the one or more components may include a nucleic acid sequence.

[0154]

[0247] In another aspect, the present disclosure provides a method for nucleic acid-based computer data storage. The method for nucleic acid-based computer data storage may include: (a) receiving computer data; (b) synthesizing a nucleic acid molecule comprising at least one nucleic acid sequence encoding the computer data; and (c) storing the nucleic acid molecule comprising at least one nucleic acid sequence. Synthesizing the nucleic acid molecule may be in the absence of base-by-base nucleic acid synthesis.

[0155]

[0248] In another aspect, the present disclosure provides a method for writing and storing information in a nucleic acid sequence. The method for writing and storing information in a nucleic acid sequence may include (a) receiving or encoding a virtual identifier library representing the information, (b) physically constructing the identifier library, and (c) storing one or more physical copies of the identifier library in one or more separate locations. An individual identifier in the identifier library may include one or more components. An individual component of the one or more components may include a nucleic acid sequence.

[0156]

[0249] In another aspect, the disclosure provides a method for storing digital information in a nucleic acid sequence, the method comprising: (a) receiving the digital information as a symbol string, wherein each symbol in the symbol string has a symbol value and a symbol position within the symbol string; and (b) forming a first identifier nucleic acid sequence, the method comprising: (1) selecting one component nucleic acid sequence from each of M layers from a set of distinguishable component nucleic acid sequences separated into M distinct layers; (2) depositing the M selected component nucleic acid sequences into compartments; and (3) physically assembling the M selected component nucleic acid sequences in (2) such that the component nucleic acid sequences from the first and second layers correspond to first and second end sequences of the identifier nucleic acid sequence, and the component nucleic acid sequence in the third layer corresponds to a third sequence of the identifier nucleic acid sequence, defining a physical order of the M layers in the first identifier nucleic acid sequence, thereby defining the first and second end sequences and (c) forming a plurality of additional identifier nucleic acid sequences, each of which has (1) a first and second end sequence and a third sequence located between the first and second end sequences, and (2) corresponds to each symbol position, wherein at least one of the first end sequence, the second end sequence, and the third sequence of at least one additional identifier nucleic acid sequence is identical to the target sequence of the first identifier nucleic acid sequence in (b), allowing a probe to select at least two identifier nucleic acid sequences corresponding to each symbol having consecutive symbol positions within the symbol string; and (d) collecting the identifier nucleic acid sequences in (b) and (c) into a pool in the form of a powder, liquid, or solid.

[0157]

[0250] In another aspect, the disclosure provides a method for storing digital information in a nucleic acid sequence, the method comprising: (a) receiving the digital information as a symbol string, each symbol in the symbol string having a symbol value and a symbol position within the symbol string, the digital information including image data represented by a collection of vectors; (b) forming a first identifier nucleic acid sequence by depositing M selected component nucleic acid sequences selected from a set of distinguishable component nucleic acid sequences separated into M distinct layers into a compartment; and (c) forming a plurality of identifier nucleic acid sequences, each of the plurality of identifier nucleic acid sequences being positioned between first and second end sequences and between the first and second end sequences. and a third sequence for positioning the at least one additional identifier nucleic acid sequence at each symbol position, wherein at least one of the first end sequence, the second end sequence, and the third sequence of the at least one additional identifier nucleic acid sequence is identical to the target sequence of the first identifier nucleic acid sequence in (b), allowing a single probe to select at least two identifier nucleic acid sequences corresponding to each symbol having an associated symbol position within the symbol string; and (d) collecting the identifier nucleic acid sequences in (b) and (c) in a pool having a powder, liquid, or solid form, wherein storing image data in the nucleic acid sequences allows any nearby pixel to be queried for its color value using a random access method.

[0158]

[0251] In another aspect, the disclosure provides a method for storing digital information in a nucleic acid sequence, the method comprising: (a) receiving the digital information as a symbol string, each symbol in the symbol string having a symbol value and a symbol position within the symbol string; (b) depositing M selected component nucleic acid sequences selected from a set of distinguishable component nucleic acid sequences separated into M distinct layers into a compartment to form a first identifier nucleic acid sequence; and (c) physically assembling a plurality of identifier nucleic acid sequences, each of the plurality of identifier nucleic acid sequences comprising a first and second terminal sequence and and a third sequence located between the first and second terminal sequences, corresponding to each symbol position, wherein at least one of the first terminal sequence, the second terminal sequence, and the third sequence of the at least one additional identifier nucleic acid sequence is identical to the target sequence of the first identifier nucleic acid sequence in (b), such that a single probe can select at least two identifier nucleic acid sequences corresponding to each symbol having an associated symbol position within the symbol string; and (d) collecting the identifier nucleic acid sequences in (b) and (c) into a pool having a powder, liquid, or solid form.

[0159]

[0252] In another aspect, the disclosure provides a method for storing digital information in a nucleic acid sequence, the method comprising: (a) receiving the digital information as a symbol string, each symbol in the symbol string having a symbol value and a symbol position within the symbol string; (b) dividing the symbol string into one or more blocks of a size equal to or less than a fixed length; (c) depositing M selected component nucleic acid sequences selected from a set of M distinct component nucleic acid sequences separated into different layers into compartments to form a first nucleic acid sequence; and (d) physically assembling a plurality of identifier nucleic acid sequences, wherein the plurality of identifier nucleic acid sequences are (b) and (c) are assembled into a pool having a powder, liquid, or solid form, each of which has a first and second terminal sequence and a third sequence located between the first and second terminal sequences, corresponding to each symbol position, and at least one of the first terminal sequence, the second terminal sequence, and the third sequence of the at least one additional identifier nucleic acid sequence is identical to the target sequence of the first identifier nucleic acid sequence in (b), allowing a single probe to select at least two identifier nucleic acid sequences corresponding to each symbol having an associated symbol position within the symbol string; and (e) collecting the identifier nucleic acid sequences in (b) and (c) into a pool having a powder, liquid, or solid form.

[0160]

[0253] In another aspect, the disclosure provides a method for storing digital information in a nucleic acid sequence, the method comprising: (a) receiving the digital information as a symbol string, each symbol in the symbol string having a symbol value and a symbol position within the symbol string; (b) depositing M selected component nucleic acid sequences selected from a set of distinguishable component nucleic acid sequences separated into M distinct layers into compartments to form a first nucleic acid sequence; and (c) physically assembling a plurality of identifier nucleic acid sequences, each of the plurality of identifier nucleic acid sequences having first and second terminal sequences and a third sequence located between the first and second terminal sequences, corresponding to each symbol position, and (d) assembling the identifier nucleic acid sequences in (b) and (c) into a pool having a powder, liquid, or solid form; and (e) using the identifier nucleic acid sequences in (d) to perform calculations on the symbol string involving Boolean logic operations including AND, OR, NOT, or NAND to produce a new pool of nucleic acid molecules.

[0161]

[0254] In another aspect, the disclosure provides a method for storing digital information in a nucleic acid sequence, the method comprising: (a) receiving the digital information as a symbol string, each symbol in the symbol string having a symbol value and a symbol position within the symbol string; (b) (1) selecting one component nucleic acid sequence from a set of distinguishable component nucleic acid sequences separated into M distinct layers, from each of the M layers; (2) depositing the M selected component nucleic acid sequences in an area to form a first identifier nucleic acid sequence; and (c) forming a first identifier nucleic acid sequence, each of the M selected component nucleic acid sequences having a first and second terminal sequence and a terminal sequence positioned between the first terminal sequence and the second terminal sequence. (b) physically assembling a plurality of identifier nucleic acid sequences corresponding to each symbol position, wherein at least one of the first end sequence, the second end sequence, and the third sequence of the at least one additional identifier nucleic acid sequence is identical to the target sequence of the first identifier nucleic acid sequence in (b), such that a single probe can select at least two identifier nucleic acid sequences corresponding to each symbol having an associated symbol position in the symbol string; and (d) collecting the identifier nucleic acid sequences in (b) and (c) into a pool having a powder, liquid, or solid form.

[0162]

[0255] In another aspect, the disclosure provides a method for storing digital information in a nucleic acid sequence, the method comprising: (a) receiving the digital information as a symbol string, each symbol in the symbol string having a symbol value and a symbol position within the symbol string; and (b) (1) selecting one component nucleic acid sequence from each of M layers from a set of distinguishable component nucleic acid sequences separated into M distinct layers; (2) depositing the M selected component nucleic acid sequences in an area; and (3) physically assembling the M selected component nucleic acid sequences in (2) to form a first identifier nucleic acid sequence comprising designated components, wherein the designated components comprise at least one target sequence. (c) physically assembling a plurality of additional identifier nucleic acid sequences, each having a designated component, wherein the designated component comprises at least one target sequence of the first identifier nucleic acid sequence in (b), thereby enabling the probe to select at least two identifier nucleic acid sequences corresponding to each symbol having consecutive symbol positions within the symbol string; and (d) collecting the identifier nucleic acid sequences in (b) and (c) into a pool having a powder, liquid, or solid form.

[0163]

[0256] Figure 57 shows the overall process of encoding information into a nucleic acid sequence, writing information into a nucleic acid sequence, reading the information written into a nucleic acid sequence, and decoding the read information. Digital information or data can be translated into one or more strings of symbols. In one example, the symbols are bits, and each bit can have a value of either "0" or "1." Each symbol can be mapped or encoded to an object (e.g., an identifier) ​​that represents that symbol. Each symbol can be represented by a distinct identifier. The distinct identifier can be a nucleic acid molecule composed of components. The components can be nucleic acid sequences. Digital information can be written into a nucleic acid sequence by generating an identifier library corresponding to the information. The identifier library can be physically created by physically constructing an identifier corresponding to each symbol in the digital information. All or any portion of the digital information can be accessed at a time. In one example, a subset of identifiers is accessed from the identifier library. The subset of identifiers can be read by sequencing and identifying the identifiers. To decode the digital data, the identified identifiers can be associated with corresponding symbols.

[0164]

[0257] A method for encoding and reading information using the approach of Figure 57 can include, for example, receiving a bitstream and using an identifier rank or nucleic acid index to map each bit (a bit having a bit value of "1") in the bitstream to a distinct nucleic acid identifier. Constructing a nucleic acid sample pool or identifier library that includes copies of identifiers corresponding to bit value 1 (and does not include identifiers with a bit value of 0). Reading the sample can include using molecular biology methods (e.g., sequencing, hybridization, PCR, etc.) to identify which identifiers are represented in the identifier library and assigning a bit value of "1" to the bits corresponding to those identifiers and a bit value of "0" otherwise (again, referencing the identifier rank to identify the bit in the original bitstream to which each identifier corresponds), and then decoding the information back into the encoded original bitstream.

[0165]

[0258] Encoding a string of N distinct bits allows an equal number of unique nucleic acid sequences to be used as possible identifiers. This approach to information encoding may use de novo synthesis of an identifier (e.g., a nucleic acid molecule) for each new item of information (N-bit string) to be stored. In other cases, the cost of synthesizing an identifier (a number equal to or less than N) anew for each new item of information to be stored can be reduced by one-time de novo synthesis and continued maintenance of all possible identifiers, such that encoding a new item of information may involve mechanically selecting and mixing together pre-synthesized (or pre-assembled) identifiers to form an identifier library. In other cases, both (1) the de novo synthesis of up to N identifiers for each new item of information to be stored, or (2) the cost of maintaining and selecting from N possible identifiers for each new item of information to be stored, or any combination thereof, can be reduced by synthesizing and maintaining several (fewer than N, and in some cases much fewer than N) nucleic acid sequences and then modifying these sequences through enzymatic reactions to generate up to N identifiers for each new item of information to be stored.

[0166]

[0259] Identifiers can be rationally designed and selected to facilitate read, write, access, copy, and delete operations. Identifiers can be designed and selected to minimize write errors, mutations, degradation, and read errors. See Chemical Methods Section H for rational design of DNA sequences, including synthetic nucleic acid libraries (e.g., identifier libraries).

[0167]

[0260] Figures 58A and 58B schematically illustrate an example method, referred to as "data-at-address," for encoding digital data into objects or identifiers (e.g., nucleic acid molecules). Figure 58A illustrates encoding a bitstream into an identifier library, where individual identifiers are constructed by concatenating or assembling a single component that specifies an identifier rank with a single component that specifies a byte value. In general, the data-at-address method uses identifiers that encode information modularly by including two objects: one object is a "byte value object" (or "data object") that identifies a byte value, and one object is a "rank object" (or "address object") that identifies the identifier rank (or the relative position of the byte in the original bitstream). Figure 58B illustrates an example of a data-at-address method in which each rank object can be combinatorially constructed from a set of components, and each byte value object can be combinatorially constructed from a set of components. The combinatorial construction of rank objects and byte value objects allows more information to be written into an identifier than if the objects were made of only a single component (see, e.g., Figure 58A).

[0168]

[0261] Figures 59A and 59B schematically illustrate another example of a method for encoding digital information into an object or identifier (e.g., a nucleic acid sequence). Figure 59A illustrates encoding a bit stream into an identifier library, where the identifier is constructed from a single component that specifies an identifier rank. The presence of an identifier at a particular rank (or address) specifies a bit value of "1," and the absence of an identifier at a particular rank (or address) specifies a bit value of "0." This type of encoding may use identifiers that encode only the rank (the relative position of the bit in the original bit stream) and use the presence or absence of those identifiers in the identifier library to encode a bit value of "1" or "0," respectively. Reading and decoding the information may involve identifying identifiers present in the identifier library and assigning a bit value of "1" to their corresponding rank, and a bit value of "0" otherwise. Figure 59B illustrates an example encoding method in which each identifier may be combinatorially constructed from a set of components, such that each possible combinatorial structure specifies a rank. Such a combinatorial structure allows more information to be written into an identifier than if an object were made of only a single component (see, e.g., Figure 59A). For example, a component set may include five distinct components. The five distinct components may be assembled to generate ten distinct identifiers, each containing two of the five components. Each of the ten distinct identifiers may have a rank (or address) corresponding to the position of a bit in the bit stream. The identifier library may include a subset of the ten possible identifiers corresponding to the positions of bit values ​​"1" and exclude a subset of the ten possible identifiers corresponding to the positions of bit values ​​"0" in a bit stream of length 10.

[0169]

[0262] Figure 60 shows a contour plot in logarithmic space of the relationship between the combination space of possible identifiers (C, x-axis) and the average number of identifiers (k, y-axis) physically constructed to store information of a given original size in bits (D, contour) using the encoding method shown in Figures 59A and 59B. This plot assumes that the original information of size D is re-encoded into a C bit string (C can be a value greater than D), where some bits k have a bit value of "1." The plot further assumes that encoding of information into nucleic acids is performed on the re-encoded bit string, and identifiers are constructed for positions with a bit value of "1" and identifiers are not constructed for positions with a bit value of "0." According to the assumption, the combination space of possible identifiers has size C to identify every position in the re-encoded bit string, and the number of identifiers used to encode a bit string of size D is such that D = log(Cchoosek), where Cchoosek can be a mathematical expression for some method of choosing k unordered results from C possibilities. Thus, as the combinatorial space of possible identifiers grows beyond the size (in bits) of a given information item, the number of physically constructed identifiers used to store that given information may decrease.

[0170]

[0263] Figure 61 shows an overview of a method for writing information to a nucleic acid sequence. Prior to writing, the information may be translated into a symbol string and encoded into multiple identifiers. Writing information may include setting up reactions to generate possible identifiers. Reactions may be prepared by depositing inputs into compartments. Inputs may include nucleic acids, components, templates, enzymes, or chemical reagents. Compartments may be wells, tubes, locations on a surface, chambers in a microfluidic device, or droplets in an emulsion. Multiple reactions may be set up in multiple compartments. Reactions may proceed to generate identifiers through programmed temperature incubation or cycling. Reactions may be selectively or universally removed (e.g., deleted). Reactions may also be selectively or universally interrupted, combined, and purified to collect identifiers into a single pool. Identifiers from multiple identifier libraries may be collected in the same pool. Individual identifiers may include barcodes or tags to identify which identifier library they belong to. Alternatively or additionally, the barcodes may include metadata about the encoded information. Supplemental nucleic acids or identifiers can also be included in the identifier pool along with the identifier library. The supplemental nucleic acids or identifiers may contain metadata for the encoded information or may function to obscure or hide the encoded information.

[0171]

[0264] The identifier rank (e.g., nucleic acid index) can constitute a method or key for ordering identifiers. The method can include a lookup table with all identifiers and their corresponding ranks. The method can also include a lookup table with the ranks of all components that make up the identifier and a function that determines the order of any identifier containing a combination of those components. Such a method can be referred to as lexicographic ordering and can be similar to the way words are alphabetized in a dictionary. In a data-at-address encoding method, the identifier rank (encoded by the identifier's rank object) can be used to identify the location of a byte (encoded by the identifier's byte value object) in the bitstream. In an alternative method, the identifier rank of the current identifier (encoded by the entire identifier itself) can be used to identify the location of a bit value "1" in the bitstream.

[0172]

[0265] The key may assign distinct bytes to unique subsets of identifiers (e.g., nucleic acid molecules) within a sample. For example, in a simple form, the key may assign each bit in a byte to a unique nucleic acid sequence that specifies the position of that bit, and then the presence or absence of that nucleic acid sequence within the sample may specify a bit value of 1 or 0, respectively. Reading the encoded information from the nucleic acid sample may involve any number of molecular biology techniques, including sequencing, hybridization, or PCR. In some embodiments, reading the encoded dataset may include reconstructing a portion of the dataset or reconstructing the entire encoded dataset from each nucleic acid sample. If the sequence can be read, a nucleic acid index can be used along with the presence or absence of a unique nucleic acid sequence, and the nucleic acid sample can be decoded into a bitstream (e.g., multiple bits, a byte, individual strings of multiple bytes, or strings of multiple bytes).

[0173]

[0266] Identifiers can be constructed by combinatorially assembling component nucleic acid sequences. For example, information can be encoded by taking a set of nucleic acid molecules (e.g., identifiers) from a defined set of molecules (e.g., combinatorial space). Each possible identifier for a defined set of molecules can be an assembly of nucleic acid sequences (e.g., components) from a pre-assembled set of components that can be separated into layers. Each individual identifier can be constructed by linking one component from every layer in a fixed order. For example, if there are M layers, and each layer can have n components, then at most C=n M unique identifiers can be constructed, up to a maximum of 2 C It is possible to decode and store up to C different information items or bits. For example, storing 1 megabit of information requires 1×10 6 Distinguishable identifiers or size C = 1 x 10 6 The identifier in this example can be assembled from a variety of components organized in different ways. The assemblies can each have n=1×10 3 Alternatively, the assembly can be made from M=2 pre-assembled layers each containing n=1×10 components. 2 The assembly can be made from M=3 layers containing components. In some embodiments, the assembly can be made from M=2, M=3, M=4, M=5, or more layers. As this example shows, the total number of components can be reduced by using a larger number of layers to encode the same amount of information. Using a smaller total number of components can be advantageous in terms of writing costs.

[0174]

[0267] In one example, one can start with two sets or layers of unique nucleic acid sequences, X and Y, each having x and y components (e.g., nucleic acid sequences), respectively. Each nucleic acid sequence from X can be assembled into each nucleic acid sequence from Y. While the total number of nucleic acid sequences held in the two sets can be the sum of x and y, the total number of nucleic acids, and therefore possible identifiers, that can be generated can be the product of x and y. If sequences from X can be assembled into sequences of Y in any order, even more nucleic acid sequences (e.g., identifiers) can be generated. For example, the number of nucleic acid sequences (e.g., identifiers) generated can be twice the product of x and y if the order of assembly is programmable. This set of all possible nucleic acid sequences that can be generated can be referred to as XY. The order of the assembled units of the unique nucleic acid sequences in XY can be controlled using nucleic acids with distinct 5' and 3' ends, and restriction enzyme digestion, ligation, polymerase chain reaction (PCR), and sequencing can be performed on the distinct 5' and 3' ends of the sequences. Such techniques can reduce the total number of nucleic acid sequences (e.g., components) used to encode N distinguishable bits by encoding information in the combination and order of the assembly product. For example, to encode 100 bits of information, two layers of 10 distinguishable nucleic acid molecules (e.g., components) may be assembled in a fixed order to generate 10*10 or 100 distinguishable nucleic acid molecules (e.g., identifiers), or one layer of 5 distinguishable nucleic acid molecules (e.g., components) and another layer of 10 distinguishable nucleic acid molecules (components) may be assembled in any order to generate 100 distinguishable nucleic acid molecules (e.g., identifiers).

[0175]

[0268] The nucleic acid sequences (e.g., components) within each layer may contain a unique (i.e., distinguishable) sequence or barcode with an intermediate common hybridization region on one end and another common hybridization region on the other end. The barcode may contain a sufficient number of nucleotides to uniquely identify every sequence within the layer. For example, there are typically four possible nucleotides at each base position within the barcode. Thus, a three base barcode may contain four nucleotides. 3= 64 nucleic acid sequences. Barcodes can be designed to be randomly generated. Alternatively, barcodes can be designed to avoid sequences that may create complications for the identifier construction chemistry or sequencing. Additionally, barcodes can be designed so that each has a minimum Hamming distance from other barcodes, thereby reducing the likelihood that mutations or reading errors at base resolution can interfere with proper identification of the barcode. See Chemical Methods Section H for rational design of DNA sequences.

[0176]

[0269] The hybridization regions at one end of a nucleic acid sequence (e.g., a component) can be different in each layer, but the hybridization regions can be the same for each member within a layer. Adjacent layers are layers that have complementary hybridization regions on the components that allow them to interact with each other. For example, any component from layer X may have complementary hybridization regions and thus be able to attach to any component from layer Y. The hybridization region on the opposite end may serve the same purpose as the hybridization region on the first end. For example, any component from layer Y may attach to any component of layer X on one end and any component of layer Z on the opposite end.

[0177]

[0270] Figures 62A and 62B show an example method, called a "product scheme," for constructing an identifier (e.g., a nucleic acid molecule) by combinatorially assembling distinguishable components (e.g., nucleic acid sequences) from each layer in a fixed order. Figure 62A shows the architecture of an identifier constructed using the product scheme. An identifier can be constructed by combining a single component from each layer in a fixed order. For M layers, each with N components, N MThere are 27 possible identifiers. Figure 62B shows an example of a combinatorial space of identifiers that can be constructed using the product method. In one example, the combinatorial space can be generated from three layers, each containing three distinct components. The components can be combined such that one component from each layer can be combined in a fixed order. The total combinatorial space of this assembly method can include 27 possible identifiers.

[0178]

[0271] Figures 63-66 illustrate chemical methods for implementing the product method (see Figure 62). The methods illustrated in Figures 63-66, along with any other method for assembling two or more distinguishable components in a fixed order, can be used to generate, for example, any one or more identifiers in an identifier library. Identifiers may be constructed using any of the implementation methods described in Figures 63-66 at any time during the methods or systems disclosed herein. In some cases, all or part of the combinatorial space of possible identifiers may be constructed before digital information is encoded or written, in which case the writing process may involve mechanically selecting and pooling identifiers (that encoded the information) from a pre-existing set. In other cases, identifiers may be constructed after one or more steps of the data encoding or writing process have occurred (i.e., as the information is being written).

[0179]

[0272] Enzymatic reactions can be used to assemble components from different layers or sets. Because components (e.g., nucleic acid sequences) from each layer have specific hybridization or attachment regions to components from adjacent layers, assembly can be performed in a one-pot reaction. For example, a nucleic acid sequence (e.g., component) X1 from layer X, a nucleic acid sequence Y1 from layer Y, and a nucleic acid sequence Z1 from layer Z can form an assembled nucleic acid molecule (e.g., identifier) ​​X1Y1Z1. Furthermore, by including multiple nucleic acid sequences from each layer, multiple nucleic acid molecules (e.g., identifiers) can be assembled in a single reaction. For example, including both Y1 and Y2 in the one-pot reaction of the previous example can produce two assembled products (e.g., identifiers) X1Y1Z1 and X1Y2Z1. This reaction multiplexing can be used to accelerate the writing time of multiple physically assembled identifiers. See Chemical Methods Section H for more information on rational design of DNA sequences as it relates to assembly efficiency. Assembly of nucleic acid sequences can be performed in a time period of about 1 day, 12 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour or less. The accuracy of the encoded data can be about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or greater than 99%.

[0180]

[0273] Identifiers may be constructed in a product-by-product fashion using overlap extension polymerase chain reaction (OEPCR), as shown in Figure 63. Each component in each layer may comprise a double-stranded or single-stranded (as shown in the figure) nucleic acid sequence with a common hybridization region on the sequence end that may be homologous and / or complementary to the common hybridization region on the sequence end of a component from an adjacent layer. Individual identifiers are composed of components X1 through X A One component (e.g., a unique sequence) from layer X (or layer 1), Y1 to Y A a second component (e.g., a unique sequence) from layer Y (or layer 2) containing Z1 to Z BA component from layer X may have a 3' end that shares complementarity with the 3' end of a component from layer Y. Thus, single-stranded components from layers X and Y can be annealed together at their 3' ends and extended using PCR to generate a double-stranded nucleic acid molecule. The generated double-stranded nucleic acid molecule can be melted to generate a 3' end that shares complementarity with the 3' end of the component from layer Z. The component from layer Z can anneal with the generated nucleic acid molecule and extended to generate a unique identifier comprising single components from layers X, Y, and Z in a fixed order. See Chemical Methods Section A for OEPCR. DNA size selection (e.g., using gel extraction; see Chemical Methods Section E) or polymerase chain reaction (PCR) using primers flanking the outermost layers (see Chemical Methods Section D) can be performed to separate the fully assembled identifier product from other by-products that may be formed during the reaction. Sequential nucleic acid capture with two probes (one for each of the two outermost layers) can also be performed to separate the fully assembled identifier product from other by-products that may be formed during the reaction (see Chemical Methods Section F).

[0181]

[0274] Identifiers can be assembled according to a product scheme using sticky end ligation, as shown in Figure 64. Distinguishable identifiers can be assembled using three layers, each containing a double-stranded component (e.g., double-stranded DNA (dsDNA)) with a single-stranded 3' overhang. For example, components X1-X A One component from layer X (or layer 1) containing Y1-Y B A second component from layer Y (or layer 2) containing Z1-Z C and a third component from layer Z (or layer 3) comprising: To combine a component from layer X with a component from layer Y, the component in layer X can comprise a common 3' overhang, labeled a in Figure 64, and the component in layer Y can comprise a common complementary 3' overhang, a *To combine components from layer Y with components from layer Z, the elements of layer Y can include a common 3' overhang, designated b in Figure 64, and the elements of layer Z can include a common complementary 3' overhang, b * The 3' overhang of the Layer X component can be complementary to the 3' end of the Layer Y component, and the other 3' overhang of the Layer Y component can be complementary to the 3' end of the Layer Z component, allowing hybridization and ligation of these components. Thus, a component from Layer X cannot hybridize to other components from Layer X or Layer Z, and similarly, a component from Layer Y cannot hybridize to other elements from Layer Y. Furthermore, a single component from Layer Y can be ligated to a single component of Layer X and a single component of Layer Z, ensuring the formation of a complete identifier. See Chemical Methods Section B for cohesive end ligation. DNA size selection (e.g., using gel extraction; see Chemical Methods Section E) or polymerase chain reaction (PCR) using primers flanking the outermost layer (see Chemical Methods Section D) can be performed to separate the identifier product from other by-products that may be formed during the reaction. Sequential nucleic acid capture with two probes (one for each of the two outermost layers) can also be performed to separate the identifier product from other by-products that may be formed during the reaction (see Chemical Methods Section F).

[0182]

[0275] Cohesive ends for sticky-end ligation can be generated by treating each layer's components with a restriction endonuclease (see Chemical Methods section C for more information on restriction enzyme reactions). In some embodiments, multiple layer components can be generated from one "parent" set of components. For example, in embodiments, a single parent set of double-stranded components can have complementary restriction sites at each end (e.g., BamHI and BglII restriction sites). Any two components can be selected for assembly and individually digested with one or the other complementary restriction enzyme (e.g., BglII or BamHI), resulting in complementary cohesive ends that can be ligated to each other, resulting in an inert scar. The product nucleic acid sequence can also contain complementary restriction sites at each end (e.g., BamHI at the 5' end and BglII at the 3' end), and such a nucleic acid sequence can be ligated to another component from the parent set following the same process. This process can be cyclical indefinitely (Figure 76). If the parent contains N components, then each cycle may correspond to adding an additional layer of N components to the product scheme.

[0183]

[0276] A method of using ligation to construct a sequence of nucleic acids comprising elements from Set X (e.g., dsDNA Set 1) and elements from Set Y (e.g., dsDNA Set 2) involves obtaining or constructing two or more pools of double-stranded sequences (e.g., dsDNA Set 1 and dsDNA Set 2), where the first set (e.g., dsDNA Set 1) contains a cohesive end (e.g., a) and the second set (e.g., dsDNA Set 2) contains a cohesive end (e.g., a) that is complementary to the cohesive end of the first set. * Any DNA from the first set (e.g., dsDNA Set 1) and any subset of DNA from the second set (e.g., dsDNA Set 2) can be combined, assembled, and then ligated together to form a single double-stranded DNA having elements from the first set and elements from the second set.

[0184]

[0277] Identifiers can be assembled in a product-wise manner using site-specific recombination, as shown in Figure 65. Identifiers can be constructed by assembling components from three different layers. Layer X (or layer 1) components have attB on one side of the molecule. x The component from layer Y (or layer 2) can contain a double-stranded molecule with a recombinase site on one side and an attP x recombinase site and attB on the other side y Layer Z (or layer 3) components can comprise a double-stranded molecule with a recombinase site, and the components of layer Z (or layer 3) can comprise an attP yEach pair can contain a recombinase site. The attB and attP sites within a pair are capable of recombination in the presence of their corresponding recombinase enzyme, as indicated by their subscripts. One component from each layer can be combined so that one component from layer X associates with one component from layer Y, and one component from layer Y associates with one component from layer Z. Application of one or more recombinase enzymes can recombine the components to generate a double-stranded identifier containing the ordered components. DNA size selection (e.g., using gel extraction) or PCR using primers flanking the outermost layer can be performed to separate the identifier product from other by-products that may be formed during the reaction. Generally, multiple orthogonal pairs of attB and attP can be used, and each pair can be used to assemble components from additional layers. For large serine recombinase families, up to six orthogonal pairs of attB and attP can be generated per recombinase, and multiple orthogonal recombinases can be implemented as well. For example, 13 layers can be assembled by using 12 orthogonal pairs of attB and attP, six from each of two large serine recombinases, such as BxbI and PhiC31. The orthogonality of the attB and attP pairs ensures that the attB site from one pair does not react with the attP site from another pair. This allows components from different layers to assemble in a fixed order. Recombinase-mediated recombination reactions can be reversible or irreversible, depending on the recombinase system being implemented. For example, the large serine recombinase family catalyzes irreversible recombination reactions without the need for any high-energy cofactors, whereas the tyrosine recombinase family catalyzes reversible reactions.

[0185]

[0278] Identifiers can be constructed in a multi-layer fashion using template-directed ligation (TDL), as shown in Figure 66A. Template-directed ligation utilizes single-stranded nucleic acid sequences, called "templates" or "staples," to facilitate the ordered ligation of components to form the identifier. The templates simultaneously hybridize to components from adjacent layers, holding them adjacent to each other (3' ends relative to 5' ends) while a ligase ligates them. In the example from Figure 66A, three layers or sets of single-stranded components are combined. Sequence a * a first layer (e.g., Layer X or Layer 1) of components sharing a common sequence a at their 3' ends that is complementary to sequence b * and c * a second layer (e.g., Layer Y or Layer 2) of components sharing common sequences b and c at their 5' and 3' ends, respectively, which are complementary to sequence d * a third layer (e.g., layer Z or layer 3) of components sharing a common sequence d at their 5' ends, which may be complementary to the sequence a; and a set of two templates, or a first staple, * b * (5'→3'), and the second staple contains the sequence c * d * A set of two "staples" containing (5'→3') nucleotides. In this example, one or more components from each layer can be selected and mixed into a reaction with staples, which can facilitate ligation of one component from each layer in a defined order to form the identifier by complementary annealing. See Chemical Methods Section B for TDL. DNA size selection (e.g., using gel extraction; see Chemical Methods Section E) or polymerase chain reaction (PCR) using primers flanking the outermost layer (see Chemical Methods Section D) can be performed to separate the identifier product from other by-products that may be formed during the reaction. Sequential nucleic acid capture with two probes (one for each of the two outermost layers) can also be performed to separate the identifier product from other by-products that may be formed during the reaction (see Chemical Methods Section F).

[0186]

[0279] Figure 66B shows a histogram of copy number (abundance) for the 256 distinct nucleic acid sequences assembled in each six-layer TDL. Each edge layer (the first and last layers) contained one component, and each of the four remaining inner layers (the remaining four layers) contained four components. Each edge layer component was 28 bases long, including a 10-base hybridization region. Each inner layer component was 30 bases long, including a 10-base common hybridization region at the 5' end, a 10-base variable (barcode) region, and a 10-base common hybridization region at the 3' end. Each of the three template strands was 20 bases long. All 256 distinct sequences were assembled in a multiplexed manner, with one reaction containing all the components and templates, T4 polynucleotide kinase (to phosphorylate the components), T4 ligase, ATP, and other appropriate reaction reagents. The reactions were incubated at 37°C for 30 minutes, then at room temperature for 1 hour. Sequencing adapters were added to the reaction products by PCR, and the products were sequenced on an Illumina MiSeq instrument. The relative copy numbers for each distinct assembled sequence are shown, out of a total of 192,910 assembled sequence reads. Other embodiments of this method may use double-stranded components, where these components are first melted to form single-stranded versions that can be annealed to staples. Other embodiments or variants of this method (i.e., TDL) can be used to construct a combinatorial space of identifiers that is more complex than can be achieved with the product method.

[0187]

[0280] A variety of other chemical implementations can be used to construct identifiers according to a product scheme, including Golden Gate assembly, Gibson assembly, and ligase cycling reaction assembly.

[0188]

[0281] Figures 67A and 67B show schematics of an example of a method, termed the "Sorting Scheme," for permuting components (e.g., nucleic acid sequences) to construct identifiers (e.g., nucleic acid molecules). Figure 67A shows the architecture of an identifier constructed using the Sorting Scheme. Identifiers can be constructed by combining single components from each layer in a programmable order. Figure 67B shows an example of a combinatorial space of identifiers that can be constructed using the Sorting Scheme. In one example, three layers, each containing one distinguishable component, can generate a combinatorial space of size 6. Components can be concatenated in any order. In general, with M layers, each with N components, the Sorting Scheme can generate a total of N M This allows for a combinatorial space of M! identifiers.

[0189]

[0282] Figure 67C shows an example of an embodiment of a permutation scheme using template-directed ligation (TDL, see Chemical Methods section B). Components from multiple layers are assembled between an anchored left-end component and an anchored right-end component, called edge scaffolds. These edge scaffolds are the same for all identifiers in the combinatorial space, and therefore these edge scaffolds can be added as part of the reaction master mix for implementation. Templates or staples are present at any possible junction between any two layers or scaffolds, such that the order in which components from different layers are incorporated into the identifier during the reaction depends on the template selected for the reaction. To allow for any possible permutation of the layers for M layers, M 2There can be +2M distinct selectable staples. M of these templates (shaded gray) form junctions between the layers and themselves and may not be included for sorting assembly as described here. However, their inclusion can allow for a larger combinatorial space using identifiers containing repetitive components, such as those shown in Figures 67D-G. DNA size selection (e.g., using gel extraction; see Chemical Methods section E) or polymerase chain reaction (PCR) using primers flanking the outermost layer (see Chemical Methods section D) can be performed to separate the identifier product from other by-products that may be formed during the reaction. Sequential nucleic acid capture with two probes, one for each of the two outermost layers, can also be performed to separate the identifier product from other by-products that may be formed during the reaction (see Chemical Methods section F).

[0190]

[0283] Figures 67D-G show an example of how the permutation scheme can be extended to include the specific case of identifiers with repeating components. Figure 67D shows an example of a method of the embodiment from Figure 67C that can be used to construct identifiers with permuted and repeating components. For example, an identifier may contain a total of three components assembled from two distinguishable components. In this example, a component from a layer may be present multiple times in the identifier. Adjacent linkages of the same component may be represented by staples with adjacent complementary hybridization regions, e.g., a in the figure. * b *This can be achieved by using a (5'→3') staple at both the 3' and 5' ends of the same component. Generally, for M layers, there are M such staples. The incorporation of repeating components in this embodiment can generate nucleic acid sequences of more than one length (i.e., containing 1, 2, 3, 4, or more components) assembled between edge scaffolds, as shown in Figure 67E. Figure 67E shows how the example embodiment from Figure 67D can result in non-targeting nucleic acid sequences assembled between edge scaffolds in addition to the identifier. The appropriate identifier cannot be separated from the non-targeting nucleic acid sequences by PCR because they share the same primer binding sites at the edges. However, in this example, because each assembled nucleic acid sequence can be designed to have a unique length (e.g., if all components have the same length), DNA size selection (e.g., using gel extraction) can be performed to separate the targeted identifier (e.g., the second sequence from the top) from the non-targeting sequences. See Chemical Methods section E for size selection. Figure 67F shows another example in which construction of identifiers with repeating components can generate multiple nucleic acid sequences with distinct lengths but equal end sequences in the same reaction. This method can use templates that assemble components in one layer with components in another layer in an alternating pattern. Similar to the method shown in Figure 67E, size selection can be used to select identifiers of a designed length. Figure 67G shows an example in which construction of identifiers with repeating components can generate multiple nucleic acid sequences with equal end sequences and, for some nucleic acid sequences (e.g., the third and fourth, and the sixth and seventh from the top), equal lengths. In this example, nucleic acid sequences that share equal lengths can be excluded from both individual identifiers, such that even when PCR and DNA size exclusion are performed, one cannot be constructed and the other cannot be constructed.

[0191]

[0284] Figures 68A-68D schematically illustrate an example of a method, referred to as the "MchooseK method," for constructing an identifier (e.g., a nucleic acid molecule) having any number of assembled components (e.g., nucleic acid sequences) K out of a larger number of M possible components. Figure 68A illustrates the architecture of an identifier constructed using the MchooseK method. Using this method, an identifier is constructed by assembling one component from each layer in any subset of all layers (e.g., selecting components from k layers out of M possible layers). Figure 68B illustrates an example of the combinatorial space of an identifier that can be constructed using the MchooseK method. In this assembly method, the combinatorial space is N for M layers, N components per layer, and an identifier length of K components. K In one example, if there are five layers, each containing one component, then up to ten distinct identifiers can be assembled, each containing two components.

[0192]

[0285] The MchooseK method can be implemented using template-directed ligation (see Chemical Methods section B), as shown in Figure 68C. Similar to the TDL embodiment for the permutation method (Figure 67C), components in this example are assembled between edge scaffolds, which may or may not be included in the reaction master mix. Components can be divided into M layers, e.g., where the left edge scaffold can be rank 1 and the right edge scaffold can be rank M+1, with M=4 layers having predefined ranks from 2 to M. The template contains the nucleic acid sequences for 3'→5' ligation of any two components having lower to higher ranks, respectively. ((M+1) 2There are (M+1) / 2 such templates. Individual identifiers for any K components from the distinguishable layers can be constructed by combining those selected components in a ligation reaction in which corresponding K+1 staples are used to join the edge scaffold and the K components in their rank order. Such a reaction setup can yield nucleic acid sequences corresponding to target identifiers between edge scaffolds. Alternatively, a reaction mix containing all templates can be combined with selected components to assemble target identifiers. This alternative method can generate a variety of nucleic acid sequences of distinguishable lengths but with the same end sequences (if all component lengths are equal), as shown in Figure 68D. Target identifiers (at the bottom) can be separated by size from the by-product nucleic acid sequences. See Chemical Methods section E for nucleic acid size selection.

[0193]

[0286] Figures 69A and 69B schematically illustrate an example of a method called a "compartmentalization scheme" for constructing identifiers with compartmentalized components. Figure 69A shows an example of a combinatorial space of identifiers that can be constructed using the compartmentalization scheme. Individual identifiers can be constructed by assembling one component from each layer in a fixed order, with any compartment (spatial classification component) placed between any two components in different layers as needed. For example, a set of components can be organized into one compartment component and four layers, each containing one component. Components from each layer can be combined in a fixed order, and single compartment components can be assembled at various locations between layers. Identifiers in this combinatorial space can include no compartment component, include a compartment between a component from the first layer and a component from the second layer, include a compartment between a component from the second layer and a component from the third layer, etc., to create a combinatorial space of eight possible identifiers. In general, with M layers, each with N components, and p compartment components, N can be constructed. K (p+1) M-1 possible identifiers can be constructed. This method can generate identifiers of various lengths.

[0194]

[0287] Figure 69B shows an example of an embodiment of a compartmentalization scheme using template-guided ligation (see Chemical Methods Section B). The template contains a nucleic acid sequence for ligating one component from each of the M layers to each other in a fixed order. For each compartment component, there is an additional pair of templates, which allow for ligation of the compartment component between components from any two adjacent layers. For example, one template of the pair (e.g., sequence g * b * (5'→3')) to allow the 3' end of layer 1 (having sequence b) to ligate to the 5' end of the compartment component (having sequence g), and the other template of the pair (e.g., sequence c * h * A template pair (having sequence h (5'→3')) allows the 3' end of a compartment component (having sequence h) to ligate to the 5' end of layer 2 (having sequence c). To insert a compartment between any two components of adjacent layers, the reaction need not include a standard template for ligating these layers together, but a template pair for ligating the compartment at that position can be selected in the reaction. In the current example, targeting of a compartment component between layer 1 and layer 2 is achieved using template c. * b * (5'→3') but template c * h * (5'→3') and template g * b * (5'→3') pairs can be used for selection for the reaction. Components can be assembled between edge scaffolds that can be included in the reaction mix (with their corresponding templates for ligation to the first and Mth layers, respectively). In general, for M layers and p compartment components, there will be a total of about M-1+2 * p *(M-1) selectable templates can be used in this method. This embodiment of the compartmentalization scheme allows reactions to generate a variety of nucleic acid sequences of distinct lengths but with the same end sequences. Target identifiers can be separated from by-product nucleic acid sequences by DNA size selection. Specifically, exactly one nucleic acid sequence product can have exactly M layer components. If the layer components are designed to be large enough compared to the compartment components, it may be possible to define a universal size selection region where identifiers can be selected (and none of the non-target by-products will be selected) regardless of the specific compartmentalization of components within the identifier, thereby allowing multiple compartmentalized identifiers from multiple reactions to be separated in the same size selection step. See Chemical Methods Section E for nucleic acid size selection.

[0195]

[0288] Figures 70A and 70B schematically illustrate an example of a method called the "unconstrained sequence scheme" or "USS" for constructing identifiers made up of any sequence of components from several possible components. Figure 70A shows an example of the combinatorial space of three-component (or four-scaffold) long identifiers that can be constructed using the unconstrained sequence scheme. The unconstrained sequence scheme constructs individual identifiers of length K components with one or more distinguishable components each taken from one or more layers, where each distinguishable component can appear in any of the K component positions in the identifier (this allows for repetition). For example, for two layers each containing one component, there are eight possible three-component long identifiers. In general, for M layers each having one component, there are M possible identifiers for components of length K. KFigure 70B shows an example of an embodiment of the unconstrained array method using template-directed ligation (see Chemical Methods section B). In this method, K+1 single-stranded, ordered scaffold DNA components (including two edge scaffolds and K-1 internal scaffolds) are present in the reaction mix. An individual identifier comprises a single component ligated between every pair of adjacent scaffolds. For example, a component ligated between scaffold A and scaffold B, a component ligated between scaffold C and scaffold D, etc. (until all K adjacent scaffold junctions are occupied by components). In the reaction, selected components from different layers are introduced to the scaffolds along with selected pairs of staples that direct them to assemble onto the appropriate scaffold. For example, staple a * L * (5'→3') and Staple A * b * The (5'→3') pair directs the ligation of layer 1 components with 5'-terminal region "a" and 3'-terminal region "b" between the L and A scaffolds. Generally, with M layers and K+1 scaffolds, 2 * M *Any USS identifier of length K can be constructed using K selectable staples. Because the staples connecting a moiety to a scaffold at its 5' end and the staples connecting the same moiety to a scaffold at its 3' end are separate, nucleic acid by-products can be formed by reacting a target identifier with an equal edge scaffold, but with fewer than K moieties (fewer than K+1 scaffolds), or with more than K moieties (more than K+1 scaffolds). Targeted identifiers can be formed using exactly K moieties (K+1 scaffolds) and therefore may be selectable by techniques such as DNA size selection if all moieties are designed to be equal length and all scaffolds are designed to be equal length. See Chemical Methods Section E for nucleic acid size selection. In certain embodiments for the unconstrained array format, where there may be one component per layer, that component may comprise exclusively a single distinct nucleic acid sequence that fulfills all three roles: (1) an identification barcode, (2) a hybridization region for staple-mediated ligation of the 5' end to the scaffold, and (3) a hybridization region for staple-mediated ligation of the 3' end to the scaffold.

[0196]

[0289] The internal scaffolds shown in Figure 70B can be designed so that they use the same hybridization sequence for both staple-mediated 5' ligation of the scaffold to one component and staple-mediated 3' ligation of the scaffold to another (not necessarily distinguishable) component. Thus, the one-scaffold, two-staple stacked hybridization event depicted in Figure 70B occurs between the scaffold and each of the staples, thus representing a statistical back-and-force hybridization event that allows both 5' and 3' component ligation. In other embodiments of the unconstrained array format, scaffolds can be designed with two linked hybridization regions—a distinguishable 3' hybridization region for staple-mediated 3' ligation and a distinguishable 5' hybridization region for staple-mediated 5' ligation.

[0197]

[0290] Figures 71A and 71B schematically illustrate an example of a method, referred to as a "component elimination scheme," for constructing identifiers by deleting nucleic acid sequences (or components) from parent identifiers. Figure 71A shows an example of a combinatorial space of possible identifiers that can be constructed using the component elimination scheme. In this example, the parent identifier can include multiple components. The parent identifier can include a number of components greater than or equal to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more. Individual identifiers can be constructed by selectively deleting any number of components from the N possible components to create identifiers of size 2. N or by removing a fixed number K components from the N possible components, thus resulting in an "NchooseK" combination space of size NchooseK. In an example using a parent identifier with 3 components, the complete combination space might be 8 and the 3choose2 combination space might be 3.

[0198]

[0291] Figure 71B shows an example of an embodiment of a component deletion scheme using double-strand targeted cleavage and repair (DSTCR). A parent sequence can be a single-stranded DNA substrate containing a component flanked by nuclease-specific target sites (which can be four bases in length or fewer), in which case the parent can be incubated with one or more double-strand-specific nucleases corresponding to the target sites. Individual components can be targeted for deletion with complementary single-stranded DNA (or cleavage template) that binds to the parent component DNA (and the flanking nuclease sites), resulting in the formation of a stable double-stranded sequence on the parent that can be cleaved at both ends by the nuclease. Another single-stranded DNA (or repair template) hybridizes with the resulting separated ends of the parent (between which the component sequence was located) and brings them together for ligation, either directly or bridged by a replacement sequence such that the ligated parent sequence no longer contains the active site targeted by the nuclease. We refer to this method as "double-stranded targeted cleavage" (DSTC). Size selection can be used to select identifiers with a specific number of components deleted. See Chemical Methods Section E for nucleic acid size selection.

[0199]

[0292] Alternatively or additionally, the parent identifier can be a double-stranded or single-stranded nucleic acid substrate containing components separated by a spacer sequence so that no two components are adjacent to the same sequence. The parent identifier can be incubated with Cas9 nuclease. Individual components can be targeted for deletion with a guide ribonucleic acid (cleavage template) that binds to the edge of the component and enables Cas9-mediated cleavage at its adjacent site. A single-stranded nucleic acid (repair template) can hybridize to the resulting separated ends of the parent identifier (e.g., where the component sequence was located between these ends), thus bringing them together for ligation. Ligation can be performed directly or by bridging the ends with a replacement sequence so that the parent ligated sequence no longer contains a spacer sequence that can be targeted by Cas9. We refer to this method as "sequence-specific targeted cleavage and repair" or "SSTCR."

[0200]

[0293] Identifiers can be constructed by inserting components into a parent identifier using a derivative of a DSTCR. The parent identifier can be a single-stranded nucleic acid substrate containing nuclease-specific target sites (which can be four or fewer bases in length) each embedded within a distinct nucleic acid sequence. The parent identifier can be incubated with one or more double-stranded-specific nucleases corresponding to the target sites. Each target site of the parent identifier can be targeted for component insertion with a complementary single-stranded nucleic acid (cleavage template) that binds to the target site of the parent identifier and the distinct surrounding nucleic acid sequence, resulting in the formation of a double-stranded site. The double-stranded site can also be cleaved by a nuclease. Another single-stranded nucleic acid (repair template) hybridizes to the resulting separated ends of the parent identifier and bridges them together for ligation, such that the ligated parent sequence no longer contains the active site targeted by the nuclease. Alternatively, a derivative of an SSTCR can be used to insert components into the parent identifier. The parent identifier can be a double-stranded or single-stranded nucleic acid, and the parent can be incubated with Cas9 nuclease. Distinguishable sites on the parent identifier can be targeted for cleavage with a guide RNA (cleavage template). Single-stranded nucleic acids (repair templates) hybridize to the separate ends of the parent identifier, bridging them together for ligation by the component sequences, such that the ligated sequence of the parent identifier no longer contains the active site targeted by the nuclease. Size selection can be used to select identifiers with a specific number of components inserted.

[0201]

[0294] Figure 72 shows a schematic representation of a parent identifier with recombinase recognition sites. Different patterns of recognition sites can be recognized by different recombinases. All recognition sites for a given set of recombinases are arranged so that the nucleic acid between them can be excised when the recombinase is applied. The nucleic acid strand shown in Figure 72 can be split into two strands depending on the subset of recombinases applied to it. 572. In some embodiments, recombinases can be used to excise, shift, invert, and transpose segments of DNA to generate unique molecules to create different nucleic acid molecules, as depicted in Figure 72. Generally, N recombinases can be used to generate 2 N possible identifiers can be constructed from the parents. In some embodiments, multiple orthogonal pairs of recognition sites from different recombinases can be arranged on the parent identifier in an overlapping manner, such that application of one recombinase influences the type of recombination event that occurs when a downstream recombinase is applied (see Roquet et al., Synthetic recombinase-based state machines in living cells, Science 353(6297):aad8559(2016), incorporated herein by reference in its entirety). Such a system may be capable of ordering N recombinases, constructing different identifiers for each N!. The recombinases may be tyrosine family recombinases, e.g., Flp and Cre, or may be recombinases of the large serine recombinase family, e.g., PhiC31, BxbI, TP901, or A118. The use of recombinases from the large serine recombinase family can be advantageous because they facilitate irreversible recombination and therefore can generate identifiers more efficiently than other recombinases.

[0202]

[0295] In some instances, a single nucleic acid sequence can be programmed to become many distinct nucleic acid sequences by applying multiple recombinases in distinct orders. Where the number of recombinases, M, can be less than 7 or can be 7 for the large serine recombinase family, applying M recombinases in different subsets in different orders can result in approximately e 1 M! distinct nucleic acid sequences can be generated. If M, the number of recombinases, can exceed 7, the number of sequences that can be generated is 3.9 MSee, for example, Roquet et al., Synthetic recombinase-based state machines in living cells, Science 353(6297):aad8559(2016), which is incorporated herein by reference in its entirety. Additional methods for generating different DNA sequences from a single consensus sequence can include targeted nucleic acid editing enzymes, such as CRISPR-Cas, TALEN, and zinc finger nucleases. Sequences generated by recombinases, targeted editing enzymes, and the like, can be used in conjunction with previous methods, such as any of the methods disclosed in any of the figures and disclosures of the present application.

[0203]

[0296] If the bitstream of information to be encoded is larger than can be encoded by any single nucleic acid molecule, the information can be divided and indexed with nucleic acid sequence barcodes. Furthermore, any subset of nucleic acid molecules of size k from a set of N nucleic acid molecules can be selected to generate log2(Nchoosek) bits of information. Barcodes can be assembled onto nucleic acid molecules in the subset of size k to encode even longer bitstreams. For example, M barcodes can be used to encode M * Log2(Nchoosek) bits of information can be generated. Given the number N of available nucleic acid molecules in a set and the number M of available barcodes, a subset of size k=k0 can be selected to minimize the total number of molecules in the pool for encoding a piece of information. A method for encoding digital information can include splitting a bitstream and encoding individual elements. For example, a bitstream containing six bits can be split into three components, each containing two bits. Each two-bit component can be barcoded to form an information cassette, which can then be grouped or pooled together to form a hyperpool of information cassettes.

[0204]

[0297] Barcodes can facilitate information indexing when the amount of digital information to be encoded exceeds the amount that can fit into a single pool. For example, layering the approach disclosed in Figure 59 by including tags with unique nucleic acid sequences encoded with nucleic acid indexes allows for encoding longer bit strings and / or information containing multiple bytes. An information cassette or identifier library can include nitrogenous bases or nucleic acid sequences containing unique nucleic acid sequences that provide position and bit value information, in addition to barcodes or tags that indicate the component of the bit stream to which a given sequence corresponds. An information cassette can include one or more unique nucleic acid sequences and barcodes or tags. The barcodes or tags on an information cassette can provide a reference to the information cassette and any sequences contained therein. For example, tags or barcodes on an information cassette can indicate which portion of a bit stream or which bit component of a bit stream the unique sequence encodes information (e.g., bit value and bit position information).

[0205]

[0298] Barcodes can be used to encode information in bits into pools that exceed the size of the combinatorial space of possible identifiers. For example, a 10-bit sequence can be divided into two sets of bytes, each containing five bits. Each byte can be mapped to a set of five possible distinguishable identifiers. Initially, the identifiers generated for each byte may be the same, but they can be kept in separate pools; otherwise, a reader of the information may not be able to tell which byte a particular nucleic acid sequence belongs to. However, by barcoding or tagging each identifier with a label corresponding to the byte to which the encoded information applies (e.g., barcode 1 can be attached to a sequence in the nucleic acid pool to provide the first five bits, and barcode 2 can be attached to a sequence in the nucleic acid pool to provide the second five bits), the identifiers corresponding to those two bytes can then be combined into a pool (e.g., a "hyperpool" or one or more identifier libraries). Each identifier library of one or more combined identifier libraries can contain a distinguishable barcode that identifies a given identifier as belonging to the given identifier library. Methods for attaching a barcode to each identifier in the identifier library can include using PCR, Gibson, ligation, or any other technique that allows a given barcode (e.g., barcode 1) to be attached to a given nucleic acid sample pool (e.g., barcode 1 to nucleic acid sample pool 1 and barcode 2 to nucleic acid sample pool 2). Sequencing methods can be used to read samples from the hyperpools, and the barcodes or tags can be used to parse the sequencing information. Methods that use identifier libraries and barcodes with a set of M barcodes and N possible identifiers (combinatorial space) can encode a bitstream having a length corresponding to the product of M and N.

[0206]

[0299] In some embodiments, the identifier libraries can be stored in an array of wells. The array of wells can be defined as having n columns and q rows, with each well containing two or more identifier libraries in a hyperpool. The encoded information in each well can constitute one large contiguous item of information, n x q in size, larger than the information contained in each well. Aliquots can be taken from one or more wells in the array of wells, and the encoding can be read using sequencing, hybridization, or PCR.

[0207]

[0300] A nucleic acid sample pool, hyperpool, identifier library, group of identifier libraries, or well containing a nucleic acid sample pool or hyperpool may contain unique nucleic acid molecules corresponding to bits of information (e.g., identifiers) and multiple complementary nucleic acid sequences. The complementary nucleic acid sequences may not correspond to encoded data (e.g., not correspond to bit values). A complementary nucleic acid sample can mask or conceal the information stored in the sample pool. The complementary nucleic acid sequences may be derived from a biological source or synthetically generated. Complementary nucleic acid sequences derived from a biological source may include randomly fragmented nucleic acid sequences or rationally fragmented sequences. Biologically derived complementary nucleic acids can hide or obscure data-containing nucleic acids in a sample pool by providing natural genetic information along with synthetically encoded information, particularly when the synthetically encoded information (e.g., the combinatorial space of the identifiers) is created to resemble natural genetic information (e.g., a fragmented genome). In one example, the identifiers are derived from a biological source and the complementary nucleic acids are derived from a biological source. A sample pool may contain multiple sets of identifiers and complementary nucleic acid sequences. Each set of identifiers and complementary nucleic acid sequences may be derived from different organisms. In one example, the identifiers are derived from one or more organisms and the complementary nucleic acid sequences are derived from a single, different organism. The complementary nucleic acid sequences may be derived from one or more organisms, and the identifiers may be derived from a single organism that is different from the organism from which the complementary nucleic acids are derived. Both the identifiers and complementary nucleic acid sequences may be derived from multiple different organisms. A key may be used to distinguish between the identifiers and complementary nucleic acid sequences.

[0208]

[0301] The supplemental nucleic acid sequence can store metadata about the written information. The metadata may include additional information for determining and / or authorizing the primary source and / or intended recipient of the primary information. The metadata may include additional information about the format of the primary information, the device and method used to encode and write the primary information, and the date and time the primary information was written to the identifier. The metadata may include additional information about the format of the primary information, the device and method used to encode and write the primary information, and the date and time the primary information was written to the nucleic acid sequence. The metadata may include additional information about modifications made to the primary information after the information was written to the nucleic acid sequence. The metadata may include annotations to the primary information or one or more references to external information. Alternatively or additionally, the metadata may be stored in one or more barcodes or tags associated with the identifier.

[0209]

[0302] The identifiers in the identifier pool can have the same, similar, or different lengths from one another. The complementary nucleic acid sequences can have a length that is less than the length of the identifiers, a length that is substantially equal to the length of the identifiers, or a length that is longer than the length of the identifiers. The complementary nucleic acid sequences can have an average length that is within 1 base, 2 bases, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, or more of the average length of the identifiers. In one example, the complementary nucleic acid sequences are the same or substantially the same length as the identifiers. The concentration of the complementary nucleic acid sequences can be less than, substantially equal to, or higher than the concentration of the identifiers in the identifier library. The concentration of the complementary nucleic acid can be about 1%, 10%, 20%, 40%, 60%, 80%, 100%, 125%, 150%, 175%, 200%, 1000%, 1×10 of the concentration of the identifiers. 4 %, 1×10 5 %, 1×10 6 %, 1×10 7 %, 1×10 8% or less, or about 1%, 10%, 20%, 40%, 60%, 80%, 100%, 125%, 150%, 175%, 200%, 1000%, 1×10 concentration of the identifier. 4 %, 1×10 5 %, 1×10 6 %, 1×10 7 %, 1×10 8 The concentration of the complementary nucleic acid may be equal to or less than about 1%, 10%, 20%, 40%, 60%, 80%, 100%, 125%, 150%, 175%, 200%, 1000%, 1×10 of the concentration of the identifier. 4 %, 1×10 5 %, 1×10 6 %, 1×10 7 %, 1×10 8 %, or more than about 1%, 10%, 20%, 40%, 60%, 80%, 100%, 125%, 150%, 175%, 200%, 1000%, 1×10 concentration of the identifier. 4 %, 1×10 5 %, 1×10 6 %, 1×10 7 %, 1×10 8 % or more. Higher concentrations may be beneficial for obfuscation or data hiding. In one example, the concentration of the capture nucleic acid sequences is substantially higher than the concentration of the identifiers in the identifier pool (e.g., 1 x 10 8 %expensive).

[0210]

[0303] In another aspect, the present disclosure provides a method for copying information encoded in a nucleic acid sequence. The method for copying information encoded in a nucleic acid sequence may include (a) providing an identifier library and (b) constructing one or more copies of the identifier library. The identifier library may include a subset of a plurality of identifiers from a larger combinatorial space. Each individual identifier of the plurality of identifiers may correspond to an individual symbol in a symbol string. The identifier may include one or more components. The components may include a nucleic acid sequence.

[0211]

[0304] In another aspect, the present disclosure provides a method for accessing information encoded in a nucleic acid sequence. The method for accessing information encoded in a nucleic acid sequence may include (a) providing an identifier library and (b) extracting from the identifier library a portion or subset of identifiers present in the identifier library. The identifier library may include a subset of a plurality of identifiers from a larger combinatorial space. Each individual identifier of the plurality of identifiers may correspond to an individual symbol in a symbol string. The identifier may include one or more components. The components may include a nucleic acid sequence.

[0212]

[0305] Information can be written to one or more identifier libraries as described elsewhere herein. Identifiers can be constructed using any of the methods described elsewhere herein. Stored data can be copied by generating copies of individual identifiers in the identifier library or in one or more identifier libraries. Portions of identifiers may be copied, or the entire library may be copied. Copying can be accomplished by amplifying identifiers in the identifier library. When one or more identifier libraries are combined, a single identifier library may be copied, or multiple identifier libraries may be copied. If an identifier library includes a supplemental nucleic acid sequence, the supplemental nucleic acid sequence may or may not be copied.

[0213]

[0306] Identifiers in an identifier library may be constructed to include one or more common primer binding sites. The one or more binding sites may be located on the edge of each identifier or may be interwoven throughout each identifier. The primer binding sites may allow an identifier library-specific primer pair or a universal primer pair to bind to the identifier and amplify the identifier. All identifiers in an identifier library or all identifiers in one or more identifier libraries may be replicated multiple times using multiple PCR cycles. Identifiers may be copied using conventional PCR, and the identifiers may be replicated exponentially with each PCR cycle. The number of identifier copies may increase exponentially with each PCR cycle. Identifiers may be copied using linear PCR, and the identifiers may be replicated linearly with each PCR cycle. The number of identifier copies may increase linearly with each PCR cycle. Identifiers may be ligated into a circular vector prior to PCR amplification. The circular vector may include a barcode at each end of the identifier insertion site. PCR primers for amplifying the identifier can be designed to prime from the vector, so that the barcoded edges are included in the amplification product along with the identifier. During amplification, recombination between identifiers can result in copied identifiers containing uncorrelated barcodes on each edge. Uncorrelated barcodes can be detectable when reading the identifier. Identifiers containing uncorrelated barcodes can be considered false positives and can be ignored during the information decoding process. See Chemical Methods Section D.

[0214]

[0307] Information can be encoded by assigning each bit of information to a unique nucleic acid molecule. For example, three sample sets (X, Y, and Z), each with two nucleic acid sequences, can be assembled into eight unique nucleic acid molecules to encode the following eight data bits: N1=X1Y1Z1 N2=X1Y1Z2 N3=X1Y2Z1 N4=X1Y2Z2 N5=X2Y1Z1 N6=X2Y1Z2 N7=X2Y2Z1 N8=X2Y2Z2 Thus, each bit in the string can be assigned to a corresponding nucleic acid molecule (e.g., N1 can designate the first bit, N2 can designate the second bit, N3 can designate the third bit, etc.). The entire bit string can be assigned to a combination of nucleic acid molecules, where nucleic acid molecules corresponding to a bit value of "1" are included in the combination or pool. For example, in UTF-8 encoding, the letter "K" can be represented by the 8-bit string code 01001011, and this 8-bit string code can be encoded by the presence of four nucleic acid molecules (e.g., X1Y1Z2, X2Y1Z1, X2Y2Z1, and X2Y2Z2 in the example above).

[0215]

[0308] Information can be accessed by sequencing or hybridization assays. For example, primers or probes can be designed to bind to common or barcoded regions of a nucleic acid sequence. This can allow for amplification of any region of a nucleic acid molecule. The amplification product can then be read by sequencing the amplification product or by hybridization assays. In the above example of encoding the letter "K," if the first half of the data is of interest, a primer specific to the barcode region of the X1 nucleic acid sequence and a primer binding to the common region of the Z set can be used to amplify the nucleic acid molecule. This can return the sequence Y1Z2, which can encode 0100. Subsequent sequences of that data can also be accessed by further amplifying the nucleic acid molecule with a primer binding to the barcode region of the Y1 nucleic acid sequence and a primer binding to the common sequence of the Z set. This can return the Z2 nucleic acid sequence, which encodes the subsequence 01. Alternatively, data can be accessed by checking for the presence or absence of specific nucleic acid sequences without sequencing. For example, amplification using a primer specific to the Y2 barcode can generate an amplification product for the Y2 barcode but not the Y1 barcode. The presence of a Y2 amplification product can signal a bit value of "1." Alternatively, the absence of a Y2 amplification product can signal a bit value of "0."

[0216]

[0309] PCR-based methods can be used to access and copy data from identifiers or nucleic acid sample pools. Common primer binding sites adjacent to identifiers within a pool or hyperpool can be used to easily copy information-containing nucleic acids. Alternatively, other nucleic acid amplification techniques, such as isothermal amplification, can be used to easily copy data from a sample pool or hyperpool (e.g., an identifier library). See Chemical Methods Section D for nucleic acid amplification. In examples where a sample contains a hyperpool, a primer that binds in the forward direction to a specific barcode on one edge of the identifier can be used with another primer that binds in the reverse direction to a common sequence on the opposite edge of the identifier to access and obtain a specific subset of information (e.g., all nucleic acids associated with a particular barcode). Various reading methods can be used to extract information from the encoded nucleic acids, for example, microarrays (or any type of fluorescent hybridization), digital PCR, quantitative PCR (qPCR), and various sequencing platforms can also be used to read the encoded sequence and, by extension, the digitally encoded data.

[0217]

[0310] Accessing information stored in nucleic acid molecules (e.g., identifiers) can be achieved by selectively removing a portion of non-targeted identifiers from an identifier library or pool of identifiers, or, for example, by selectively removing all identifiers in an identifier library from a pool of multiple identifier libraries. As used herein, "access" and "query" can be used interchangeably. Accessing data can also be achieved by selectively capturing targeted identifiers from an identifier library or pool of identifiers. Targeted identifiers can correspond to data of interest within a longer information item. A pool of identifiers may also include supplemental nucleic acid molecules. Supplemental nucleic acid molecules may contain metadata about the encoded information and may be used to conceal or mask identifiers corresponding to the information. Supplemental nucleic acid molecules may or may not be extracted during access to targeted identifiers. Figures 73A-73C schematically outline an example method for accessing a portion of information stored in a nucleic acid sequence by accessing certain specific identifiers from a larger number of identifiers. Figure 73A shows an example method for accessing identifiers containing specified components using polymerase chain reaction, affinity-tagged probes, and degradation-targeted probes. For PCR-based access, a pool of identifiers (e.g., an identifier library) may contain identifiers with a common sequence at each end, a variable sequence at each end, or either a common sequence or a variable sequence at each end. The common sequence or variable sequence may be a primer binding site. One or more primers may bind to the common or variable regions at the edges of the identifiers. Primed identifiers can be amplified by PCR. Amplified identifiers can significantly outnumber unamplified identifiers. During reading, amplified identifiers can be identified. Identifiers from an identifier library can contain a sequence at one or both of their ends that is distinguishable from that library, thus allowing selective access to a single library from a pool or group of more than one identifier library.

[0218]

[0311] In the case of affinity tag-based access, a process sometimes referred to as nucleic acid capture, components constituting the identifiers in the pool may share complementarity with one or more probes. One or more probes may bind or hybridize to the identifiers to be accessed. The probes may include affinity tags. The affinity tags may be captured on a solid support such as a membrane, a well, a column, or beads. When beads are used as the solid support, the affinity tags may bind to the beads to form complexes comprising the beads, at least one probe, and at least one identifier. The beads may be magnetic, and together with a magnet, the beads can collect and separate the identifiers to be accessed. Prior to reading, the identifiers may be removed from the beads under denaturing conditions. Alternatively or additionally, the beads may collect non-targeted identifiers and sequester them from the remainder of the pool, which may be washed, transferred to a separate container, and read. When a column is used, the affinity tags may be bound to the column. The identifiers to be accessed may be bound to the column for capture. Subsequently, the column-bound identifiers may be eluted or denatured from the column prior to reading. Alternatively, non-targeted identifiers can be selectively targeted to a column, while targeted identifiers can flow through the column. Identifiers bound to a solid support can be removed from the solid support by exposure to conditions such as acid, base, oxidation, reduction, heat, light, metal ion catalysis, displacement, or elimination chemical reactions, or by enzymatic cleavage. In certain embodiments, the accessed identifier can be attached to the solid support through a cleavable linkage moiety. For example, the solid support can be functionalized to provide a cleavable linker for covalently binding to the targeted identifier. The linker moiety can be six atoms or longer. In some embodiments, the cleavable linker can be a TOPS (two oligonucleotides per synthesis) linker, an amino linker, a chemically cleavable linker, or a photocleavable linker. Accessing the targeted identifiers can involve simultaneously applying one or more probes to the pool of identifiers, or sequentially applying one or more probes to the pool of identifiers.For nucleic acid capture, see Chemical Methods Section F.

[0219]

[0312] In the case of degradation-based access, the components constituting the identifiers in the pool may share complementarity with one or more degradation-targeting probes. The probes can bind or hybridize to distinguishable components of the identifiers. The probes can be targeted by degradative enzymes such as endonucleases. In one example, one or more identifier libraries can be combined. A set of probes can hybridize with one of the identifier libraries. The set of probes can include RNA, which can guide a Cas9 enzyme. The Cas9 enzyme can be introduced into one or more identifier libraries. Identifiers hybridized with the probes can be degraded by the Cas9 enzyme. Identifiers to be accessed may not be degraded by the degradative enzyme. In another example, the identifiers can be single-stranded, and the identifier library can be combined with a single-strand-specific endonuclease, such as S1 nuclease, that selectively degrades identifiers that are not to be accessed. Identifiers to be accessed can be hybridized with a complementary set of identifiers to protect them from degradation by the single-strand-specific endonuclease. The identifier to be accessed may be separated from the degradation products by size selection, such as by size-selective chromatography (e.g., agarose gel electrophoresis). Alternatively or additionally, the undegraded identifier may be selectively amplified (e.g., using PCR), so that the degradation products are not amplified. Primers that hybridize to each end of the undegraded identifier, and therefore do not hybridize to each end of the degraded or cleaved identifier, can be used to amplify the undegraded identifier.

[0220]

[0313] Figure 73B shows an example of a method for performing an "OR" or "AND" operation using the polymerase chain reaction to access identifiers containing multiple components. In one example, if two forward primers bind to distinct sets of identifiers on the left end, "OR" amplification of the intersection of these sets of identifiers can be achieved by using the two forward primers together in a multiplex PCR reaction with a reverse primer that binds to all of the identifiers on the right end. In another example, if one forward primer binds to a set of identifiers on the left end and one reverse primer binds to a set of identifiers on the right end, "AND" amplification of the intersection of these two sets of identifiers can be achieved by using the forward and reverse primers together as a primer pair in a PCR reaction.

[0221]

[0314] Figure 73C shows an example of a method for using affinity tags to perform an "OR" or "AND" operation to access identifiers containing multiple components. In one example, if affinity probe "P1" captures all identifiers with component "C1" and another affinity probe "P2" captures all identifiers with component "C2," then P1 and P2 can be used simultaneously to capture the set of all identifiers with C1 or C2 (corresponding to an "OR" operation). In another example, using the same components and probes, P1 and P2 can be used sequentially to capture the set of all identifiers with C1 and C2 (corresponding to an "AND" operation).

[0222]

[0315] In another aspect, the present disclosure provides a method for reading information encoded in a nucleic acid sequence. The method for reading information encoded in a nucleic acid sequence may include (a) providing an identifier library, (b) identifying identifiers present in the identifier library, (c) generating a symbol string from the identifiers present in the identifier library, and (d) compiling information from the symbol string. The identifier library may include a subset of a plurality of identifiers from the combinatorial space. Each individual identifier of the subset of identifiers may correspond to an individual symbol in the symbol string. The identifier may include one or more components. The components may include nucleic acid sequences.

[0223]

[0316] Information can be written to one or more identifier libraries as described elsewhere herein. Identifiers can be constructed using any of the methods described elsewhere herein. Stored data can be copied and stored data can be accessed using any of the methods described elsewhere herein.

[0224]

[0317] The identifier may include information about the location of the coded symbol, the value of the coded symbol, or both the location and value of the coded symbol. The identifier may include information about the location of the coded symbol, and the presence or absence of the identifier in the identifier library may indicate the value of the symbol. The presence of the identifier in the identifier library may indicate a first symbol value (e.g., a first bit value) in the binary string, and the absence of the identifier in the identifier library may indicate a second symbol value (e.g., a second bit value) in the binary string. Basing the bit value on the presence or absence of the identifier in the identifier library in a binary manner may reduce the number of identifiers to be assembled and therefore reduce writing time. In one example, the presence of the identifier may indicate a bit value of "1" at the mapped position, and the absence of the identifier may indicate a bit value of "0" at the mapped position.

[0225]

[0318] Generating a symbol (e.g., a bit value) for a piece of information can include identifying the presence or absence of an identifier to which the symbol (e.g., bit) can be mapped or encoded. Determining the presence or absence of an identifier can include sequencing the identifier or using a hybridization array to detect the presence of the identifier. In one example, decoding and reading the encoded sequence can be performed using a sequencing platform. Exemplary sequencing platforms include U.S. Patent Application No. 14 / 465,685, filed August 21, 2014, entitled "METHOD OF NUCLEIC ACID AMPLIFICATION," which was published December 18, 2014 as U.S. Patent Application Publication No. 2014-0371100A1; U.S. Patent Application No. 13 / 886,234, filed May 2, 2013, entitled "METHOD OF NUCLEIC ACID AMPLIFICATION," which was published September 5, 2013 as U.S. Patent Application Publication No. 2013-0231254A1; and U.S. Patent Application No. 13 / 886,234, filed May 2, 2013, entitled "METHOD OF NUCLEIC ACID AMPLIFICATION," which was published October 8, 2009 as U.S. Patent Application Publication No. 2009-0253141A1. No. 12 / 400,593, filed March 9, 2009, entitled "SEQUENCES," each of which is incorporated herein by reference in its entirety.

[0226]

[0319] In one example, decoding of nucleic acid-encoded data can be achieved by base-by-base sequencing of nucleic acid strands, such as Illumina® Sequencing, or by utilizing sequencing techniques that indicate the presence or absence of specific nucleic acid sequences, such as capillary electrophoretic fragmentation analysis. Sequencing may also utilize the use of reversible terminators. Sequencing may also utilize the use of natural or unnatural (e.g., engineered) nucleotides or nucleotide analogs. Alternatively or additionally, decoding of nucleic acid sequences can be performed using a variety of analytical techniques, including, but not limited to, any method that generates an optical, electrochemical, or chemical signal. A variety of sequencing techniques can be used, including but not limited to polymerase chain reaction (PCR), digital PCR, Sanger sequencing, high-throughput sequencing, sequencing-by-synthesis, single molecule sequencing, sequencing-by-ligation, RNA-Seq (Illumina), next-generation sequencing, digital gene expression (Helicos), clonal single microarray (Solexa), shotgun sequencing, Maxim-Gilbert sequencing, or massively parallel sequencing.

[0227]

[0320] Various readout methods can be used to extract information from the encoded nucleic acids. In one example, microarrays (or any type of fluorescent hybridization), digital PCR, quantitative PCR (qPCR), and various sequencing platforms can also be used to read the encoded sequence and, by extension, the digitally encoded data.

[0228]

[0321] The identifier library may further include complementary nucleic acid sequences that provide metadata about the information, that conceal or mask the information, or that both provide metadata and mask the information. The complementary nucleic acids may be identified simultaneously with the identification of the identifier. Alternatively, the complementary nucleic acids may be identified before or after the identifier is identified. In one example, the complementary nucleic acids are not identified during reading of the encoded information. The complementary nucleic acid sequences may be indistinguishable from the identifier. An identifier index or key may be used to differentiate complementary nucleic acid molecules from the identifier.

[0229]

[0322] Data encoding and decoding efficiency can be improved by re-encoding an input bit string to reduce the number of nucleic acid molecules used. For example, if an encoding method receives an input string with a high occurrence of "111" subsequences that can be mapped to three nucleic acid molecules (e.g., identifiers), it can be re-encoded to a "000" subsequence that can be mapped to an empty set of nucleic acid molecules. Alternative input subsequences of "000" can also be re-encoded to "111." This re-encoding method can reduce the total amount of nucleic acid molecules used to encode the data because the number of "1"s in the dataset can be reduced. In this example, the total size of the dataset can be increased to accommodate a codebook that specifies new mapping instructions. An alternative method for improving encoding and decoding efficiency can be to re-encode an input string to reduce its variable length. For example, "111" can be re-encoded to "00," which can reduce the size of the dataset and reduce the number of "1"s in the dataset.

[0230]

[0323] By specifically designing identifiers to facilitate detection, the speed and efficiency of decoding nucleic acid-encoded data can be controlled (e.g., increased). For example, a nucleic acid sequence (e.g., an identifier) ​​designed for ease of detection can include a nucleic acid sequence containing a majority of nucleotides that are easier to call and detect based on their optical, electrochemical, chemical, or physical properties. Engineered nucleic acid sequences can be single-stranded or double-stranded. Engineered nucleic acid sequences can also contain synthetic or non-natural nucleotides that enhance the detectable properties of the nucleic acid sequence. Engineered nucleic acid sequences can contain all natural nucleotides, all synthetic or non-natural nucleotides, or a combination of natural, synthetic, and non-natural nucleotides. Synthetic nucleotides can include nucleotide analogs, such as peptide nucleic acids, locked nucleic acids, glycol nucleic acids, and threose nucleic acids. Non-natural nucleotides can include dNaM, an artificial nucleoside containing a 3-methoxy-2-naphthyl group, and d5SICS, an artificial nucleoside containing a 6-methylisoquinoline-1-thion-2-yl group. The engineered nucleic acid sequences may be designed for a single enhanced property, such as enhanced optical properties, or the engineered nucleic acid sequences may be designed with multiple enhanced properties, such as enhanced optical and electrochemical properties or enhanced optical and chemical properties. See Chemical Methods Section H regarding DNA design.

[0231]

[0324] Engineered nucleic acid sequences may contain reactive natural, synthetic, and non-natural nucleotides that do not enhance the optical, electrochemical, chemical, or physical properties of the nucleic acid sequence. The reactive components of the nucleic acid sequence may allow for the addition of chemical moieties that confer enhanced properties to the nucleic acid sequence. Each nucleic acid sequence may contain a single chemical moiety or multiple chemical moieties. Examples of chemical moieties include, but are not limited to, fluorescent moieties, chemiluminescent moieties, acidic or basic moieties, hydrophobic or hydrophilic moieties, and moieties that alter the oxidation state or reactivity of the nucleic acid sequence.

[0232]

[0325] Sequencing platforms may be specifically designed for decoding and reading information encoded in nucleic acid sequences. Sequencing platforms may be dedicated to sequencing single-stranded nucleic acid molecules or double-stranded nucleic acid molecules. Sequencing platforms may decode nucleic acid-encoded data by reading individual bases (e.g., base-by-base sequencing) or by detecting the presence or absence of the entire nucleic acid sequence (e.g., component) incorporated into a nucleic acid molecule (e.g., an identifier). Sequencing platforms may include the use of promiscuous reagents, longer read lengths, and the use of detection of specific nucleic acid sequences by adding detectable chemical moieties. Using more promiscuous reagents during sequencing may increase read efficiency by enabling faster base calling, thereby reducing sequencing time. The use of longer read lengths may allow longer sequences of encoded nucleic acids to be decoded per read. Adding detectable chemical moiety tags may allow the detection of the presence or absence of a nucleic acid sequence by the presence or absence of the chemical moiety. For example, each nucleic acid sequence encoding a bit of information can be tagged with a chemical moiety that produces a unique optical, electrochemical, or chemical signal. The presence or absence of that unique optical, electrochemical, or chemical signal can indicate a "0" or "1" bit value. A nucleic acid sequence can contain a single chemical moiety, or it can contain multiple chemical moieties. The chemical moiety can be added to a nucleic acid sequence prior to using the nucleic acid sequence to encode data. Alternatively, or in addition, the chemical moiety can be added to a nucleic acid sequence after encoding the data but before decoding the data. The chemical moiety tag can be added directly to the nucleic acid sequence, or the nucleic acid sequence can contain a synthetic or non-natural nucleotide anchor to which the chemical moiety tag can be added.

[0233]

[0326] A unique code can be applied to minimize or detect encoding and decoding errors. Encoding and decoding errors can occur due to false negatives (e.g., nucleic acid molecules or identifiers not included in random sampling). One example of an error detection code can be a checksum sequence that counts the number of identifiers in a consecutive set of possible identifiers included in an identifier library. During reading of the identifier library, the checksum can indicate the expected number of identifiers obtained from that consecutive set, and identifiers can continue to be sampled for reading until the expected number is met. In some embodiments, a checksum sequence can be included for every R consecutive sets of identifiers, where R can be equal to or greater than 1, 2, 5, 10, 50, 100, 200, 500, or 1000 in size, or can be less than 1000, 500, 200, 100, 50, 10, 5, or 2. The smaller the value of R, the better the error detection. In some embodiments, the checksum can be a complementary nucleic acid sequence. For example, a set containing seven nucleic acid sequences (e.g., components) can be divided into two groups: nucleic acid sequences for constructing identifiers in a product manner (components X1-X3 in layer X and Y1-Y3 in layer Y) and nucleic acid sequences for complementary checksums (X4-X7 and Y4-Y7). Checksum sequences X4-X7 can indicate whether 0, 1, 2, or 3 sequences from layer X assemble with each member of layer Y. Alternatively, checksum sequences Y4-Y7 can indicate whether 0, 1, 2, or 3 sequences from layer Y assemble with each member of layer X. In this example, the original identifier library with identifiers {X1Y1, X1Y3, X2Y1, X2Y2, X2Y3} can be supplemented to include checksums to result in the following pool: {X1Y1, X1Y3, X2Y1, X2Y2, X2Y3, X1Y6, X2Y7, X3Y4, X6Y1, X5Y2, X6Y3}. Checksum sequences can also be used for error correction. For example, the absence of X1Y1 and the presence of X1Y6 and X6Y1 in the above dataset can allow for the inference that the X1Y1 nucleic acid molecule is missing from the dataset.The checksum sequence can indicate whether the identifier is missing from the sampled or accessed portion of the identifier library. If the checksum sequence is missing, an accessing method such as PCR or affinity-tagged probe hybridization can amplify and / or isolate it. In some embodiments, the checksum may not be a complementary nucleic acid sequence. In that case, the checksum can be directly encoded into the information so that it is represented by the identifier.

[0234]

[0327] Data encoding and decoding noise can be reduced by constructing identifiers as palindromes, for example, by using palindromic pairs of components rather than single components in a product manner. Pairs of components from different layers can then be assembled together in a palindromic manner (e.g., YXY rather than XY for components X and Y). This palindromic method can be extended to a larger number of layers (e.g., ZYXYZ rather than XYZ), and may allow for the detection of erroneous cross-reactivity between identifiers.

[0235]

[0328] Adding excess (e.g., large excess) complementary nucleic acid sequences to the identifier can prevent recovery of the encoded identifier by sequencing. Prior to decoding the information, the identifier may be enriched by the complementary nucleic acid sequences. For example, the identifier may be enriched by a nucleic acid amplification reaction using primers specific to the identifier end. Alternatively or additionally, the information may be decoded without enriching the sample pool by sequencing using specific primers (e.g., sequencing-by-synthesis). In both decoding methods, it may be difficult to enrich or decode the information without the decoding key or without knowing something about the composition of the identifier. Alternative access methods, such as the use of affinity tag-based probes, may also be utilized.

[0236]

[0329] Systems for encoding digital information into nucleic acids (e.g., DNA) can include systems, methods, and devices for converting files and data (e.g., raw data, compressed zip files, integer data, and other forms of data) into bytes and encoding the bytes into nucleic acids, typically segments or sequences of DNA, or combinations thereof.

[0237]

[0330] In one aspect, the present disclosure provides a system for encoding binary sequence data using nucleic acids. The system for encoding binary sequence data using nucleic acids may include a device and one or more computer processors. The device may be configured to construct an identifier library. The one or more computer processors may be individually or collectively programmed to (i) translate information into a symbol string, (ii) map the symbol string to a plurality of identifiers, and (iii) construct an identifier library including at least a subset of the plurality of identifiers. Each identifier of the plurality of identifiers may correspond to an individual symbol of the symbol string. Each identifier of the plurality of identifiers may include one or more components. Each component of the one or more components may include a nucleic acid sequence.

[0238]

[0331] In another aspect, the present disclosure provides a system for reading binary sequence data using nucleic acids. The system for reading binary sequence data using nucleic acids may include a database and one or more computer processors. The database may store an identifier library encoding information. The one or more computer processors may be individually or collectively programmed to (i) identify identifiers in the identifier library, (ii) generate a plurality of symbols from the identifiers identified in (i), and (iii) compile information from the plurality of symbols. The identifier library may include a subset of the plurality of identifiers. Each individual identifier in the plurality of identifiers may correspond to an individual symbol in a symbol string. The identifier may include one or more components. The components may include nucleic acid sequences.

[0239]

[0332] A non-limiting embodiment of a method of using the system for encoding digital data can include receiving digital information in the form of a byte stream, parsing the byte stream into individual bytes, mapping bit positions within the bytes using a nucleic acid index (or identifier rank), and encoding sequences corresponding to either a bit value of 1 or a bit value of 0 into an identifier. Obtaining the digital data can include sequencing a nucleic acid sample or pool containing sequences of nucleic acids (e.g., identifiers) that map to one or more bits, referencing the identifier rank to determine whether the identifier is present in the nucleic acid pool, and decoding the position and bit value information for each sequence into bytes containing the sequence of digital information.

[0240]

[0333] A system for encoding, writing, copying, accessing, reading, and decoding information encoded in or written to a nucleic acid molecule may be a single integrated unit or multiple units configured to perform one or more of the above-mentioned operations. A system for encoding and writing information to a nucleic acid molecule (e.g., an identifier) ​​may include a device and one or more computer processors. The one or more computer processors may be programmed to parse the information into a string of symbols (e.g., a string of bits). The computer processor may generate a rank of the identifiers. The computer processor may categorize the symbols into two or more categories. One category may include symbols represented by the presence of a corresponding identifier in the identifier library, and the other category may include symbols represented by the absence of a corresponding identifier in the identifier library. The computer processor may direct the device to assemble an identifier corresponding to a symbol represented by the presence of the identifier in the identifier library.

[0241]

[0334] The device may include multiple regions, sections, or compartments. Reagents and components for assembling the identifier may be stored in one or more regions, sections, or compartments of the device. Layers may be stored in separate regions of a section of the device. Layers may contain one or more unique components. Components within one layer may be unique and not overlap with components in another layer. Regions or sections may include containers, and compartments may include wells. Each layer may be stored in a separate container or compartment. Each reagent or nucleic acid sequence may be stored in a separate container or compartment. Alternatively or additionally, reagents may be combined to form a master mix for identifier construction. The device may transfer reagents, components, and templates from one section of the device to be combined with each other. The device may provide conditions for completing the assembly reaction. For example, the device may provide heating, agitation, and detection of reaction progress. The constructed identifier may be subjected to one or more subsequent reactions to add barcodes, common sequences, variable sequences, or tags to one or more ends of the identifier. The identifiers can then be directed to the regions or compartments to generate an identifier library. One or more identifier libraries can be stored in each region, section, or individual compartment of the device. The device can transfer fluids (e.g., reagents, components, templates) using pressure, vacuum, or suction.

[0242]

[0335] The identifier library can be stored on the device or transferred to a separate database. The database can contain one or more identifier libraries. The database can provide conditions for long-term storage of the identifier library (e.g., conditions to reduce degradation of the identifiers). The identifier library can be stored in powder, liquid, or solid form. Aqueous solutions of the identifiers can be lyophilized for more stable storage (see Chemical Methods Section G for more information on lyophilization). Alternatively, the identifiers can be stored in the absence of oxygen (e.g., anaerobic storage conditions). The database can provide UV photoprotection, reduced temperature (e.g., refrigeration or freezing), and protection from degradative chemicals and enzymes. Prior to transfer to the database, the identifier library can be lyophilized or frozen. The identifier library can contain ethylenediaminetetraacetic acid (EDTA) to inactivate nucleases and / or buffers to maintain the stability of the nucleic acid molecules.

[0243]

[0336] The database may be coupled to, include, or be separate from a device that writes, copies, accesses, or reads information to the identifiers. A portion of the identifier library can be removed from the database before copying, accessing, or reading. The device that copies information from the database can be the same device as the device that writes the information or a different device. The device that copies information can extract an aliquot of the identifier library from the device and combine the aliquot with reagents and components to amplify some or all of the identifier library. The device can control the temperature, pressure, and agitation of the amplification reaction. The device can include compartments, and one or more amplification reactions can occur in the compartment containing the identifier library. The device can copy more than one pool of identifiers simultaneously.

[0244]

[0337] The copied identifiers may be transferred from the copy device to the access device. The access device may be the same device as the copy device. The access device may include separate regions, sections, or compartments. The access device may have one or more columns, bead reservoirs, or magnetic regions for separating the identifiers bound to the affinity tags (see Chemical Methods Section F for nucleic acid capture). Alternatively, or in addition, the access device may have one or more size selection units. The size selection unit may include agarose gel electrophoresis or any other method for size-selecting nucleic acid molecules (see Chemical Methods Section E for more information on nucleic acid size selection). Copying and extraction may be performed in the same region of the device or in different regions of the device (see Chemical Methods Section D for nucleic acid amplification).

[0245]

[0338] The accessed data can be read in the same device, or the accessed data can be transferred to another device. The reading device can include a detection unit for detecting and identifying the identifier. The detection unit can be part of a sequencer, hybridization array, or other unit for identifying the presence or absence of the identifier. The sequencing platform can be specifically designed for decoding and reading information encoded in nucleic acid sequences. The sequencing platform can be dedicated to sequencing single-stranded or double-stranded nucleic acid molecules. The sequencing platform can decode nucleic acid-encoded data by reading individual bases (e.g., base-by-base sequencing) or by detecting the presence or absence of an entire nucleic acid sequence (e.g., a component) incorporated within the nucleic acid molecule (e.g., an identifier). Alternatively, the sequencing platform can be a system such as Illumina® sequencing or capillary electrophoretic fragmentation analysis. Alternatively, or in addition, decoding of the nucleic acid sequence can be performed using a variety of analytical techniques implemented by the device, including, but not limited to, any method that produces an optical, electrochemical, or chemical signal.

[0246]

[0339] Storing information in nucleic acid molecules can have various applications, including, but not limited to, long-term information storage, confidential information storage, and medical information storage. In one example, a person's medical information (e.g., medical history and medical records) can be stored in a nucleic acid molecule and kept by the person. The information can be stored externally (e.g., in a wearable device) or internally (e.g., in a subcutaneous capsule). When a patient is brought to a clinic or hospital, a sample can be obtained from the device or capsule, and the information can be decoded using a nucleic acid sequencer. Storing personal medical records in nucleic acid molecules can provide an alternative to computer- and cloud-based storage systems. Storing personal medical records in nucleic acid molecules can reduce the incidence or prevalence of medical record hacking. The nucleic acid molecules used in capsule-based medical record storage can be derived from the human genome sequence. Using the human genome sequence can reduce the immunogenicity of the nucleic acid sequence in the event of capsule damage and leakage.

[0247]

[0340] The present disclosure provides computer systems programmed to implement the methods of the present disclosure. Figure 75 shows a computer system 1901 programmed or otherwise configured to encode digital information into a nucleic acid sequence and / or read (e.g., decode) information derived from a nucleic acid sequence. The computer system 1901 can adjust various aspects of the encoding and decoding procedures of the present disclosure, such as, for example, bit value and bit position information for a given bit or byte from an encoded bitstream or bytestream.

[0248]

[0341] The computer system 1901 includes a central processing unit (CPU, also referred to herein as a "processor" and a "computer processor") 1905, which may be a single-core processor or a multi-core processor, or multiple processors for parallel processing. The computer system 1901 also includes memory or memory locations 1910 (e.g., random access memory, read-only memory, flash memory), electronic storage 1915 (e.g., a hard disk), a communication interface 1920 (e.g., a network adapter) for communicating with one or more other systems, and peripherals 1925, such as cache, other memory, data storage, and / or electronic display adapters. The memory 1910, storage 1915, interface 1920, and peripherals 1925 communicate with the CPU 1905 through a communication bus (solid lines) such as a motherboard. The storage 1915 may be a data storage unit (or data repository) for storing data. The computer system 1901 may be operably coupled to a computer network ("network") 1930 using the communication interface 1920. Network 1930 may be the Internet, an Internet and / or extranet, or an intranet and / or extranet in communication with the Internet. Network 1930 may, in some cases, be a telecommunications and / or data network. Network 1930 may include one or more computer servers, thereby enabling distributed computing such as cloud computing. Network 1930 may, in some cases, utilize computer system 1901 to implement a peer-to-peer network, thereby enabling devices coupled to computer system 1901 to act as clients or servers.

[0249]

[0342] The CPU 1905 is capable of executing sequences of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1910. The instructions may be directed to the CPU 1905, which may then cause the CPU 1905 to be programmed or otherwise configured to implement the methods of the present disclosure. Examples of operations performed by the CPU 1905 may include fetch, decode, execute, and writeback.

[0250]

[0343] The CPU 1905 may be part of a circuit, such as an integrated circuit. One or more other components of the system 1901 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0251]

[0344] The storage device 1915 may store files, such as drivers, libraries, and saved programs. The storage device 1915 may store user data, such as user preferences and user programs. The computer system 1901 may include one or more additional data storage units that, in some cases, are external to the computer system 1901, such as located on a remote server that communicates with the computer system 1901 over an intranet or the Internet.

[0252]

[0345] Computer system 1901 is capable of communicating with one or more remote computer systems over network 1930. For example, computer system 1901 is capable of communicating with a user's remote computer system or other devices and / or mechanisms (e.g., a sequencer or other system for chemically determining the order of nitrogenous bases in a nucleic acid sequence) that the user can use in the process of analyzing data encoded or decoded in a nucleic acid sequence. Examples of remote computer systems include personal computers (e.g., portable PCs), slate or tablet PCs (e.g., Apple® iPad®, Samsung® Galaxy Tab), telephones, smartphones (e.g., Apple® iPhone®, Android-enabled devices, Blackberry®), or personal digital assistants. A user can access computer system 1901 via network 1930.

[0253]

[0346] The methods described herein may be implemented by machine-executable (e.g., computer processor) code stored in an electronic storage location of the computer system 1901, such as, for example, memory 1910 or electronic storage 1915. The machine-executable or machine-readable code may be provided in the form of software. During use, the code may be executed by the processor 1905. In some cases, the code may be retrieved from storage 1915 and stored in memory 1910 for immediate access by the processor 1905. In some circumstances, the electronic storage 1915 may be omitted, and the machine-executable instructions may be stored in memory 1910.

[0254]

[0347] The code may be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or it may be compiled at run time. The code may be provided in a programming language that can be selected to allow the code to be executed in a pre-compiled or as-compiled fashion.

[0255]

[0348] Aspects of the systems and methods presented herein, such as computer system 1901, can be embodied in programming. Various aspects of the technology can be considered "products" or "articles of manufacture," generally in the form of machine- (or processor-) executable code and / or associated data carried or embodied on some type of machine-readable medium. The machine-executable code can be stored in electronic storage, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. A "storage" type medium can include any or all of a computer's tangible memory, processor, etc., or its associated modules, such as various semiconductor memories, tape drives, disk drives, etc., that can provide non-transitory storage at any time for software programming. All or portions of the software may, from time to time, be communicated over the Internet or various other telecommunications networks. Such communication, for example, enables the software to be loaded from one computer or processor to another, e.g., from a management server or host computer to an application server's computer platform. Thus, other types of media that may carry software elements include light waves, radio waves, and electromagnetic waves, such as those used across physical interfaces between local devices through wired and optical landline networks and through various air links. The physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered media bearing software. As used herein, unless limited 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.

[0256]

[0349] Thus, machine-readable media, such as computer-executable code, may take many forms, including but not limited to tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, such as storage devices in any computer, such as those that can be used to implement databases, etc., as shown in the figures. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire, including the electrical wires that comprise the bus bars within a computer system, and fiber optics. 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. Thus, common forms of computer readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tape, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punched card paper tape, any other physical storage media with patterns of holes, RAM, ROM, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or cartridges, carrier waves carrying data or instructions, cables or links which transport such carrier waves, or any other medium from which a computer can 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.

[0257]

[0350] The computer system 1901 may include or communicate with an electronic display 1935 including a user interface (UI) 1940 to provide sequence output data including, for example, chromatographs, sequences, and bits, bytes, or bitstreams encoded or read by a machine or computer system encoding or decoding nucleic acids, raw data, files, and compressed or uncompressed zip files that are encoded or decoded into DNA storage data. Examples of UIs include, without limitation, graphical user interfaces (GUIs) and web-based user interfaces.

[0258]

[0351] The methods and systems of the present disclosure can be implemented via one or more algorithms. The algorithms can be implemented via software when executed by the central processing unit 1905. Prior to encoding the digital information, the algorithms can be used, for example, with the DNA index and the raw data or the data compressed or restored into a zip file to determine a customized method for coding the digital information into the raw data or the data compressed into a zip file.

[0259] chemical method

[0352] A—Overlap Extension PCR (OEPCR) Assembly. In OEPCR, components are assembled in a reaction containing a polymerase and dNTPs (deoxynucleotide triphosphates, including dATP, dTTP, dCTP, dGTP, or variants or analogs thereof). Components can be single-stranded or double-stranded nucleic acids. Assembled components adjacent to each other can have complementary 3' ends, complementary 5' ends, or homology between the 5' end of one component and the 3' end of the adjacent component. These terminal regions are referred to as "hybridization regions" and are intended to facilitate the formation of hybridized junctions between components in OEPCR, where the 3' end of one input component (or its complement) hybridizes to the 3' end of its intended adjacent component (or its complement). An assembled double-stranded product can then be formed by polymerase extension. This product can then be assembled into more components through subsequent hybridization and extension. Figure 63 shows an example schematic of OEPCR for assembling three nucleic acids.

[0260]

[0353] In some embodiments, OEPCR can involve cycling between three temperatures: a melting temperature, an annealing temperature, and an extension temperature. The melting temperature is intended to convert double-stranded nucleic acids into single-stranded nucleic acids and eliminate the formation of secondary structures or...

Claims

1. 1. A system for translating digital information into a nucleic acid sequence, comprising: a source reservoir configured to hold a fluid comprising a pool of nucleic acid molecules; a main channel comprising a plurality of conductive plates and a counter electrode disposed opposite one another along a first dimension of the main channel; a destination reservoir; and an input channel in fluid communication with the source reservoir and the main channel, the input channel configured to dispense a first volume of fluid from the source reservoir to the main channel, the first volume comprising a first plurality of nucleic acid molecules; an output channel in fluid communication with the main channel and the destination reservoir, the output channel configured to distribute a second volume of fluid from the main channel to the destination reservoir; A system including:

2. The system of claim 1 , wherein the main channel comprises a reaction chamber.

3. The system of claim 1 , wherein the main channel is a reaction chamber.

4. The system of any one of claims 1 to 3, comprising a plurality of cells, each cell comprising one of the plurality of conductive plates and a portion of the counter electrode.

5. The system of claim 1 or 2, wherein each cell includes a base layer and a first dielectric layer, the first dielectric layer being disposed between the base layer and the plurality of conductive plates.

6. The system of claim 1 or 2, wherein each cell includes a base layer and a first dielectric layer, the plurality of conductive plates being disposed between the base layer and the first dielectric layer.

7. The system of any one of claims 1 to 6, wherein the base layer comprises a semiconductor layer.

8. The system of claim 7 , wherein the base layer comprises a non-semiconductor layer attached to the semiconductor layer.

9. The system of any one of claims 1 to 8, wherein the base layer comprises a conductive layer.

10. The system of any one of claims 1 to 9, wherein the base layer comprises an insulating layer.

11. The system of any one of claims 1 to 10, wherein the base layer is transparent.

12. The system of claim 11 , wherein the substrate comprises glass.

13. The system of any preceding claim, wherein the substrate comprises a heater element.

14. The system of claim 13 , wherein the heater element comprises a resistive heating element.

15. The system of any one of claims 4 to 14, wherein each cell includes a second dielectric layer, said second dielectric layer being disposed on said counter electrode.

16. 16. The system of claim 15, wherein the plurality of conductive plates and the second dielectric layer are disposed opposite one another along a first dimension of the main channel.

17. The system of any one of claims 4 to 16, wherein each cell comprises a plurality of nucleic acid adaptors fixed to the conductive plate.

18. The system of any one of claims 1 to 17, further comprising a third dielectric layer, said third dielectric layer disposed on said plurality of said conductive plates.

19. 20. The system of claim 18, wherein each cell comprises a plurality of nucleic acid adaptors fixed to the third dielectric layer.

20. 20. The system of claim 4, wherein each cell includes one of the plurality of conductive plates and one of a plurality of counter electrodes, each of the plurality of counter electrodes being disposed on an opposing one of the plurality of conductive plates.

21. The system of any one of claims 16 to 18, wherein the cells are arranged in a two-dimensional array along second and third dimensions of the main channel, the array having rows and columns of cells.

22. The system of any preceding claim, wherein each conductive plate is electrically connected to a voltage source.

23. A system according to any preceding claim, comprising a control system including a plurality of switches, each switch being electrically connected to one of the plurality of conductive plates and to the voltage source.

24. 24. The system of claim 23, wherein the voltage across each cell of the array is individually controllable by actuating one of the plurality of switches.

25. The system of any one of claims 1 to 24, wherein the fluid is a dielectric fluid.

26. The system of any one of claims 1 to 24, wherein the fluid is a conductive fluid.

27. The system of any one of claims 1 to 26, comprising a buffer reservoir in fluid communication with the main channel.

28. The system of any one of claims 1 to 27, comprising a ligase reservoir in fluid communication with the main channel.

29. A system according to any preceding claim, comprising a pump configured to pump the volume of fluid into one or more of the input channel, the main channel and the output channel.

30. A system according to any preceding claim, comprising a valve for controlling the flow of said volume of liquid through said main channel.

31. 31. The system of any one of claims 1 to 30, comprising a plurality of source reservoirs, each of said plurality of source reservoirs having a volume of fluid containing a population of substantially identical nucleic acid molecules.

32. 32. The system of claim 31 , wherein each of the multiple source reservoirs contains a different population of substantially identical nucleic acid molecules.

33. A system according to any preceding claim, comprising a plurality of destination reservoirs in fluid communication with the main channel.

34. The system of any one of claims 1 to 33, wherein the nucleic acid molecule encodes digital information.

35. The system of any one of claims 1 to 34, wherein the nucleic acid molecules comprise identifier nucleic acid molecules that encode digital information from a string of length L.

36. The system of any one of claims 1 to 35, wherein the nucleic acid molecule comprises a plurality of component nucleic acid molecules of an identifier nucleic acid molecule that encodes digital information from a string of length L.

37. 37. The system of claim 35 or 36, wherein each individual identifier nucleic acid molecule corresponds to a symbol value and a symbol position within the symbol string, and the pool of identifier nucleic acid molecules corresponds to a subset of identifier nucleic acid sequences in an identifier library that are capable of encoding any symbol string having length L.

38. The system of any one of claims 1 to 37, wherein the main channel comprises a plurality of fluidly connected reaction chambers.

39. The system of any one of claims 21 to 38, wherein the two-dimensional array is divided into two or more blocks.

40. 40. The system of claim 39, wherein each of the plurality of fluidly connected reaction chambers contains a block.

41. The system of any one of claims 1 to 40, wherein the second volume of liquid comprises a second plurality of nucleic acid molecules.

42. The system of any one of claims 4 to 41, wherein the second plurality of nucleic acid molecules comprises nucleic acid molecules released from a cell.

43. 1. A system for decoding digital information into a nucleic acid sequence, comprising: A system according to any one of claims 5 to 42, a sequencing device disposed on the first dielectric layer, the sequencing device having an inlet in fluid communication with the main channel and an outlet in fluid communication with the cavity; and a base electrode disposed downstream of the outlet; A system including:

44. 44. The system of claim 43, wherein the sequencing device comprises a nanopore.

45. 44. The system of claim 43, wherein the sequencing device comprises a nanochannel.

46. 44. The system of claim 43, wherein the sequencing device comprises a nanopore or nanochannel formed in a solid-state membrane.

47. The system of any one of claims 43 to 46, wherein the cavity is disposed in the base layer of the main channel.

48. The system of any one of claims 43 to 47, wherein the main channel and the cavity contain an electrolyte solution.

49. The system of any one of claims 44 to 48, wherein the nanopore or nanochannel comprises alpha hemolysin (αHL) or Mycobacterium smegmatis porin A (MspA).

50. 50. The system of any one of claims 43 to 49, wherein the electric field generated by the base electrode and the counter electrode has a differential potential across the nanopore or nanochannel of greater than 100 mV.

51. The system of any one of claims 43 to 50, comprising a plurality of nanopores or nanochannels.

52. 52. The system of claim 51, wherein each block includes one of the plurality of sequencing devices.

53. 1. A system for decoding digital information into a nucleic acid sequence, comprising: A system according to any one of claims 5 to 42, a sequencing device comprising a nanochannel having an inlet in fluid communication with the main channel and an outlet in fluid communication with the cavity; a central electrode disposed downstream of the outlet; a block electrode disposed such that a cell is disposed between the block electrode and the central electrode; a nanochannel sensor configured to detect a change in current while a nucleic acid translocates through the nanochannel; A system including:

54. 54. The system of claim 53, wherein the electric field generated by the blocking electrode and the central electrode has a differential potential across the nanochannel of greater than 100 mV.

55. 1. A system for decoding digital information into a nucleic acid sequence, comprising: A system according to any one of claims 5 to 42, a sequencing device including a zero mode waveguide reader; A system including:

56. 56. The system of claim 55, wherein the dielectric layer is transparent.

57. 57. The system of claim 55 or 56, wherein the dielectric layer comprises a waveguide channel.

58. 58. The system of claim 57, wherein the waveguide channel comprises a polymerase immobilized therein.

59. 59. The system of claim 57 or 58, wherein the waveguide channel comprises a set of primers and fluorescently labeled nucleotides.

60. 60. The system of any one of claims 55 to 59, comprising a detector configured to detect a fluorescent signal generated by incorporation of a fluorescently labeled nucleotide during synthesis of a complementary strand of a single-stranded DNA molecule.

61. 1. A method for encoding digital information into a nucleic acid sequence, comprising: obtaining a pool of nucleic acid molecules suspended in a fluid in a source reservoir; flowing an input volume comprising a plurality of nucleic acid molecules from the source reservoir through an input channel into a main channel, the main channel comprising a plurality of cells, each cell comprising one of a plurality of conductive plates and a portion of a counter electrode, the plurality of conductive plates and the counter electrode being disposed opposite one another along a first dimension of the main channel, each conductive plate comprising a plurality of nucleic acid adaptors immobilized thereon; applying a binding voltage to a first cell of the plurality of cells, thereby binding a portion of the plurality of nucleic acid molecules to the nucleic acid adapters of a first conductive plate of the plurality of conductive plates; The method includes:

62. 62. The method of claim 61, comprising obtaining a plurality of pools of nucleic acid molecules, each of the plurality of pools of nucleic acid molecules suspended in a fluid within one of a plurality of source reservoirs.

63. 63. The method of claim 61 or 62, comprising, after applying the coupling voltage, flowing a volume of buffer from a buffer reservoir through the main channel and into a waste reservoir.

64. flowing a second input volume from a second source reservoir through the input channel into the main channel, the second input volume comprising a second plurality of nucleic acid molecules; applying the binding voltage to the first cell of the plurality of cells, thereby binding a portion of the second plurality of nucleic acid molecules to the portion of the plurality of nucleic acid molecules bound to the nucleic acid adapter; 64. The method of claim 62 or 63, comprising:

65. flowing a third input volume from a third source reservoir through the input channel into the main channel, the third input volume comprising a third plurality of nucleic acid molecules; applying the binding voltage to the first cell of the plurality of cells, thereby binding a portion of the third plurality of nucleic acid molecules to the portion of the second plurality of nucleic acid molecules; 65. The method of claim 64, comprising:

66. flowing a second input volume from a second source reservoir through the input channel into the main channel, the second input volume comprising a second plurality of nucleic acid molecules; applying the binding voltage to a second cell of the plurality of cells, thereby binding a portion of the second plurality of nucleic acid molecules to the nucleic acid adapters of a second conductive plate of the plurality of conductive plates; 64. The method of claim 62 or 63, comprising:

67. flowing a third input volume from a third source reservoir through the input channel into the main channel, the third input volume comprising a third plurality of nucleic acid molecules; applying the binding voltage to a second cell of the plurality of cells, thereby binding a portion of the third plurality of nucleic acid molecules to the portion of the second plurality of nucleic acid molecules; 67. The method of claim 66, comprising:

68. flowing a quality control (QC) input volume, the QC input volume comprising a plurality of tagged nucleic acid molecules comprising a fluorophore, from a QC source reservoir through the input channel into the main channel; applying the combined voltage to the first cell of the plurality of cells; The method of any one of claims 61 to 67, comprising:

69. flowing a quality control (QC) input volume, the QC input volume comprising a plurality of tagged nucleic acid molecules comprising a fluorophore, from a QC source reservoir through the input channel into the main channel; applying the combined voltage to two or more cells of the plurality of cells; The method of any one of claims 61 to 67, comprising:

70. flowing a second quality control (QC) input volume from a second QC source reservoir through the input channel into the main channel, the second QC input volume comprising a plurality of tagged nucleic acid molecules comprising a second fluorophore; applying the combined voltage to the first cell of the plurality of cells; 70. The method of claim 68 or 69, comprising:

71. flowing a second quality control (QC) input volume from a second QC source reservoir through the input channel into the main channel, the second QC input volume comprising a plurality of tagged nucleic acid molecules comprising a second fluorophore; applying the combined voltage to two or more cells of the plurality of cells; 70. The method of claim 68 or 69, comprising:

72. flowing a second quality control (QC) input volume from a second QC source reservoir through the input channel into the main channel, the second QC input volume comprising a plurality of tagged nucleic acid molecules comprising a second fluorophore; applying the combined voltage to each one of the plurality of cells; 70. The method of claim 68 or 69, comprising:

73. 73. The method of any one of claims 68 to 72, comprising measuring the amount of fluorescence in one or more of the plurality of cells using a fluorescence detector.

74. 74. The method of any one of claims 61 to 73, comprising flowing a fluid containing a ligase from a ligase reservoir into the main channel.

75. 75. The method of any one of claims 61 to 74, wherein joining the nucleic acid molecules comprises sticky end ligation.

76. 76. The method of any one of claims 61 to 75, wherein joining the nucleic acid molecules comprises blunt end ligation.

77. 77. A method according to any one of claims 61 to 76, comprising flowing a volume of buffer from a buffer reservoir through the main channel and into a waste reservoir after each application of the coupling voltage.

78. 78. The method of any one of claims 61 to 77, comprising applying a release voltage to one or more of the plurality of cells, thereby releasing the bound nucleic acid molecule.

79. 78. The method of any one of claims 61 to 77, comprising establishing an electric field in one or more of said plurality of cells, thereby releasing said bound nucleic acid molecules.

80. 78. The method of any one of claims 61-77, comprising heating one or more of the plurality of cells, thereby releasing the bound nucleic acid molecules.

81. flowing a volume of a fluid comprising an enzyme from an enzyme reservoir through the main channel and reacting the enzyme with the nucleic acid adaptors, thereby releasing the portions of the plurality of nucleic acid molecules; The method of any one of claims 61 to 77, comprising:

82. 82. A method according to any one of claims 78 to 81, comprising flowing an output volume from the main channel through an output channel to a destination reservoir.

83. 83. The method of any one of claims 61 to 82, wherein the nucleic acid molecule comprises an identifier nucleic acid molecule that stores digital information from a string of length L.

84. 83. The method of any one of claims 61 to 82, wherein the nucleic acid molecule comprises a plurality of component nucleic acid molecules of an identifier nucleic acid molecule that stores digital information from a string of length L.

85. 85. The method of claim 83 or 84, wherein each individual identifier nucleic acid molecule corresponds to a symbol value and a symbol position within the symbol string, and wherein the pool of identifier nucleic acid molecules corresponds to a subset of identifier nucleic acid sequences in an identifier library that are capable of encoding any symbol string having length L.

86. 1. A method for processing digital information in a nucleic acid sequence, comprising: (i) obtaining a pool of nucleic acid molecules suspended in a fluid in a source reservoir; (ii) flowing an input volume comprising a plurality of nucleic acid molecules from the source reservoir through an input channel into a main channel, the main channel comprising a plurality of cells, each cell comprising one of a plurality of conductive plates and a portion of a counter electrode, the plurality of conductive plates and the counter electrode being disposed opposite one another along a first dimension of the main channel, each conductive plate comprising a plurality of nucleic acid adaptors immobilized thereon; (iii) applying a binding voltage to a first cell of the plurality of cells, thereby binding a portion of the plurality of nucleic acid molecules to the nucleic acid adapters of a first conductive plate of the plurality of conductive plates; (iv) flowing a volume of buffer from a buffer reservoir through the main channel and into a waste reservoir; (v) flowing a fluid containing a ligase from a ligase reservoir into the main channel; (vi) flowing a second input volume from a second source reservoir through the input channel into the main channel, the second input volume comprising a second plurality of nucleic acid molecules; (vii) applying the binding voltage to the first cell of the plurality of cells, thereby binding a portion of the second plurality of nucleic acid molecules to the portion of the plurality of nucleic acid molecules bound to the nucleic acid adaptor; (viii) flowing a quantity of buffer from a buffer reservoir through said main channel and into a waste reservoir; (ix) flowing a fluid containing a ligase from a ligase reservoir into the main channel; The method includes:

87. (x) flowing an nth input volume from an nth source reservoir through the input channel into the main channel, the nth input volume comprising a nth plurality of nucleic acid molecules; (xi) applying the binding voltage to the first cell of the plurality of cells, thereby binding a portion of the nth plurality of nucleic acid molecules to the binding portion of the (n-1)th plurality of nucleic acid molecules; (xii) flowing a quantity of buffer from a buffer reservoir through said main channel and into a waste reservoir; (xiii) flowing a fluid containing a ligase from a ligase reservoir into the main channel; performing steps x through xiii n times, where n is 3 or greater; 87. The method of claim 86, comprising:

88. 88. The method of claim 86 or 87, comprising (xiv) storing the main channel in a storage device.

89. 89. The method of any one of claims 86 to 88, comprising applying a release voltage to the cell, thereby releasing the bound nucleic acid molecule.

90. 90. The method of claim 89, comprising flowing an output volume from the main channel through an output channel to a destination reservoir.

91. 91. The method of claim 90, comprising storing the destination reservoir in a storage device.

92. After one or both of steps (ix) or (xiii), flowing a quality control (QC) input volume, the QC input volume comprising a plurality of tagged nucleic acid molecules comprising a fluorophore, from a QC source reservoir through the input channel into the main channel; applying the combined voltage to the first cell of the plurality of cells; The method of any one of claims 86 to 91, comprising:

93. applying a release voltage to the cell, thereby releasing the bound nucleic acid molecule; directing the released nucleic acid molecules to a sequencing device having an inlet in fluid communication with the main channel and an outlet in fluid communication with a cavity; directing the released nucleic acid molecules through the sequencing device and into the cavity; measuring a plurality of voltages at the sequencing device; performing base calling based on the measured voltages; and The method of any one of claims 86 to 92, comprising:

94. 94. The method of claim 93, wherein the released nucleic acid molecule is or comprises a single-stranded DNA molecule.

95. 95. The method of claim 93 or 94, comprising applying a voltage between the counter electrode and a cavity electrode disposed downstream of the outlet of the sequencing device.

96. applying a release voltage to a plurality of cells, thereby releasing the bound nucleic acid molecule from each of the plurality of cells; flowing an operator solution volume from an operator source reservoir through an input channel into a main channel, the operator solution volume including a plurality of operator nucleic acid molecules, the operator nucleic acid molecules corresponding to logical operators; performing a chemical reaction between the operator nucleic acid molecule and the released nucleic acid molecule, thereby generating a plurality of resultant nucleic acid molecules; The method of any one of claims 86 to 92, comprising:

97. directing the resulting nucleic acid molecules to a sequencing device having an inlet in fluid communication with the main channel and an outlet in fluid communication with a cavity; directing the resulting nucleic acid molecule through the sequencing device into the cavity; measuring a plurality of voltages at the sequencing device; performing base calling based on the measured voltages; and 97. The method of claim 96, comprising:

98. 98. The method of any one of claims 86 to 97, wherein the nucleic acid molecule comprises an identifier nucleic acid molecule encoding digital information corresponding to a string of length L.

99. 98. The method of any one of claims 86 to 97, wherein the nucleic acid molecule comprises a plurality of component nucleic acid molecules of an identifier nucleic acid molecule that stores digital information from a string of length L.

100. 100. The method of claim 98 or 99, wherein each individual identifier nucleic acid molecule corresponds to a symbol value and a symbol position within the symbol string, and wherein the pool of identifier nucleic acid molecules corresponds to a subset of identifier nucleic acid sequences in an identifier library that are capable of encoding any symbol string having length L.

101. A device for reading a nucleic acid sequence, comprising: a nanochannel disposed in the substrate and configured to receive an input nucleic acid molecule comprising an input strand; a sensor device disposed on or within the nanochannel, the sensor device including an electronic sensing device having an electronic gate with a gate voltage that can be modulated with a charge of a translocating readout component of the input nucleic acid molecule to vary a source-drain current at the gate; The device that contains

102. A device according to claim 101, wherein the device is part of a system according to any one of claims 5 to 42.

103. The device of claim 101, wherein the device is disposed in the main channel.

104. A device according to any one of claims 101 to 103, wherein the sensor device is or comprises a metal oxide semiconductor field effect transistor (MOSFET).

105. A device according to any one of claims 101 to 103, wherein the sensor device is or comprises an electrolytic oxide field effect transistor (EOSFET).

106. The device of any one of claims 101 to 105, wherein the reading component comprises a single-stranded nucleic acid molecule configured to hybridize to a section of the single-stranded nucleic acid molecule.

107. 107. The device of any one of claims 101 to 106, comprising a plurality of read components, each read component configured to hybridize to a complementary section of the input strand to form the input nucleic acid molecule.

108. 108. The device of any one of claims 101 to 107, wherein a first read component is configured to hybridize to one or more sections of the input strand having a first input sequence and a second read component is configured to hybridize to one or more sections of the input strand having a second input sequence.

109. 109. The device of claim 108, wherein the first read component causes a first change in source-drain current at the gate when it transitions through the gate, and the second read component causes a second change in source-drain current at the gate when it transitions through the gate.

110. 110. The device of claim 109, wherein the first change is different from the second change.

111. 109. The device of any one of claims 101 to 108, comprising a read initiation component and a read termination component, the read initiation component comprising a single-stranded nucleic acid molecule configured to hybridize to a first end of the input strand, and the read termination component comprising a single-stranded nucleic acid molecule configured to hybridize to a second end of the input strand.

112. A device according to any one of claims 101 to 111, wherein the input chain encodes digital information.

113. 113. A device according to any one of claims 101 to 112, wherein the input strand comprises one or more identifier components, each identifier component being a component of a nucleic acid identifier encoding digital information.

114. A device according to any one of claims 101 to 113, wherein the input strand comprises a first input sequence corresponding to a first identifier component and a second input sequence corresponding to a second identifier component.

115. 115. The device of any one of claims 101-114, comprising a read overlap component comprising a single stranded nucleic acid molecule configured to hybridize to at least a portion of the first identifier component and the second identifier component.

116. 116. The device of claim 115, wherein the read overlap components include a non-complementary nucleic acid section forming a flap and a nucleic acid section having a secondary molecular structure at an end of the flap.

117. 116. The device of claim 115, wherein the read overlap components include a non-complementary component forming a flap and a component having a secondary molecular structure hybridized to the flap.

118. A device according to any one of claims 101 to 117, wherein the sensor device is or includes one or more electronic signal processing devices.

119. A device according to any one of claims 101 to 118, wherein the reading component is a single-stranded nucleic acid molecule comprising a section having a secondary molecular structure.

120. The device of any one of claims 101 to 118, wherein the readout component comprises a peptide aptamer.

121. The device of any one of claims 101 to 118, wherein the readout component comprises a dendrimer.

122. A device according to any one of claims 101 to 118, wherein the readout component comprises a protein.

123. A device according to any one of claims 101 to 122, comprising a plurality of sensor devices.

124. A device according to any one of claims 101 to 122, wherein the sensor device comprises a plurality of electronic sensing devices.

125. 125. The device of claim 123 or 124, wherein the plurality of sensor devices or the plurality of electronic sensing devices are arranged in series along the path of the nanochannel.

126. 125. The device of claim 124, wherein the plurality of sensor devices or the plurality of electronic sensing devices are configured to simultaneously read two or more read components.

127. A method for reading a nucleic acid sequence, comprising: Providing a device according to any one of claims 101 to 126; Translocating the readout component through the nanochannel; The method includes:

128. A device for reading a nucleic acid sequence, comprising: a nanochannel disposed in the substrate and configured to receive an input nucleic acid molecule comprising an input strand; a sensor device disposed on or within the nanochannel, the sensor device including an optical sensing device configured to detect an optical signal from a readout component during translocation of the input nucleic acid molecule; The device that contains

129. A device according to claim 128, which is part of a system according to any one of claims 5 to 42.

130. 130. The device of claim 129, wherein the device is disposed in the main channel.

131. A device according to any one of claims 128 to 130, wherein the light sensing device is a fluorescence measuring device.

132. A device according to any one of claims 128 to 131, wherein the light sensing device comprises one or more of an optical system, a camera, or a photon counter.

133. 133. The device of any one of claims 128 to 132, wherein the reading component comprises a single stranded nucleic acid molecule configured to hybridize to a section of the single stranded nucleic acid molecule and a light emitting element.

134. 134. The device of any one of claims 128 to 133, comprising a plurality of read components, each read component configured to hybridize to a complementary section of the input strand to form the input nucleic acid molecule.

135. 135. The device of any one of claims 128 to 134, wherein a first read component is configured to hybridize to one or more sections of the input strand having a first input sequence and a second read component is configured to hybridize to one or more sections of the input strand having a second input sequence.

136. 136. The device of claim 135, wherein the first read component includes a light emitting element configured to emit a first light signal, and the second read component includes a light emitting element configured to emit a second light signal.

137. 137. The device of claim 136, wherein the first optical signal is different from the second optical signal.

138. 138. The device of claim 137, wherein the first optical signal has a greater intensity than the second optical signal.

139. 138. The device of claim 137, wherein the first light signal has a different color than the second light signal.

140. A device according to any one of claims 128 to 139, wherein the input chain encodes digital information.

141. 141. A device according to any one of claims 128 to 140, wherein the input strand comprises one or more identifier components, each identifier component being a component of a nucleic acid identifier encoding digital information.

142. 142. A device according to any one of claims 128 to 141, wherein the input strand comprises a first input sequence corresponding to a first identifier component and a second input sequence corresponding to a second identifier component.

143. 143. The device of any one of claims 128-142, comprising a read overlap component comprising a single stranded nucleic acid molecule configured to hybridize to at least a portion of the first identifier component and the second identifier component.

144. 144. The device of claim 143, wherein the read-overlapping components include a non-complementary nucleic acid section forming a flap and a light-emitting element attached to an end of the flap.

145. 145. The device of claim 144, wherein the read overlap components include a non-complementary component forming a flap and a component hybridized to the flap and having a secondary molecular structure that includes a light emitting element.

146. A device according to any one of claims 128 to 145, wherein the sensor device comprises a plurality of light sensing devices.

147. The device of any one of claims 128 to 146, wherein the light-emitting element is a fluorophore.

148. A device according to any one of claims 128 to 147, wherein the sensor device is or includes one or more electronic signal processing devices.

149. A device according to any one of claims 128 to 148, comprising a plurality of sensor devices.

150. A device according to any one of claims 128 to 148, wherein the sensor device comprises a plurality of light sensing devices.

151. 151. The device of claim 149 or 150, wherein the plurality of sensor devices or the plurality of optical sensing devices are arranged in series along the path of the nanochannel.

152. 151. The device of claim 150, wherein the plurality of sensor devices or the plurality of light sensing devices are configured to simultaneously read two or more reading components.

153. A method for reading a nucleic acid sequence, comprising: Providing a device according to any one of claims 128 to 152; Translocating the readout component through the nanochannel; and The method includes:

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