Image registration in preliminary analysis

Abstract

Systems, methods, and computer storage media are provided for image registration in preliminary analysis. In some embodiments, coordinates of a community in a reference coordinate system are determined, where the community is obtained from a flow cell image in one or more reference cycles. The one or more template images are generated in the reference coordinate system by registering the community to one or more template images using the coordinates of the community. A flow cell image is obtained in the cycle. Then, a plurality of transforms of the flow cell image are determined based on the one or more template images, each of the plurality of transforms corresponding to a sub-tile of the flow cell image and configured to register the sub-tile of the flow cell image to the one or more template images.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 349,421, filed on June 6, 2022, which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates generally to image registration, and more particularly to image registration of flow cell images acquired in different sequencing cycles and from different channels for accurate base calling during DNA sequencing. Background Art

[0004] In next generation sequencing (NGS) or NGS-like applications (such as sequencing by synthesis, sequencing by binding, or affinity sequencing), in order to identify the sequence of the target nucleic acid, a new chain is synthesized one nucleotide base at a time. During each cycle, 3'-closed nucleotides are connected to the chain at complementary locations, thereby ensuring that only one base will be connected to any given chain during a single cycle. At the imaging step of each sequencing cycle, one or more images are recorded. The base calling algorithm is applied to the image to "read" the continuous signal from each cluster or community, and the optical signal is converted into an identification of the nucleotide base sequence added to each DNA fragment. However, between two sequencing cycles, the platform of the flow cell moves relative to the objective lens, so the clusters and communities do not appear in exactly the same position in the image. Even if the platform remains at an accurate position within each cycle, the cluster or community may drift to a different position on the flow cell. In addition, slight changes caused by filters and dyes may cause image transformations between channels. Therefore, the accuracy of base calling for a given cluster or community may be reduced. There is a need for image registration that can incorporate flow cell images across cycles and / or channels into a common coordinate system, ie, accurate base calling through image registration. Summary of the invention

[0005] Provided herein are systems, devices, methods and / or computer program product embodiments and / or combinations and sub-combinations thereof that enable image registration and / or image registration rescue of flow cell images during preliminary analysis of DNA sequencing analysis. Flow cell images can come from different sequencing cycles and / or different channels. As a specific application of this aspect, provided herein are embodiments of methods, systems and media for image registration and / or image registration rescue, thereby enabling accurate base calls to be made depending on the image intensity, position and / or size of a cluster or colony.

[0006] Other embodiments of these embodiments include corresponding computer systems, devices, and computer program products recorded on computer storage devices, which are configured, alone or in combination, to perform the actions or operations of the method. For a computer system that has been configured or is to be configured to perform an operation or action, the computer system has installed thereon software, firmware, hardware, or a combination thereof that causes the computer system to perform the operation or action in operation. For a computer program product that has been configured or is to be configured to perform an operation or action, the computer program product includes instructions that, when executed by a hardware processor, cause the hardware processor to perform the operation or action.

[0007] Further embodiments, features, and advantages of the present disclosure, as well as the structure and operation of various embodiments of the present disclosure are described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The accompanying drawings, which are incorporated herein and form a part of this specification, illustrate embodiments of the present disclosure and, together with the description, further serve to explain the principles of the present disclosure and enable those skilled in the art to make and use the embodiments.

[0009] Figure 1 Shown is a block diagram of a system for DNA sequencing, image registration, and image registration rescue of flow cell images, according to some embodiments.

[0010] Figure 2 Schematic diagram of a flow cell image, sub-tiles, and regions with colonies or clusters according to some embodiments is shown.

[0011] Figure 3 A schematic diagram of a portion of a flow cell having multiple tiles is shown according to some embodiments.

[0012] Figure 4 A block diagram of a computer system for performing image registration according to some embodiments is shown.

[0013] Figure 5 A flow chart of a method for performing image registration of flow cell images according to some embodiments is shown.

[0014] Figure 6A-6B A schematic diagram showing image transformation and corresponding 2D displacement according to some embodiments is shown.

[0015] Figure 7 is a schematic diagram showing an example linear single-stranded library molecule.

[0016] Figure 8 is a schematic diagram showing an example linear single-stranded library molecule.

[0017] Fig. 9is a schematic diagram of various example configurations of multivalent molecules.

[0018] Fig.10 is a schematic diagram of an example multivalent molecule comprising a universal core linked to multiple nucleotide arms.

[0019] Fig.11 is a schematic diagram of an example multivalent molecule comprising a dendrimer core attached to multiple nucleotide arms.

[0020] Fig.12 A schematic diagram of an example multivalent molecule comprising a core linked to a plurality of nucleotide arms is shown, wherein the nucleotide arms comprise biotin, a spacer, a linker, and nucleotide units.

[0021] Fig.13 is a schematic diagram of an exemplary nucleotide arm comprising a core linking portion, a spacer, a linker, and a nucleotide unit.

[0022] Fig.14 Shown are the chemical structures of example spacers (top), as well as the chemical structures of various example linkers, including an 11-atom linker, a 16-atom linker, a 23-atom linker, and an N3 linker (bottom).

[0023] Fig.15 The chemical structures of various example linkers, including Linkers 1-9, are shown.

[0024] Fig.16 The chemical structures of various exemplary linkers joined / linked to nucleotide units are shown.

[0025] Fig.17 The chemical structures of various exemplary linkers joined / linked to nucleotide units are shown.

[0026] Fig.18 The chemical structures of various exemplary linkers joined / linked to nucleotide units are shown.

[0027] Fig.19 The chemical structures of various exemplary linkers joined / linked to nucleotide units are shown.

[0028] Fig. 20 The chemical structure of an example biotinylated nucleotide arm is shown. In this example, the nucleotide unit is linked to the linker via a propargylamine attachment at the 5 position of a pyrimidine base or at the 7 position of a purine base.

[0029] Fig.21 Shown are example flow cell images of four different color channels from a particular flow cycle, according to some embodiments.

[0030] Fig. 22An example index allocation rate improvement using an embodiment of the image registration rescue method herein is shown.

[0031] Fig.23 An example index mismatch rate improvement using an embodiment of the image registration rescue method herein is shown.

[0032] Fig.24A A flow chart of a method for performing image registration rescue of flow cell images is shown according to some embodiments.

[0033] Fig. 24B A flow chart of a method for performing image registration rescue of flow cell images is shown according to some embodiments.

[0034] Fig.25 is a schematic diagram of an example test target according to some embodiments.

[0035] Fig.26 A schematic diagram showing an example beaded flow cell having a surface coated with fluorescent beads.

[0036] Fig. 27 A schematic diagram of an exemplary embodiment of a low binding solid support of the present disclosure is provided, wherein the support comprises alternating layers of a glass substrate and a hydrophilic coating covalently or non-covalently attached to the glass, and the support further comprises chemically reactive functional groups that serve as attachment sites for oligonucleotide primers.

[0037] Fig.28 is a schematic diagram illustrating an example embodiment of a padlock probe according to some embodiments.

[0038] Fig.29 is a schematic diagram showing a workflow for generating a circularized padlock probe in a cell according to some embodiments, the workflow comprising generating a first cDNA and a second cDNA from a first target RNA molecule and a second target RNA molecule (respectively), hybridizing the first padlock probe and the second padlock probe (respectively) with the first cDNA molecule and the second cDNA molecule to generate a first circularized padlock probe and a second circularized padlock probe (respectively).

[0039] Fig.30 is a schematic diagram illustrating a rolling circle and sequencing workflow within a cell according to some embodiments, the workflow comprising generating a first concatemer and a second concatemer by rolling circle amplification using a first covalently closed circularizing molecule and a second covalently closed circularizing molecule (respectively).

[0040] Fig.31 is a schematic diagram illustrating an example workflow for sequencing concatemers generated within cells, according to some embodiments.

[0041] Fig.32 is a schematic diagram illustrating an example workflow for sequencing concatemers generated within cells, according to some embodiments.

[0042] Fig.33 is a schematic diagram illustrating an example workflow for sequencing concatemers generated within cells, according to some embodiments.

[0043] Fig.34 is a schematic diagram illustrating an example workflow for sequencing concatemers generated within cells, according to some embodiments. DETAILED DESCRIPTION

[0044] Provided herein are systems, apparatus, methods and / or computer program product embodiments and / or combinations and sub-combinations thereof, which enable image registration and / or image registration rescue of flow cell images to perform accurate base calls. Image registration and registration rescue techniques can be used for flow cell images obtained by various imaging and / or sequencing techniques. These techniques can be used for flow cell images obtained from various sequencing samples (including in situ samples). The techniques disclosed herein can be used for base calls in next generation sequencing, and base calls will be used as the primary example of the application of these techniques described herein. However, this imaging analysis technique may also be useful in other applications using point detection and / or charge coupled device (CCD) imaging.

[0045] In DNA sequencing, identifying the center of a cluster or colony (which is usually formed on a bead) and generating base calls for the identified cluster or colony is sometimes referred to as a preliminary analysis. Preliminary analysis may also involve the formation of a template for a flow cell. The template may include the estimated positions of all detected clusters or colonies in a common coordinate system. Templates may be generated by identifying the cluster or colony positions in all images in the first few cycles of the sequencing process. Ideally, during the sequencing cycle, the cluster or colony remains in the same position so that base calls can be made about the same cluster or colony. However, the platform of the sequencer and / or flow cell may not remain in the same position in each cycle, for example, relative to the objective lens. In addition, in different cycles, the cluster or colony may drift relative to the flow cell. The changes between the different color channels caused by the filter and the fluorescent dye may further cause the transformation of the cluster or colony in the image acquired across different channels. Clusters or colonies can be as small as 1-2 pixels, and the colony tends to be distributed in a relatively random pattern on the flow cell image. A slight drift of a few pixels can significantly reduce the accuracy and reliability of the base calls of a given cluster or colony. For example, in cycle k, the first colony may have drifted to the position where the second colony was once located in cycle k-1, and without image registration, the base calls of the second colony in cycle k may be incorrect. The technology disclosed herein can be used for image registration of flow cell images, so that base calls can be accurately and reliably performed in preliminary analysis. There are multiple algorithms for image registration. These existing algorithms may have various defects. For example, image registration by deriving nonlinear transformations of flow cell images between cycles may add additional complexity to the algorithm, and cause undesirable delays in registration and downstream operations. Additionally, image registration using the entire flow cell image may be computationally intensive and prone to delays and failures. Image registration methods that add external fiducial markers to flow cell images may not reflect image offsets and other transformations between channels or across cycles, and fiducial markers may interfere with image intensity from colonies or clusters, which requires additional processing of such interference.

[0046] The technology disclosed herein advantageously utilizes multiple transformations corresponding to the sub-picture blocks of the flow cell image to estimate the image transformation of the entire flow cell image. The technology disclosed herein is rooted in the characteristics of the flow cell image across cycles and the transformation between channels, so the transformation of the sub-picture blocks can provide a reasonable estimate of the transformation of the flow cell image without causing excessive computational burden and the delay of subsequent sequencing analysis. Determining multiple transformations advantageously saves computing time, system data storage space, and reduces computational complexity, while achieving accurate and efficient image registration results. When determining each individual transformation of the sub-picture block, the technology disclosed herein also advantageously utilizes the information in the adjacent sub-picture blocks. Like this, compared with the method for determining the transformation based on the sub-picture block itself, the technology of this article allows to improve image registration without increasing computational complexity or computing time. Even if the image is low nucleotide diversity, these technologies also enable reliable and accurate image registration.

[0047] Sequencing system

[0048] Figure 1 A block diagram of a computer-implemented system 100 according to one or more embodiments disclosed herein is shown. The system 100 has a sequencing system 110, which includes a flow cell 112, a sequencer 114, an imager 116, a data storage device 122, and a user interface 124. The sequencing system 110 can be connected to a cloud 130. The sequencing system 110 can include one or more of the following: a dedicated processor 118, a field programmable gate array (FPGA) 120, and a computer system 126.

[0049] In some embodiments, the flow cell 112 is configured to capture DNA fragments and form a DNA sequence for base calling on the flow cell. The flow cell 112 may include a carrier disclosed herein. The carrier may be a solid phase carrier. As disclosed herein, the carrier may include a surface coating thereon. The surface coating may be a polymer coating as disclosed herein.

[0050] The flow cell 112 can include a plurality of tiles or imaging areas thereon, and each tile can be divided into a sub-tile grid. Each sub-tile can include a plurality of clusters or colonies thereon. As a non-limiting example, the flow cell can have 424 tiles, and each tile can be divided into a 6x 9 grid, so there are 54 sub-tiles. The flow cell image disclosed herein can be an image of signals including a plurality of clusters or colonies. The flow cell image can include one or more signal tiles or one or more signal sub-tiles. In some embodiments, each tile or sub-tile can include millions of colonies or clusters. As a non-limiting example, a tile can include 1,000 to 10 million clusters or colonies. Each colony can be a collection of many DNA fragment copies. In some embodiments, the flow cell image can be an image including at least a portion of a tile and approximately all signals thereon. The flow cell image can be acquired from a channel using an imager 116 during an imaging or sequencing cycle.

[0051] In the case where a 3D sample (e.g., cell or tissue) is fixed on a flow cell, the flow cell image can be at multiple z levels, which are orthogonal to the image plane of the flow cell image. Therefore, the flow cell image can include multiple z levels to cover the entire sample in a 3D manner. The z-axis can extend from the objective lens of the optical system disclosed herein to a carrier, e.g., a flow cell. Each z level of the flow cell image can be separated from an adjacent z level by a predetermined distance, e.g., about 0.1um to about 15um. Each z level of the flow cell image can be separated from an adjacent level by 1um to 10um. At each z level, a flow cell image can be acquired from one or more sequencing cycles and / or one or more channels. Each flow cell image can include at least a portion of one or more tiles or sub-tiles of the flow cell in its field of view. Figure 3 A portion of a flow cell 112 is shown with a plurality of tiles 290. The image plane is defined by the x-axis and the y-axis. And the z-axis is orthogonal to the xy plane. Although Figure 3 As shown, the flow cell image, sample and z-axis are described in a Cartesian coordinate system, but any other coordinate system can be used to define the spatial position and relationship of the colony or cluster and its image herein. Other coordinate systems can include, but are not limited to, polar coordinate systems, cylindrical coordinate systems, or spherical coordinate systems.

[0052] Sequencer 114 can be configured to flow a mixture of nucleotides onto flow cell 112, cut blockers from nucleotides between flow steps, and perform other steps for forming a DNA sequence on flow cell 112. Nucleotide can have a fluorescent element connected, which emits light or energy at a wavelength indicating the type of nucleotide. Each type of fluorescent element can correspond to a specific nucleotide base (e.g., A, G, C, T). The fluorescent element can emit light of visible wavelengths. In certain embodiments, sequencer 114 and flow cell 112 can be configured to perform various sequencing methods disclosed herein, e.g., affibody sequencing.

[0053] For example, each nucleotide base can be assigned a color. Different types of nucleotides can have different colors. For example, adenine (A) can be red, cytosine (C) can be blue, guanine (G) can be green, and thymine (T) can be yellow. The color or wavelength of the fluorescent element for each nucleotide can be selected so that the nucleotides can be distinguished from each other based on the wavelength of light emitted by the fluorescent element.

[0054] Imager 116 can be configured to capture images of flow cell 112 after each flow step. In one embodiment, imager 116 is a camera configured to capture digital images, such as a CMOS or CCD camera. The camera can be configured to capture images of the wavelengths of the fluorescent elements combined with nucleotides. These images can be referred to as flow cell images.

[0055] In some embodiments, imager 116 may include one or more optical systems disclosed herein. The optical system may be configured to capture optical signals from the flow cell and generate corresponding digital images. The digital images may then be used for base calling.

[0056] In one embodiment, images of the flow cell can be captured in groups, where each image in the group is taken at a wavelength or spectrum that matches or includes only one of the fluorescent elements. In another embodiment, the images can be acquired as a single image that captures all wavelengths of the fluorescent elements.

[0057] The resolution of the imager 116 controls the level of detail in the flow cell image, including the pixel size. In existing systems, this resolution is very important because it controls the accuracy of the point finding algorithm in identifying the center of the colony. In some embodiments, the image resolution of the flow cell image disclosed herein can be about 10 nanometers (nm) to hundreds of nm or higher. One way to improve the accuracy of point finding is to improve the resolution of the imager 116, or to improve the processing of the image taken by the imager 116. Detection of the center of the colony in pixels other than those detected by the point finding algorithm can be performed. These methods can allow the accuracy of colony center detection to be improved without increasing the resolution of the imager 116. The resolution of the imager can even be lower than that of existing systems with comparable performance, which can reduce the cost of the sequencing system 110. In some embodiments, the resolution of the imager may be the same as that of the existing system, but due to image processing, its performance is superior to that of the existing system.

[0058] The image quality of the flow cell image controls the base calling quality. One way to improve the accuracy of base calling is to improve the imager 116, or to improve the processing of the image taken by the imager 116, to obtain better image quality. The method described herein can align the flow cell image to a common coordinate system, so that the base calling of a cluster or a community will be more accurate than when such registration is not performed. These methods can allow accurate and efficient image registration. In addition, since the method disclosed herein is less computationally intensive than conventional methods, the heat dissipation of the computer / processor may be easier to manage, thereby unlikely to cause the undesirable transformation of the appropriate chemical properties of the sequencing technology disclosed herein. These methods can be performed in parallel in the system 100 implemented by a computer, without interfering with or delaying the existing sequencing workflow of the system 100. The result of the image registration can be used to perform actual base calling in the current cycle in the sequencing workflow. In addition, some or all of the operations disclosed herein can be advantageously performed by FPGA, and data can be transferred between CPU and FPGA to reduce the total operating time of the method that does not use FPGA operation. Furthermore, the image intensities and corresponding localizations of the selected populations are extracted to estimate the transformation of the entire flow cell image rather than directly registering multiple flow cell images, which may require saving the images before and / or after registration. Furthermore, the transformation matrix may be saved rather than the image, which may save required memory space and increase the efficiency of the image registration process, thereby advantageously enabling image registration and subsequent analysis to be performed while a sequencing run is ongoing.

[0059] The sequencing system 110 can be configured to perform image registration of flow cell images across different cycles and / or channels. The operations or actions disclosed herein can be performed by a dedicated processor 118, FPGA 120, computing system 126, or a combination thereof. One or more operations or actions in the methods 500, 2400 disclosed herein can be performed by a dedicated processor 118, FPGA 120, computing system 126, or a combination thereof. In some embodiments, it can be determined based on one or more of the following which operations or actions will be performed by a dedicated processor 118, FPGA 120, computing system 126, or a combination thereof: the computation time for a particular operation, the complexity of the computation in a particular operation, the need for data transmission between hardware devices, or a combination thereof. Image registration disclosed herein can be performed after acquiring the flow cell image but before the actual base calling of the flow cell image is performed in the cycle.

[0060] The computing system 126 may include one or more general-purpose computers that provide operating systems such as Windows TM or Linux TM An interface for running various programs in an operating system such as . Such an operating system typically provides users with great flexibility.

[0061] In some embodiments, a special processor 118 may be configured to perform operations in the image registration method. A special processor may not be a general purpose processor, but a custom processor with specific hardware or instructions for performing these steps. A special processor runs specific software directly without an operating system. The lack of an operating system reduces overhead, but at the expense of the flexibility that the processor can perform. A special processor can use a custom programming language that can be designed to operate more efficiently than software running on a general purpose computer. This can increase the speed of executing steps and allow real-time processing.

[0062] In some embodiments, FPGA 120 can be configured to perform the operations of the image registration method herein. FPGAs are programmed as hardware that will only perform specific tasks. Special programming languages ​​can be used to convert software steps into hardware components. Once the FPGA is programmed, the hardware directly processes the digital data provided to it without running software. In contrast, FPGAs use logic gates and registers to process digital data. Since the operating system does not require any overhead, FPGAs typically process data faster than general-purpose computers. Similar to dedicated processors, this comes at the expense of flexibility.

[0063] The lack of software overhead may also allow FPGAs to operate faster than dedicated processors, although this will depend on the exact processing to be performed and the specific FPGA and dedicated processor.

[0064] A group of FPGAs 120 can be configured to perform these steps in parallel. For example, many FPGAs 120 can be configured to perform processing steps for selected areas in an image, a set of images, a sub-tile, or one or more images. Each FPGA 120 can perform its own portion of the processing steps simultaneously, thereby reducing the time required to process data. This can allow the processing steps to be completed in real time. Further discussion of the use of FPGAs is provided below.

[0065] Performing processing steps in real time can allow the system to use less storage because data can be processed as it is received. This is an improvement over conventional systems that may need to store data before processing it, which may require more storage or access to a computer system located in the cloud 130.

[0066] In some embodiments, the data storage device 122 is used to store information used in the image registration method. This information may include the image itself or information derived from the image captured by the imager 116 (e.g., pixel intensity, color, etc.). The DNA sequence determined based on base calling can be stored in the data storage device 122. Parameters for identifying the location of the colony can also be stored in the data storage device 122. The original and / or processed image intensity of each colony can be stored in the data storage device. The data storage device 122 can also store the area and / or sub-tile corresponding to each colony. The transformation matrix of each area and / or sub-tile of different cycles and / or channels can also be stored in the data storage device 122.

[0067] User interface 124 may be used by a user to operate the sequencing system or access data stored in data storage device 122 or computer system 126 .

[0068] Computer system 126 can control the general operation of the sequencing system and can be coupled to user interface 124. The computer system can also perform steps in image registration and ongoing operations, and / or subsequent operations including but not limited to base calling. In some embodiments, computer system 126 is computer system 400, such as Figure 4 Computer system 126 may store information about the operation of sequencing system 110, such as configuration information, instructions for operating sequencing system 110, or user information. Computer system 126 may be configured to transfer information between sequencing system 110 and cloud 130.

[0069] As discussed above, the sequencing system 110 may have a dedicated processor 118, an FPGA 120, or a computer system 126. The sequencing system may use one, two, or all of these elements to complete the necessary processing described above. In some embodiments, when these elements are present together, the processing tasks are split between them. For example, the FPGA 120 may be used to perform some or all of the following: pre-processing operations, image registration, and subsequent operations, while the computer system 126 may perform other processing functions for the sequencing system 110, such as base calling. Those skilled in the art will appreciate that various combinations of these elements will allow for various system embodiments that balance the efficiency and speed of processing with the cost of the processing elements.

[0070] Cloud 130 may be a network, a remote storage device, or some other remote computing system separate from sequencing system 110. Connection to cloud 130 may allow access to data stored external to sequencing system 110 or allow software in sequencing system 110 to be updated.

[0071] Image Registration

[0072] Figure 5 A flow chart of an example embodiment of a computer-implemented method 500 for image registration of flow cell images acquired in different sequencing cycles and from different channels for accurate base calling during DNA sequencing is shown in accordance with some embodiments. The method 500 may include some or all of the operations disclosed herein. The operations may be performed in the order described herein, but not limited to.

[0073] Method 500 may be performed by one or more processors disclosed herein. In some embodiments, the processor may include one or more of the following: a processing unit, an integrated circuit, or a combination thereof. For example, the processing unit may include a central processing unit (CPU) and / or a graphics processing unit (GPU). The integrated circuit may include chips such as a field programmable gate array (FPGA). In some embodiments, the processor may include computing system 400.

[0074] In some embodiments, some or all of the operations in method 500 may be performed by an FPGA. In an embodiment, when some operations are performed by an FPGA, the data after the operations are performed by the FPGA may be transmitted by the FPGA to the CPU so that the CPU may use such data to perform subsequent operations in method 500. Similarly, data may also be transmitted from the CPU to the FPGA for processing by the FPGA. In some embodiments, all of the operations in method 500 may be performed by the CPU. Alternatively, the operations performed by the CPU may be performed by other processors (such as a dedicated processor) or a GPU. In some embodiments, all of the operations in method 500 may be performed by an FPGA.

[0075] In some embodiments, method 500 is configured to align or register flow cell images across different sequencing cycles and / or different color channels to a donor coordinate system. The common coordinate system may be a reference coordinate system disclosed herein. The common coordinate system may be predetermined. Figure 3 As shown, the common coordinate system can be a Cartesian coordinate system. Various other coordinate systems can also be used. Other coordinate systems can include, but are not limited to, polar coordinate systems, cylindrical coordinate systems, or spherical coordinate systems.

[0076] The flow cell image can be acquired from one of 1, 2, 3, 4 or more channels of the imager 116 using the optical system disclosed herein. Each flow cell image can include one or more tiles (e.g., imaging areas), and each tile can be divided into multiple sub-tiles. Each sub-tile can include multiple communities or clusters. Each sub-tile can include multiple areas, each of which includes several communities or clusters. For example, communities or clusters can be extracted from corresponding areas of flow cell images of 4 different channels in a given flow cycle. As another example, communities or clusters can be extracted from a flow cell image of a single channel. Example techniques for extracting multiple communities in a flow cell are described in U.S. Patent No. 11,200,446, which is hereby incorporated by reference in its entirety. Flow cell images as disclosed herein can be acquired using, for example, Figure 1 An image acquired by the flow cell 112 is shown.

[0077] The flow cell 112 may include a sample fixed thereon. The sample may include a plurality of nucleic acid template molecules. The sample may include a two-dimensional (2D) sample or a three-dimensional (3D) volume sample. The nucleic acid template molecules may be randomly or in various patterns distributed on the flow cell 112. In some embodiments, a plurality of colonies or clusters herein may be extracted from a specific region of a tile (e.g., each sub-tile). In the case of each sub-tile, a colony may be extracted in a predetermined pattern or randomly.

[0078] In certain embodiments, the colony or cluster sequenced in the flow cycle can have a certain nucleotide diversity. Even if the colony or cluster is low diversity or unbalanced diversity in the sequencing cycle, method 500 still allows image registration of flow cell images. The nucleotide diversity of nucleotide acid molecule population (e.g., colony or cluster) can refer to the relative proportion of nucleotide A, G, C and T / U present in each flow cycle. The best high or balanced diversity data can generally have the roughly equal proportion of all four nucleotides represented in each flow cycle of sequencing operation. Low or unbalanced diversity data can generally include some nucleotides of high proportion and other nucleotides of low proportion in some flow cycles of sequencing operation, for example, less than 10% of the total number of all 4 kinds of nucleotides. Therefore, compared with the image corresponding to some nucleotides of low proportion, the image corresponding to some nucleotides of high proportion can have more signal points (colony or cluster). As an example of low or unbalanced diversity data in the flow cycle, in a certain flow cycle, base A, T, C, G can be respectively about 1%, about 2%, about 1% and about 95% of the total number of colonies. As another example of low or unbalanced diversity data, the bases A, T, C, and G in the population in multiple flow cycles may be about 2%, about 5%, about 10%, and about 83%, respectively. In embodiments where low or unbalanced diversity data is present in a particular cycle and imaging is performed for sequencing analysis, image registration using prior art techniques may fail because the image from one or more channels is too dim compared to the images acquired from other channels (e.g., the signal points of the population are too sparse and / or dim).

[0079] In addition to base biases that affect diversity, complexity may also be a factor that affects image registration. Typically, complexity can indicate the source of the sample. Single samples can include DNA fragments or molecules from the same sample region or the same sample source in the genome. Multiple samples can include DNA fragments or molecules from different sample sources, such as liver, kidney, heart, cancer tissue, etc., or from one or more sample regions in the genome. When complexity is lower than a certain number (e.g., 8 or 16), the signal may be low diversity. For example, in a 2-cycle sequence, all communities are AT or TG or GC or CA. Each base accounts for 25% of the total number of bases in this cycle, but its complexity is less than 8, and the sequence is not completely random. In some embodiments, method 500 is configured to register the flow cell image, even if the community is low diversity and / or low complexity.

[0080] In some embodiments, method 500 is performed during a cycle N that is different from a reference cycle. A template image can be generated in a reference cycle, and a community of one or more channels within the reference cycle can be included in the template image in a reference coordinate system, while base calls for cycle N have not yet been performed. In some embodiments, cycle N is the current cycle. N can be any non-zero integer. For example, N can be any integer from 1 to 300 or from 1 to 400.

[0081] In certain embodiments, method 500 is performed during cycle N, and is being performed or has not yet performed sequencing and image acquisition in subsequent cycles (e.g., cycle N+1). In certain embodiments, method 500 is performed simultaneously with sequence operation, to advantageously shorten the total time of sequencing and preliminary analysis. In certain embodiments, method 500 is performed in parallel with sequence operation, to advantageously reduce the storage space required for preserving flow cell image. For example, after image registration of cycle N, the transformation of cycle N and the list of colonies or clusters with its intensity and position information can be preserved, which requires less storage space than the storage space required for actual flow cell image. In certain embodiments, method 500 can be performed after sequencing operation is completed.

[0082] In some embodiments, method 500 can allow for image registration of flow cell images of in situ samples. In situ samples can include cell samples disclosed herein, with depth along the Figure 3 The z-axis of the image plane of the flow cell image is perpendicular to the z-axis of the image plane of the flow cell image. The in-situ sample can have a 3D volume, and the colonies or clusters can be distributed in the 3D volume therein. In order to image the optical signals from the colonies or clusters, the flow cell image can be acquired at multiple z positions spaced apart from each other in the axial direction. In some embodiments, the operations of method 500 can be performed for each flow cell image at a different z level.

[0083] In some embodiments, method 500 may include operation 510 of determining coordinates of a population or cluster in a reference coordinate system.

[0084] The colony can be from a sub-tile of the flow cell image within a reference cycle, and more specifically, from one or more selected areas of the sub-tile. The flow cell image can be from different channels in 1, 2, 3, 4 or more channels of the system 100. As a non-limiting example, the reference cycle can be any cycle in the first 5 or 6 cycles. In certain embodiments, the reference cycle can be any cycle greater than 0. In certain embodiments, the reference cycle is the first cycle.

[0085] The region in the sub-tile can be selected in various ways to include at least a portion of the sub-tile. For example, the region can be selected as the central pixel of the enclosed sub-tile. For images from cell samples or other various samples unevenly distributed above the FOV, the region of each sub-tile can be selected at different positions in the sub-tile. In some embodiments, the region is selected to ensure that there are at least a threshold number of cells or colonies in the selected region. For example, operation 510 can include an operation to determine the number of cells and / or colonies in the region. The region including the central pixel, for example, 128x128, can be selected as a default. In response to determining that the central region does not include cells and / or colonies that meet a predetermined threshold, method 500 may include an operation to move the selected region until an area that meets the predetermined threshold is found. The selected region may be located at the same position across different color channels and / or cycles. The predetermined number of colonies or clusters can be 20, 40, 60, 80 or more. The predetermined number of cells can be 1, 2, 5, 8, 10 or more.

[0086] The area of ​​each sub-tile can be selected at different positions of the sub-tile. In some embodiments, based on various criteria, such as maximum signal variance, highest average signal strength or maximum signal strength standard deviation, etc., the area is selected in the selected area of ​​multiple candidate areas in the same sub-tile. The area selected by various criteria may be more likely to contain stronger colonies and / or cell signals than other areas with lower signal variance, and may help to align. In some embodiments, some or all possible options for selecting the area in the sub-tile are included as candidate areas. For example, for a 100x100 area size, a sliding window across all rows and columns of the sub-tile can provide all possible options for selecting a 100x100 area. In some embodiments, the selected area can be located at different positions of different sub-tiles. For the same sub-tile in different cycles or across different color channels, the selected area can remain the same. In some embodiments, each sub-tile may include one or more selected areas. Two different sub-tiles may include different numbers of selected areas. The size of the selected area may be the same or different. For example, sub-tile 1 may have a single selected region including its center pixel, sub-tile 2 may have 3 selected regions, none of which include any center pixel, and the sizes of the 3 selected regions may be different. Such selection based on a predetermined threshold may also be used for 2D samples with sparse populations or clusters within the sub-tile (e.g., less than 100 populations in a 128 x 128 area) or weak signals within the sub-tile (e.g., less than 10%, 20%, or other percentages of the average signal intensity compared to another area).

[0087] Selecting regions within each subtile can help ensure that the Fourier transform of the selected region contains at least some signal from the community and not just background and / or noise.

[0088] The selected area may be of various two-dimensional shapes, such as a rectangle, a circle or a square. As a non-limiting example, the selected area may include one or more central pixels of the sub-tile. Figure 2 A non-limiting example of a selected region 230 is shown. Region 230 may include a plurality of communities 232, such as Figure 2 The size of the region can be determined to balance trade-offs between different factors, including but not limited to computational complexity, time consumption, population density on the flow cell, and accuracy of image registration. For example, selecting a 64 x 64 region may be computationally simpler than selecting a 128 x 128 region, but may not be as accurate for certain samples fixed on the flow cell.

[0089] Once a region is selected, the coordinates of the colonies in the region can be determined by registering the selected region in the flow cell image across channels. Operation 510 can include the operation of registering the selected region in the flow cell image across channels. The regional registration across different channels (e.g., from the same cycle) can be based on multiple fiducial markers outside the sample fixed on the flow cell. In certain embodiments, multiple fiducial markers are distributed in a predetermined pattern to predetermine the size of the mark, the distance between the marks and the intensity of the mark. In certain embodiments, multiple fiducial markers can be randomly distributed, for example, as a coating on the flow cell. The flow cell image can be obtained from different channels with the same fiducial marker, but without any colonies, for example, the flow cell image of the test target herein. In certain embodiments, the fiducial marker can include an intensity level comparable to the colony intensity to simulate the presence of the colony. In certain embodiments, the fiducial marker can include an intensity level comparable to the colony intensity to simulate the presence of the colony.

[0090] Images of such fiducial markers can be used as a supplement to flow cell images acquired during a sequencing run, and can be used for registration and registration rescue of flow cell images and / or calibration of imager 116. In some embodiments, such images of fiducial markers can be used to register offsets and other transformations across channels within a single flow cycle, and such offsets and other transformations can be kept consistent across different flow cycles (e.g., several cycles that are consecutive to each other in a sequence run). For example, in immediately adjacent cycles (e.g., cycles 50 to 55), the offsets and transformations between channel 1 and channel 2 can be considered the same.

[0091] When the selected region is low diversity data, the image registration rescue operation in method 2400 may be performed. Image registration rescue may be performed on images from a cycle with low diversity data based on fiducial landmarks.

[0092] In some embodiments, images with only fiducial markers are not acquired in certain cycles when image registration or registration rescue is not needed across channels. In some embodiments, fiducial markers are used only to acquire additional images within a cycle to register images across different channels, for example, within a reference cycle or within the first 1-5 cycles.

[0093] In some embodiments, images with fiducial markers (e.g., test targets or flow cells with beads) are acquired in one or more cycles that are not reference cycles. In some embodiments, images with fiducial markers are acquired in one or more cycles prior to any cycle of a sequencing run. In some embodiments, images with external fiducial markers (e.g., test targets or flow cells with beads) are acquired in one or more cycles where data diversity is low.

[0094] In some embodiments, rather than using fiducial markers for inter-channel registration, the image registration information from the previous cycle or any rescue cycle can be used to register images of channels with low diversity data (e.g., channels with less than 10% of the total population in all channels). Using the image registration information from the previous cycle to image the fiducial markers in a reference cycle, a low diversity cycle, or in a fixed pattern every few cycles can reduce the total imaging time and data to be processed, while still achieving accurate and reliable image registration results using the methods herein, compared to acquiring additional images with fiducial markers in each cycle.

[0095] Various methods can be used to register flow cell images from multiple channels based on such fiducial markers, so that a fiducial marker with image intensity I centered at position (x1, y1) is located at position (xr, yr) with intensity I in the reference coordinate system, where And Mr is the transformation matrix. Similarly, the inverse transformation matrix Mr can also be determined -1 , so that Multiple fiducial markers (e.g., at least 3) can be used to estimate the transformation matrix Mr of the selected region. The transformation matrix Mr of the selected region can be used as the transformation matrix of the corresponding sub-tile. Image registration across images of different color channels can be 2D and can include translation, scaling, rotation, and / or shearing of flow cell images between different channels. In some embodiments, when a colony or cluster appears in such a corresponding channel, the colony or cluster can be used as a fiducial marker for registering the flow cell image between channels.

[0096] In some embodiments, method 500 includes determining the coordinates of the colonies or clusters in a reference coordinate system by multiplying the coordinates of the colonies or clusters by a transformation matrix Mr, i.e., (xrp, yrp) = Mr*(xp, yp), where xrp and yrp are the coordinates of each colony in the reference coordinate system, and xp and yp are the coordinates of the colonies in each flow cell image.

[0097] A common reference coordinate system for all flow cell images in a reference cycle can be determined. For example, the reference coordinate system can be the coordinate system of the flow cell images from one channel. As another example, the reference coordinate system can be based on external fiducial markers or other objects external to the flow cell images.

[0098] In some embodiments, method 500 includes an operation 520 of generating one or more template images in a reference coordinate system by registering the colony to the one or more template images using coordinates of the colony. Figure 2 210 is a schematic diagram showing one or more template images generated in a reference coordinate system in a reference cycle. In some embodiments, the size of the template image 210 is substantially the same as a single tile. In this embodiment, as shown in FIG. Figure 2 As shown, tile 210 includes a 5x5 grid of sub-tiles 220. Region 230 is selected in each sub-tile and includes the center pixel of the corresponding sub-tile. In this embodiment, the reference coordinate system has an origin 212 located at its upper left pixel. In some embodiments, each tile may include multiple template images, and each template image may include a separate region, such as region 230. Each template image may include multiple communities or clusters 232 therein.

[0099] In some embodiments, the size of the template image can be approximately the same as the size of the flow cell image so that Figure 2-3 Different tiles 210 in the flow cell image and all colonies from multiple channels can be registered to the same template image. The template image can contain colonies that will not be used in at least some operations described herein to reduce the computational burden without sacrificing accuracy. In some embodiments, more than one template image can be generated, and each template image 230 corresponds to at least a portion of a sub-tile of a flow cell image from a channel.

[0100] The template image herein can be initialized as a virtual image having a black or dark background and no signal from the community. For example, the template image can be initialized to zero, or include a minimum image intensity at all pixels.

[0101] After the coordinates of the colony are determined by image registration of flow cell images across different channels in operation 510, the intensity of the colony can be added to the template image at the position determined by the coordinates, and its size and shape are determined based on the registration. The template image can be a virtual image that combines the image intensities of the colony obtained from 2, 3, 4 or more channels in a reference cycle. The pixels of the template that do not contain colonies remain black or dark, so that the template image can have a cleaner background without the noise that appears in the actual flow cell image.

[0102] In some embodiments, method 500 includes an operation of obtaining image intensity, size, shape, or a combination thereof of a colony from at least a portion of one or more sub-tiles in a reference loop so that such information can be used to include the colony in a template image. In some embodiments, the colony can have a fixed shape and / or size. In some embodiments, a point spread function determined by an optical system is used to determine the fixed shape and / or size of the colony. In some embodiments, the colony has a fixed spot size based on σ of a Gaussian point spread function. In some embodiments, the size of one or more colonies is 1-9 pixels. In some embodiments, the size of one or more colonies is 1-3 pixels.

[0103] The template image may include communities from different channels as well as channel information. As an example, the channel information may be provided in the form of labels, or in a specific order of how the communities are included.

[0104] In some embodiments with multiple template images, each template image 230 may cover an area within a sub-tile, and such template images may, but need not, include all of the communities within the sub-tile.

[0105] In some embodiments, the template image can be a list of entries, which is simpler and more efficient to process than a 2D or 3D virtual image. For example, each colony or cluster within the template image can have its coordinates, such as a center pixel, and a corresponding intensity in a list entry. And such coordinates can be in a reference coordinate system. Intensities in different color channels can also be included in the same entry.

[0106] In some embodiments, method 500 includes operation 530 of obtaining a flow cell image in a cycle following the reference cycle. Operation 530 may include passively receiving or actively requesting the flow cell image from the optical system after the flow cell image is generated by the optical system disclosed herein. Operation 530 may include acquiring the flow cell image using the optical system. The optical system may include Figure 1 In the imager 116 in.

[0107] In some embodiments, operation 530 may include the operation of providing a plurality of nucleic acid template molecules fixed on a carrier. Each nucleic acid template molecule may include an insertion sequence of interest. The insertion sequences in different template molecules may be different. Each template molecule may correspond to an optical signal community in a flow cell image.

[0108] In certain embodiments, operation 530 can comprise the operation of generating flow cell image by carrying out one or more circulation sequencing reactions to a plurality of nucleic acid template molecules fixed on the carrier.Flow cell image can be generated or obtained by sequencing system disclosed herein.Carrying out one or more circulation sequencing reactions can comprise: use multiple nucleotide reagents to contact multiple nucleotide acid template molecules, and the multiple nucleotide reagents comprise the mixture of different types of nucleotide bases A, G, C and T / U.Independent nucleotide reagents can comprise the different detectable color marks corresponding to every different types of nucleotide bases.

[0109] In some embodiments, performing one or more cycles of sequencing reactions may include: contacting a plurality of nucleotide acid template molecules with a mixture of a plurality of sequencing primers, a plurality of polymerases, and different types of affimers. A single affimer in the mixture may include a core connected to a plurality of nucleotide arms, and each arm of the single affimer includes a nucleotide base of the same type. In some embodiments, performing one or more cycles of sequencing reactions may include: imaging an optical color signal emitted by a nucleotide reagent bound to a plurality of template molecules in each of the one or more cycles. Imaging the optical signal may be performed by an optical system, such as the imager 116 disclosed herein. In some embodiments, performing one or more cycles of sequencing reactions may include: acquiring a flow cell image in each of the one or more cycles, the flow cell image including an optical color signal emitted by a nucleotide reagent bound to a plurality of template molecules.

[0110] In some embodiments, the flow cell image comprises optical signals emitted from nucleotide reagents bound to an unbalanced diversity of nucleotide bases A, G, C, and T / U in a plurality of nucleic acid template molecules immobilized on a carrier in one or more cycles. In some embodiments, the plurality of colonies comprises an unbalanced diversity of nucleotide bases A, G, C, and T / U, and wherein the unbalanced diversity comprises: (1) a percentage of the number of one or more types of nucleotide bases relative to (2) a total number of nucleotide bases, and in the cycle N, the percentage is less than 20%, 15%, 10%, or 5%. The plurality of colonies corresponds to the plurality of nucleic acid template molecules.

[0111] In some embodiments, operation 530 may include providing a cell sample having a plurality of concatemer molecules immobilized on a carrier, wherein each concatemer molecule corresponds to a target RNA of the cell sample.

[0112] In some embodiments, operation 530 may include generating a flow cell image by performing one or more cycles of sequencing reaction on the multiple concatemer molecules fixed on the carrier by the sequencing system. Performing one or more cycles of sequencing reaction may include: contacting multiple concatemer molecules with multiple nucleotide reagents, and the multiple nucleotide reagents include a mixture of different types of nucleotide bases A, G, C and T / U. Performing one or more cycles of sequencing reaction may include: contacting multiple concatemer molecules with a mixture of multiple sequencing primers, multiple polymerases and different types of affimers. The individual affimers in the mixture include a core connected to multiple nucleotide arms, and each arm of the individual affimers includes the same type of nucleotide bases. Performing one or more cycles of sequencing reaction may include: in each cycle of one or more cycles, imaging the optical color signal emitted from the nucleotide reagent bound to the multiple concatemer molecules by the optical system. In some embodiments, performing one or more cycles of sequencing reaction may include: in each cycle of one or more cycles, acquiring a flow cell image by the optical system, the flow cell image including the optical color signal emitted from the nucleotide reagent bound to the multiple concatemer molecules.

[0113] The flow cell image may include some or all of the same communities in the template image of the reference cycle. Specifically, the flow cell image may include some or all of the same communities in an area corresponding to a selected area in the reference cycle.

[0114] Figure 2 At the bottom, a flow cell image acquired in a cycle different from the reference cycle is shown. Flow cell image 240 is acquired with a plurality of sub-tiles 250. Selected region 260 in this cycle may be located at the same relative position relative to origin 242 as selected region 230 in the reference cycle relative to origin 212. In this cycle, flow cell image 210 in the reference may have been transformed into transformed image 211, and selected region 230 is correspondingly transformed into region 231, and has some overlap with region 260. Image transformations herein may be 2D, and may include translation, scaling, rotation, and / or shearing.

[0115] In some embodiments, method 500 is configured to align a template image in a reference cycle (eg, 210 or 230 ) and a transformed image in another cycle (eg, 211 or 231 ) with a reference coordinate system.

[0116] In some embodiments, method 500 may include selecting regions 230 and 260 to more easily and conveniently determine image registration rather than directly using regions 231 or 211 in image registration. Region 260 may include at least a portion of colony 232 in a template image in a reference cycle, for example, 230.

[0117] In some embodiments, method 500 includes operation 540 of determining a plurality of transformations of flow cell image 240 based on one or more template images 210 or 230. Figure 2 As shown, each of the multiple transformations can correspond to a sub-tile 250 of the flow cell image 240 and is configured to align the sub-tile 250 of the flow cell 240 image to a corresponding portion of the template image 210 (if the template image includes the entire tile) or to the corresponding template image 230 (if multiple template images exist within the tile).

[0118] In some embodiments, operation 540 may include determining each transformation corresponding to a sub-tile of the flow cell image. More specifically, each transformation may correspond to a selected region of each sub-tile in some or all of the sub-tiles. The region may be selected from the sub-tiles in various ways to include at least a portion of the sub-tiles. The region may be a predetermined two-dimensional shape, such as a rectangle, a circle, or a square. As a non-limiting example, the selected region may include one or more center pixels of the sub-tile, such as Figure 2 260 in the figure. The size of the region can be determined to balance the trade-off between computational complexity and the accuracy of image registration. For example, selecting a 64x 64 region may be computationally simpler than selecting a 128x 128 region, but may not be as accurate. In some embodiments, the selected region includes some or all of the colonies 232 registered in the template image in the reference cycle, so that the same colonies in the template image and the flow cell image and their relative positions can be used to determine the transformation. In some embodiments, the size of the template image (e.g., 230) and the region 260 may be the same or approximately the same. In some embodiments, the size of the template image 210 or 220 and the selected region 260 may be different.

[0119] Figure 6A-6B A schematic diagram of image transformation and corresponding 2D displacement according to some embodiments is shown. In some embodiments, a cross correlation between the selected region and the template image may be calculated to determine the 2D displacement of the selected region relative to the template image. Fig. 6A A reference image (left) is shown, which is transformed into a different image (middle) after 2D shearing, scaling and rotation. The 2D displacements 601 at the four corners of the reference image can be determined, for example, using the methods disclosed herein and calculation of cross-correlations. And the 2D displacements at the four corners can be used to estimate the transformation between the two images.

[0120] In some embodiments, the cross correlation may be calculated in the spatial domain. In some embodiments, the cross correlation may be calculated in the spatial frequency domain after the Fourier transform (FT). Method 500 may include generating a corresponding Fourier transformed image (FTI) of the template image and a Fourier transform of the selected region. The Fourier transform herein may be calculated using a discrete FT (DFT), a fast FT (FFT), etc. The cross correlation may be determined based on the FTI and the Fourier transform of the selected region. As a non-limiting example, the cross correlation may be an element-by-element multiplication of the FTI with the FT of the selected region, plus a complex conjugate or rotation of one of them. Then, an element-by-element multiplication of the inverse FT may be obtained. In some embodiments, the cross correlation may be a 2D image having a peak intensity at its coordinates [xp, yp]. In some embodiments, the 2D displacement may be determined based on the coordinates [xp, yp] compared to the coordinates of the peak obtained from the cross correlation of the two original images without transformation. The 2D displacement of the selected region 260 may be used to estimate the 2D displacement of the entire sub-image block. In some embodiments, the result of calculating the cross correlation in the spatial domain or the Fourier domain may be equivalent. In some embodiments, calculations in the Fourier domain are simpler and more efficient than calculations in the spatial domain. In some embodiments, a Fourier transformed image (FTI) of a template image is stored in a storage device herein to reduce the computation time for image registration. In some embodiments, a Fourier transformed image (FTI) of a template image rather than a template image in the spatial domain is stored in a storage device herein to reduce the storage space required for image registration.

[0121] In some embodiments, the image transformation of a sub-tile can be determined based on 2D displacements from some or all adjacent sub-tiles, regardless of whether it has 2D displacements itself. In some embodiments, 2D displacements from all immediate neighbors can be used. For example, in order to determine the transformation of sub-tile 253, 2D displacements from 3 adjacent sub-tiles and 2D displacements from itself can be used. For sub-tile 251, a total of 6 2D displacements can be used, including directly adjacent sub-tiles and itself. For sub-tile 252, a total of 9 2D displacements can be used, including adjacent sub-tiles and itself. In some embodiments, 2D displacements from some but not all adjacent sub-tiles can be used. In some embodiments, except for 1-2 outlier neighbors, 2D displacements from all adjacent sub-tiles can be used to determine the transformation. Outlier neighbors can be excluded using predetermined criteria, for example, differing from other 2D displacements by more than 30% or 50%.

[0122] Figure 6B601 is an image showing 2D displacements within an example tile of a flow cell image. In this embodiment, the tile has a 6 x 9 grid of sub-tiles, and each sub-tile has a 2D displacement 601 determined using the techniques disclosed herein. The magnitude of each displacement along the x or y axis is less than about 5 pixels. The pixel size can vary depending on the imaging parameters, and an example pixel can be 0.1um to 0.9um. The 2D displacement 601 can be used to calculate a transformation of the tile by calculating the transformation of each sub-tile separately, for example, an affine matrix. In this embodiment, the affine matrix can be calculated using the methods disclosed herein.

[0123] In some embodiments, sub-pixel resolution of the 2D displacement 601, e.g., about 0.01, 0.02, 0.03, or 0.05 pixels, can be achieved using various methods including interpolation, upsampling, etc. In some embodiments, sub-pixel resolution can be achieved by fitting the peaks with a selected filter (e.g., a 3x 3 or 5x 5 Gaussian filter).

[0124] In some embodiments, the image transformation of the sub-tile can be represented by a transformation matrix. The transformation matrix can be determined as follows:

[0125]

[0126] Where n is the number of sub-tiles, a1=x1+dx1, b1=y1+dy1, a2=x2+dx2, b2=y2+dy2, ... an=xn+dxn, bn=yn+dyn, d1...dn is the 2D displacement corresponding to the sub-tile, and where dxn and dyn are the displacement components of the 2D displacement dn on the x-axis and y-axis respectively, (a1, b1)...(an,bn) are the transformed coordinates, (x1,y1)...(xn,yn) are the coordinates before the transformation, and where M is the 3x 3 transformation matrix of the sub-tile.

[0127] In some embodiments, the transformation matrix can be defined as the inverse matrix of M, that is, M -1 , so equation (1) can be expressed differently as

[0128]

[0129] In some embodiments, the transformation matrix M is an estimate in equations (1) and (3) based on the 2D displacement. In some embodiments, the value of n may affect the accuracy of the estimate. In some embodiments, more than one region can be selected within the sub-tile for cross-correlation calculation, and more than one 2D displacement can be calculated for each sub-tile and used to estimate the transformation of the sub-tile. In these embodiments, n in equation (1) can be replaced with a larger number, for example, when 2 regions are selected for each sub-tile, it can be replaced with 2*n, and the transformation matrix M can be estimated using equations (1) and (2).

[0130] In some embodiments, (a1, b1) ... (an, bn) in equations (1)-(3) are the coordinates of the selected area after transformation (e.g., the coordinates of the center pixel of the corresponding area), and (x1, y1) ... (xn, yn) are the coordinates of the selected area before transformation, e.g., the coordinates of the center pixel. In some embodiments, n is a number not less than 3. The larger n is, the more information is used to estimate the transformation matrix M. In some embodiments, n is not greater than 9.

[0131] In some embodiments, the transformation of one or more sub-tiles is linear. In some embodiments, the transformation of all sub-tiles is linear. In some embodiments, the transformation matrix is ​​a matrix in which M31 and M32 are equal to 0 and M33 is 1. In some embodiments, one or more of the transformations of each sub-tile is an affine transformation, and the transformation matrix of the entire flow cell image is an affine matrix.

[0132] In some embodiments, the transformation matrix M is an estimate in equations (1) and (3) based on the size, positioning, and number of selected regions of each selected region. In some embodiments, the size of the selected region may affect the accuracy of the estimate. In some embodiments, the size of the selected region may be approximately 128 x 128. In some embodiments, the size of the selected region may be approximately 32 x 32, 48 x 48, 64 x 64, 96 x 96, 160 x 160, 192 x 192, 256 x 256, or various sizes. As disclosed herein, the transformation of each sub-tile may be calculated using a selected region within the sub-tile, and the selected region may be equal to or smaller than the sub-tile. In either case, given the inherent characteristics of image transformation across sequencing cycles, the transformation estimated using the region may be used to estimate the transformation of the entire sub-tile. Image transformations between cycles and / or between adjacent pixels may be relatively small, for example, may be less than about 8%, 5%, or less than about 1% of scaling, rotation, and / or shearing. In some embodiments, the transformations disclosed herein may include image translations having a difference between cycles and / or between adjacent pixels greater than about 5%.

[0133] After determining multiple transformations of the individual sub-tiles, the transformation of the entire flow cell image can be accurately and reliably estimated by transforming the individual sub-tiles using the multiple transformations and combining the transformed sub-tiles into a transformed flow cell image. The techniques disclosed herein advantageously estimate the transformation of the flow cell image by determining multiple transformations of the individual sub-tiles of the flow cell image. The multiple transformations can be linear, and even if the transformations are non-linear, the transformation of the flow cell image can be accurately and reliably estimated. The techniques disclosed herein advantageously eliminate the need to calculate the transformation of the entire flow cell image, which can be more computationally intensive, more time consuming, and more prone to failure than estimating multiple transformations of the sub-tiles.

[0134] In some embodiments, the computer-implemented method 500 further includes an operation of saving the plurality of transformations by a processor disclosed herein. In some embodiments, the computer-implemented method 500 further includes an operation of transmitting the plurality of transformations to a processing unit (eg, a CPU) for subsequent operation.

[0135] In some embodiments, the computer-implemented method 500 further includes registering the sub-tiles to one or more template images using a plurality of transformations. This operation may be performed by a processing unit such as a CPU. In any given loop different from the reference loop, each sub-tile may be registered or transformed to one or more template images by multiplying the sub-tile by a transformation matrix corresponding to the sub-tile.

[0136] In some embodiments, the computer-implemented method 500 may include operations, such as operations 510 through 540 , of performing one or more pre-processing steps on the flow cell images of the reference cycle and / or other cycles prior to registering the images from the cycles.

[0137] In some embodiments, the operation of performing one or more pre-processing steps can be performed by FPGA. In some embodiments, the data after the operation can be transmitted by FPGA to CPU so that CPU can use such data to perform subsequent operations in method 500.

[0138] In some embodiments, the pre-processing steps of the one or more flow cell images in the reference cycle can be performed before operations 510, 520 or after operation 520. In some embodiments, the one or more pre-processing steps of the flow cell images in the reference cycle can be performed after the operation of receiving the flow cell images in the reference cycle from the optical system disclosed herein. In some embodiments, the one or more pre-processing steps of the flow cell images in the reference cycle can be performed before the operation of obtaining the image intensity, size, shape, or a combination thereof of the colonies from multiple sub-tiles of the flow cell image in the reference cycle.

[0139] In some embodiments, one or more pre-processing steps of the flow cell images in cycles other than the reference cycle may be performed after operation 530 or 540. In some embodiments, one or more pre-processing steps of the flow cell images in cycles other than the reference cycle may be performed after the operation of registering the sub-tiles of the flow cell images to one or more template images. In some embodiments, one or more pre-processing steps of the flow cell images in cycles other than the reference cycle may be performed before the operation of extracting the image intensities of the plurality of communities from the sub-tiles of the flow cell images. In some embodiments, one or more pre-processing steps of the flow cell images in cycles other than the reference cycle may be performed before the operation of performing base calls using the image intensities of the sub-tiles of the flow cell images.

[0140] One or more pre-processing steps may include background subtraction. Background subtraction is configured to remove at least some background signals that may interfere with the signal of interest, i.e., the image intensity of the colony. Background signals may be noise caused by multiple sources, including flow cell 112, imager 115, sequencer 114, and other sources. Background subtraction may be adjusted to avoid excessive subtraction.

[0141] One or more pre-processing steps may include image sharpening to optimize the image intensity of the colonies in view of their surroundings in the flow cell image. For example, sharpening may be performed using a Laplacian of Gaussian (LoG) filter.

[0142] One or more pre-processing steps may include intensity offset adjustment, which may remove intensity offset that was not removed during background subtraction.

[0143] One or more pre-processing steps may include color correction to remove interference to one channel from other channels or colors.

[0144] One or more pre-processing steps may include lag and lead corrections configured to correct image intensity within a particular cycle by eliminating intensity bias caused by sequencing of DNA fragments that are out of sync with other fragments due to lagging or leading.

[0145] One or more pre-processing steps may include intensity normalization to normalize image intensities of populations from different channels to be within a predetermined range.

[0146] The one or more pre-processing steps may include: background subtraction; image sharpening; or a combination thereof.

[0147] In some embodiments, the computer-implemented method 500 further includes extracting the image intensity of multiple colonies from the sub-tiles registered to the template image. This operation can be performed by processing units such as CPU or FPGA. In some embodiments, the colonies and their corresponding intensities are extracted from the flow cell image as different data formats that are simpler and more efficient to process. For example, each colony can have 4 different intensities, and each intensity comes from different channels. Such intensity can be extracted into a list, and each entry of the list corresponds to a colony. The list can be generated after image registration to reflect the positional information of the same colony in different cycles. Therefore, the image intensity of the same colony in different cycles can be located in different lists corresponding to a cycle respectively.

[0148] In some embodiments, the computer-implemented method 500 further comprises making base calls using image intensities of sub-tiles of the flow cell image after registration so that base calls can be accurately made with respect to the same population across different channels and in different cycles.

[0149] In some embodiments, method 500 includes operation 540, i.e., determining multiple transformations of the flow cell image. Operation 540 may include determining each transformation in the transformation without using any adjacent sub-tiles disclosed herein. Conversely, more than 2 regions may be selected within the sub-tile, and the 2D displacement of each region in the region may be determined. The transformation of the sub-tile may be determined using equations (1) and (2) using the 2D displacement obtained from the region within the same sub-tile. The size of the region within the sub-tile may be smaller than region 260 in the adjacent sub-tile. For example, region 260 may be approximately 128 x 128, and the region within the sub-tile may be 3, 4, 5 or even more regions, and each region may include a matrix of approximately 64 x 64. The other operations of method 500 may remain unchanged for image registration using or not using adjacent sub-tiles when generating the transformation.

[0150] Example Image Registration

[0151] The sequence run is carried out on the sequencing system 110, wherein the original position sample is fixed on the flow cell device. The flow cell image is being acquired. After the first sequencing cycle ends and the sequencing run is still in progress, the flow cell image from 4 different color channels is processed using the image preprocessing step. The matrix size of each image is 5472x 3648, and there are 424 flow cell images with 7 to 10 different z positions. Each flow cell image is divided into 54 sub-picture blocks with a 6x 9 matrix, and each sub-picture block has 608x 608 pixels. The first cycle is a reference cycle. After determining that the number of colonies in the 128x 128 region at the same position in each sub-picture block of each color image meets a predetermined threshold, the region is selected as the region for determining the displacement of the entire sub-picture block. The selected region meets the predetermined threshold of 100 colonies therein. The candidate colony position in the selected region of the individual sub-picture block is determined, and all candidate colony spots are transformed into a reference coordinate system to obtain corresponding virtual coordinates. The transformation to the reference coordinate system is based on an external benchmark marker. A 200 x 200 template was generated, including all colony positions from the 4 color channels in the first cycle. Each flow cell image in subsequent cycles was registered to the template by determining the best cross-correlation in the Fourier domain with the corresponding area of ​​the corresponding sub-tile in the first cycle. Specifically, the 2D displacement that gave the highest cross-correlation was determined and used to obtain the affine matrix for each sub-tile using the displacements from the adjacent sub-tiles.

[0152] Image registration to the rescue

[0153] In some embodiments, the sample immobilized on a support (eg, a flow cell) can be of low nucleotide diversity as disclosed herein. Fig.21 Shown are example flow cell images from four different color channels in a flow cycle. One of the channels, for example, the channel 4 in the lower right, has a signal spot (i.e., colony or cluster) less than the signal spot of the image from other channels due to lack of nucleotide diversity in a specific flow cycle. The colony emitting optical signals in channel 4 may be less than 10% of the total colony of the emission signals in all 4 different channels. In certain embodiments, compared with the image registration of images from other channels with better nucleotide diversity, the image registration (e.g., registration to a reference coordinate system) of the flow cell image from channel 4 may be less reliable. In certain embodiments, the method and system for image registration rescue can be utilized to improve the image registration accuracy and / or reliability of such channels in the flow cycle of low nucleotide diversity.

[0154] When sequencing data contains imbalance or low nucleotide diversity in one or more flow cycles, the method and system for image registration rescue can be advantageous. The method and system for image registration rescue is particularly advantageous for sequences where, in one or more flow cycles, the index read is first sequenced, and then any insertion read is sequenced, and the nucleotide diversity of the index read may be lower. The method and system for applying image registration rescue can advantageously improve the index allocation and / or matching in low diversity data, so that the sequencing results are demultiplexed to different samples more accurately and reliably. The method and system for image registration rescue can also improve base call accuracy and reliability by improving the image registration of samples with low nucleotide diversity in the insertion sequence in at least some cycles. In certain embodiments, when a custom threshold is met, the method and system for image registration rescue can be selectively turned on or off, so that the method and system will not cause unnecessary delays to the flow cell image processing to obtain base call results.

[0155] Figures 24A-24B A flow chart of an example embodiment of a method 2400 for image registration rescue of flow cell images, especially flow cell images of low diversity data, as disclosed herein is shown. The method 2400 may include some or all of the operations disclosed herein. The operations may be performed in the order described herein, but not limited to.

[0156] Method 2400 may be performed by one or more processors disclosed herein. In some embodiments, the processor may include one or more of the following: a processing unit, an integrated circuit, or a combination thereof. For example, the processing unit may include a central processing unit (CPU) and / or a graphics processing unit (GPU). The integrated circuit may include chips such as a field programmable gate array (FPGA). In some embodiments, the processor may include computing system 400.

[0157] In some embodiments, some or all of the operations in method 2400 may be performed by an FPGA. In an embodiment, when some operations are performed by an FPGA, the data after the operations are performed by the FPGA may be transmitted by the FPGA to the CPU so that the CPU may use such data to perform subsequent operations in method 2400. Similarly, data may also be transmitted from the CPU to the FPGA for processing by the FPGA. In some embodiments, all of the operations in method 2400 may be performed by the CPU. Alternatively, the operations performed by the CPU may be performed by other processors (such as a dedicated processor) or a GPU. In some embodiments, all of the operations in method 2400 may be performed by an FPGA.

[0158] In some embodiments, method 2400 is configured to align or register flow cell images across different sequencing cycles and / or different channels to a donor coordinate system. The common coordinate system can be a reference coordinate system disclosed herein. The common coordinate system can be a coordinate system of a flow cell image from one channel (e.g., a channel where the colony accounts for more than 15% of the total colony). The common coordinate system can be predetermined.

[0159] The flow cell image can be acquired from one of 1, 2, 3, 4 or more channels using the imager 116 disclosed herein. Each flow cell image can include one or more tiles (imaging areas), and each tile can be divided into multiple sub-tiles. Each sub-tile can include multiple communities. Each sub-tile can include multiple areas, each of which includes several communities. For example, communities can be extracted from corresponding areas of flow cell images of 4 different channels in a cycle. As another example, communities can be extracted from flow cell images from a single channel. The flow cell image as disclosed herein can be as follows: Figure 1 An image of at least a portion of flow cell 112 is shown.

[0160] In some embodiments, method 2400 is performed during cycle N that is different from the reference cycle. In some embodiments, method 2400 is performed during cycle N, while sequencing and image acquisition in subsequent cycles (e.g., cycle N+1) are being performed or have not yet been performed. In some embodiments, method 2400 is performed simultaneously with the sequencing run to advantageously shorten the total time for sequencing and preliminary analysis. In some embodiments, method 2400 can be performed after the sequencing run is completed. The template image can be generated in the reference cycle, and the community of one or more channels in the reference cycle can be included in the template image in the reference coordinate system, while the base call of cycle N has not yet been performed. In some embodiments, cycle N is the current cycle. N can be any non-zero integer. For example, for short read sequencing, N can be any integer from 1 to 150.

[0161] In some embodiments, the image from the cycle with low diversity data can be image registration rescued based on the fiducial marker.The fiducial marker can be outside the flow cell.In some embodiments, the fiducial marker is distributed in a predetermined pattern to predetermine the size of the mark, the distance between the marks and the intensity of the mark.In some embodiments, the fiducial marker can be randomly distributed on the flow cell, for example, as a coating.The flow cell image can be obtained from different channels with the same fiducial marker, but without any community, for example, the flow cell image of the test target.In some embodiments, the fiducial marker can include an intensity level comparable to the community intensity to simulate the presence of the community.The image of such fiducial marker can be used as a supplement to the flow cell image obtained during the sequencing run, and can be used for the registration of the flow cell image and the registration rescue and / or the calibration of the imager 116.In some embodiments, such images of fiducial markers can be used to register the offset or other transformations across different channels within the flow cycle, and such offsets and transformations can be kept consistent in different flow cycles (for example, several cycles that are continuous with each other in the sequence run). For example, in immediately adjacent loops (eg, loops 50 to 55), the offset and transformation between channel 1 and channel 2 may be considered the same.

[0162] When the selected region is low diversity data, the image registration rescue operation in method 2400 may be performed. Image registration rescue may be performed on images from a cycle with low diversity data based on fiducial landmarks.

[0163] In some embodiments, the fiducial marker is only used to generate additional images in the cycle, so as to register images across different channels, for example, in the reference cycle or in the first 1-5 cycles. In an embodiment, the fiducial marker appears in the flow cell image of one or more cycles together with the colony and the cluster, and can be distinguished from the colony or the cluster. In some embodiments, when it is not necessary to register or register rescue across channels in some cycles, images with only fiducial markers but not colonies are not obtained in such cycles. In some embodiments, when it is not necessary to register or register rescue across channels in some cycles, the fiducial marker may still appear in the flow cell image with colonies and clusters. In some embodiments, images with fiducial markers (for example, test targets or flow cells with beads) are obtained in one or more cycles that are not reference cycles. In some embodiments, images with fiducial markers are obtained in one or more cycles before any cycle of sequencing operation. In some embodiments, images with fiducial markers (for example, test targets or flow cells with beads) are obtained in one or more cycles where data diversity is low.

[0164] In some embodiments, rather than using fiducial markers for inter-channel registration, the image registration information from the previous cycle or any rescue cycle can be used to register images of channels with low diversity data (e.g., channels with less than 10% of the total population in all channels). Using the image registration information from the previous cycle to image the fiducial markers in a reference cycle, a low diversity cycle, or in a fixed pattern every few cycles can reduce the total imaging time and data to be processed, while still achieving accurate and reliable image registration results using the methods herein, compared to acquiring additional images with fiducial markers in each cycle.

[0165] Image registration of images across channels can be 2D and can include translation, scaling, rotation, and / or shearing of flow cell images across different channels. Various methods can be used to register flow cell images from multiple channels based on such external fiducial markers, such that a marker with image intensity I centered at position (x1, y1) is located at position (xr, yr) with intensity I in a reference coordinate system, where And Mr is the transformation matrix. Similarly, the inverse transformation matrix Mr can also be determined -1 , so that In some embodiments, multiple fiducial markers may be used to determine the transformation matrix Mr, similar to equations (1)-(3). In some embodiments, the multiple fiducial markers need to be at least 3 marks.

[0166] In some embodiments, image registration rescue can be performed based on a flow cell with beads or a flow cell with external fiducial markers. Fig.21 The flow cell images acquired in channel 4 of the image may have additional fiducial markers of a specific shape, size, or pattern. Registration of images with low signal levels or low colony counts (less than 10% of the total colony count) across channels or reference coordinate systems can be rescued directly using registration of the same external fiducial markers from different channels, such as by image registration method 500. When the flow cell images are acquired in the absence of any fiducial markers, image registration rescue can be performed using method 2400.

[0167] In some embodiments, method 2400 may include operation 2410, i.e., obtaining a first flow cell image from a first channel in a first flow cycle and obtaining a second flow cell image from a second channel in a first flow cycle. In some embodiments, the flow cell images, e.g., the first flow cell image and the second flow cell image, are obtained from the imager 116 of the sequencing system 110. In some embodiments, the first flow cell image is obtained in cycle N. In some embodiments, the number of colonies in the first flow cell image may be less than 1%, 2%, 5%, 10%, or 15% of the total number of colonies in the flow cell images obtained in all channels in the first flow cycle. Therefore, the sample of the first flow cycle (e.g., cycle N) has low nucleotide diversity. For example, the first flow cell image from the first channel may correspond to nucleotide A, and the total number of A is less than 10% of the total number of A, T, C, and G in cycle N. The number of colonies in the second flow cell image may be more than 1%, 2%, 5%, 10%, or 15% of the total number of colonies in the flow cell images obtained among all channels in the first flow cycle. For example, the second flow cell image may correspond to nucleotide T, and the total number of Ts in this cycle is greater than 20% of the total number of A, T, C, and G (i.e., the total number of populations) in cycle N. The first flow cell image may have a lower population number or signal level than one or more flow cell images from other channels in the same flow cycle. Due to the lower population number and lower signal level in the first flow cell image, direct image registration of the first flow cell image to one or more template images or another image (e.g., the second flow cell image) using conventional methods may be inaccurate and unreliable.

[0168] In certain embodiments, flow cell images, for example, the first flow cell image and the second flow cell image of this article, belong to samples comprising nucleotide acid template molecules. The sample can be fixed on a solid phase carrier disclosed herein, for example, a flow cell or a flow cell with beads. The first flow cycle can be in cycle 1 to cycle 30 of a sequencing run. The first flow cycle can be in cycle 2 to cycle 200 of a sequencing run. The first flow cycle can be in cycle 2 to the last cycle of a sequencing run, for example, in cycle 400.

[0169] In some embodiments, the carrier can be part of a flow cell as disclosed herein, and the sample can be fixed on the flow cell. In some embodiments, the carrier can be part of a flow cell with beads. The flow cell with beads can include a surface coated with fluorescent beads, and the fluorescent beads are chemically fixed to the surface. In some embodiments, the fluorescent beads are randomly distributed on the surface. In some embodiments, the fluorescent beads are distributed on the surface in a repeating pattern. Similar to the test targets disclosed herein, the fluorescent beads can provide a fiducial marker that enables image registration across different channels in the same flow cycle. In some embodiments, the fluorescent beads can provide a fiducial marker that enables image registration across different channels. In some embodiments, the fluorescent beads include one, two, three, four, five or six different types of beads, and the beads emit different colors in response to light excitation. In some embodiments, the fluorescent beads emit fluorescence of one or more wavelengths in response to light excitation. The fluorescent beads emit fluorescence of multiple wavelengths in response to light excitation, and each wavelength in the multiple wavelengths corresponds to a different channel in all channels.

[0170] In some embodiments, operation 2410 may include an operation of providing a nucleic acid template molecule fixed on a carrier. In some embodiments, each nucleic acid template molecule may include at least a first insertion sequence and a first sample index sequence. The first sample index sequence may include a first universal sample index sequence, and the first universal sample index may identify a sample source of the insertion sequence.

[0171] In some embodiments, operation 2410 may include an operation for performing a sequencing reaction of one or more flow cycles. Such a sequencing reaction may be performed by a sequencing system (e.g., Figure 1 100) is performed. The sequencing reaction may be performed on the first sample index sequence before the insertion sequence is subjected to one or more cycles of sequencing reaction, so as to generate a flow cell image comprising a first flow cell image and a second flow cell image in the first flow cycle. The sequencing reaction may be performed on the insertion sequence before the index sequence is subjected to one or more cycles of sequencing reaction, so as to generate a flow cell image comprising a first flow cell image and a second flow cell image in the first flow cycle. In some embodiments, the first flow cycle may be performed on the first sample index sequence of each nucleic acid template molecule. In some embodiments, the first flow cycle may be performed on the first insertion sequence of each nucleic acid template molecule.

[0172] In some embodiments, operation 2410 may include an operation of generating a flow cell image comprising a first flow cell image, a second flow cell image, or both by performing one or more cycles of sequencing reactions on a plurality of nucleic acid template molecules fixed on a carrier. Performing one or more cycles of sequencing reactions may include: contacting a plurality of nucleotide acid template molecules with a plurality of nucleotide reagents, the plurality of nucleotide reagents comprising a mixture of different types of nucleotide bases A, G, C, and T / U. A separate nucleotide reagent may include different detectable color labels corresponding to each different type of nucleotide base. In some embodiments, performing one or more cycles of sequencing reactions may include: contacting a plurality of nucleotide acid template molecules with a mixture of a plurality of sequencing primers, a plurality of polymerases, and a plurality of different types of affimers. A separate affimer in the mixture may include a core connected with a plurality of nucleotide arms, and each arm of the separate affimer includes a nucleotide base of the same type. In some embodiments, performing one or more cycles of sequencing reactions includes: in each cycle in one or more cycles, imaging an optical color signal emitted by a nucleotide reagent bound to a plurality of template molecules. Imaging an optical signal may be performed by an optical system, for example, an imager 116 disclosed herein. In some embodiments, performing one or more cycles of a sequencing reaction may include: in each of the one or more cycles, acquiring a flow cell image comprising optical color signals emitted from nucleotide reagents bound to a plurality of template molecules.

[0173] In some embodiments, the flow cell image comprises optical signals emitted from nucleotide reagents bound to an unbalanced diversity of nucleotide bases A, G, C, and T / U in a plurality of nucleic acid template molecules immobilized on a carrier in one or more cycles. In some embodiments, the plurality of colonies comprises an unbalanced diversity of nucleotide bases A, G, C, and T / U, and wherein the unbalanced diversity comprises: (1) a percentage of the number of one or more types of nucleotide bases relative to (2) a total number of nucleotide bases, and in the cycle N, the percentage is less than 20%, 15%, 10%, or 5%. The plurality of colonies corresponds to the plurality of nucleic acid template molecules.

[0174] In some embodiments, operation 2410 may include providing a cell sample having a plurality of concatemer molecules immobilized on a carrier, wherein each concatemer molecule corresponds to a target RNA of the cell sample.

[0175] In some embodiments, operation 2410 may include an operation of generating a flow cell image (e.g., including a first flow cell image, a second flow cell image, or both) by performing one or more cycles of sequencing reactions on a plurality of concatemer molecules immobilized on a carrier by a sequencing system. Performing one or more cycles of sequencing reactions may include: contacting the plurality of concatemer molecules with a plurality of nucleotide reagents, the plurality of nucleotide reagents including a mixture of different types of nucleotide bases A, G, C, and T / U. Performing one or more cycles of sequencing reactions may include: contacting the plurality of concatemer molecules with a mixture of a plurality of sequencing primers, a plurality of polymerases, and a different type of affimers.

[0176] The individual affimers in the mixture comprise a core to which a plurality of nucleotide arms are attached, and each arm of the individual affimers comprises a nucleotide base of the same type. Performing one or more cycles of sequencing reactions may comprise: in each of the one or more cycles, imaging, by an optical system, an optical color signal emitted from a nucleotide reagent bound to a plurality of concatemer molecules. In some embodiments, performing one or more cycles of sequencing reactions may comprise: in each of the one or more cycles, acquiring, by an optical system, a flow cell image comprising an optical color signal emitted from a nucleotide reagent bound to a plurality of concatemer molecules.

[0177] In some embodiments, the method 2400 may include an operation 2420 of obtaining a first rescue image from a first channel in a rescue flow cycle and obtaining a second rescue image from a second channel in a rescue flow cycle.

[0178] The number of colonies in each of the first rescue image and the second rescue image may exceed 1%, 2%, 5%, 10% or 15% of the total number of colonies in the flow cell images acquired in all channels in the rescue flow cycle. In other words, the first rescue image and the second rescue image are acquired in channels corresponding to nucleotides with high nucleotide content. For example, the first rescue image and the second rescue image correspond to A and T, respectively, and the number of A or T in each rescue image may exceed 15%, 18% or 20% of the total number of colonies among all different channels.

[0179] The first flow cycle may be different from the rescue flow cycle. In some embodiments, the rescue flow cycle is a cycle completed before the first flow cycle. In some embodiments, the rescue flow cycle is completed immediately before the first flow cycle, such that the image registration across different channels is substantially the same in the first flow cycle and the rescue cycle. In some embodiments, the first flow cell image and the second flow cell image belong to a sample fixed on a carrier, and the first rescue image and the second rescue image belong to the same sample fixed on the carrier.

[0180] In some embodiments, the rescue cycle can be before cycle 1 of the sequencing run, and the first rescue image and the second rescue image are of a test target external to the sample on the carrier. In some embodiments, the first rescue image and the second rescue image are of a test target in the absence of a sample fixed on the carrier. The test target can include a fiducial marker. The fiducial marker can emit a fluorescent signal that can be detected in all channels during the rescue flow cycle.

[0181] The test target may include a coating of a predetermined geometric shape or pattern that is repeated in space. The predetermined geometric pattern or shape may be repeated in one or two dimensions. In some embodiments, the test target lacks a flow cell and a liquid. In some embodiments, the test target includes one or more substrates having a predetermined refractive index. The test target may include a top substrate having a predetermined refractive index. In some embodiments, the test target may include a bottom substrate. At least a portion of the first substrate or the second substrate includes a coating having a predetermined geometric pattern or shape. In some embodiments, the thickness of the top substrate or the bottom substrate is configured to simulate the presence of a first assumed flow cell. In some embodiments, the thickness of the top substrate is configured to allow imaging of the bottom surface of the first channel of the assumed first flow cell. The predetermined geometric shape or pattern of the coating may include an optically opaque portion and a transparent portion.

[0182] Fig.25 Non-limiting examples of test targets here are shown. The left schematic diagram shows an example solid test target, which has a first substrate (top) and a second substrate (bottom) and an opaque layer between the first substrate and the second substrate. The opaque layer can be coated on the bottom surface of the first substrate or the top surface of the second substrate. The opaque layer forms a micro pattern. The first substrate is transparent, which allows light to be transmitted from its bottom surface and see the micro pattern. The test target lacks a flow cell and a liquid. The thickness of the first substrate is adjusted to simulate the existence of a hypothetical flow cell containing a fluid / liquid, wherein the hypothetical flow cell can be located between the first substrate and the second substrate. For example, the first substrate is thicker and has an additional thickness. The right schematic diagram shows a hypothetical flow cell, which includes a channel with a thickness of [T-channel], and the channel contains a fluid / liquid with a refractive index of [n-fluid]. The test target can be positioned on a sequencing system to perform or facilitate sequencing analysis, such as image registration or image registration rescue, by obtaining and analyzing images of fluorescent beads.

[0183] In some embodiments, the flow cell disclosed herein may include a carrier disclosed herein. The carrier may be solid phase. At least a portion of the carrier may be transparent. The carrier may include one or more substrates. At least a portion of the one or more substrates may be transparent. Fig.26An example embodiment of a flow cell 900 is shown. The flow cell 900 includes a carrier 901 and other elements, such as a coating. The carrier can include a top substrate 910 and a bottom substrate 910. Each substrate 910 can have a predetermined thickness, and different substrates can have different thicknesses. The substrate can define one or more channels 920 of the device 900. The channel can allow a fluid (e.g., a liquid or a gas) to flow therethrough. The flow cell can include one or more inlets 920 and one or more outlets 930 in one or more substrates 910. Fig.26 An example device 900 is shown in which two substrates form two channels, and each channel has an inlet and an outlet. However, in other embodiments, the number of substrates, channels, inlets and outlets may be different. In some embodiments, the number of substrates, channels, inlets and outlets may be any integer greater than 0. Fig.26 An example device 900 is shown having two planar substrates, the surfaces of which have no curvature. However, the substrates need not be planar.

[0184] In some embodiments, one or more channels 920 can extend from an inlet 930 to an outlet 940 such that fluid can flow from the inlet 930 through the one or more channels 920 to the outlet 940. As an example, sequencing reagents can be introduced into the flow cell through the inlet, flow through the channel, and then exit from the outlet. The channel 920 can include a top inner surface 921 and a bottom inner surface 922. One or more of the surfaces can be coated with a fiducial marker, e.g., a fluorescent bead.

[0185] Benchmarks (e.g., fluorescent beads) can be fixed to the surface. Fluorescent beads can be covalently or chemically fixed to the surface. Fluorescent beads can be fixed to or fixedly attached to the surface by forming coatings 950, 951 on surfaces 921, 922 so that fluorescent beads remain fixed (fixed) or fixed (immobilized) relative to surfaces 921, 922. Coatings 950, 951 can be directly applied to internal surfaces 921, 922 and contact the internal surfaces. Alternatively, coatings 950, 951 can be indirectly applied to internal surfaces 921, 922 or not directly contact the internal surfaces. In some embodiments, coatings 950, 951 can be applied when there are certain compounds between surfaces 921, 922 and coatings 950, 951. For example, coatings 950, 951 can be applied on another coating that is directly applied to surfaces 921, 922 and contacted with the surface. In some embodiments, the surface is passivated by another coating (not shown). Such another coating can immobilize surface capture primers, nucleic acid template molecules, or both for capturing polynucleotides on the surface 921, 922. In some embodiments, the surface 921, 922 comprises polynucleotides captured thereon.

[0186] In some embodiments, the coating 951, 952 to which fluorescent beads are attached can be mixed with one or more other coatings so that the mixed coating can be directly applied to the interior surface 921, 922 and contact the interior surface. The mixed coating can fix the fluorescent beads on the surface. In addition, the mixed coating can also fix surface capture primers, nucleic acid template molecules, or both for capturing polynucleotides on the surface 921, 922. In some embodiments, the fixed coating can capture polynucleotides on the surface 921, 922, and the application of sequencing reagents can facilitate the use of various sequencing methods (e.g., sequencing by affinity) to sequence the polynucleotides disclosed herein.

[0187] In some embodiments, the flow cell 900 can be used on a sequencing system for DNA sequencing. The flow cell 900 can receive various sequencing reagents through an inlet 930 before a sequencing cycle, and allow the reagents to flow through one or more channels 920 and exit through an outlet 940. In some embodiments, during or after the application of sequencing reagents to the flow cell 900, the fluorescent beads remain fixed relative to the surfaces 921, 922.

[0188] In some embodiments, the fluorescent beads are chemically fixed to the surface. In some embodiments, the fluorescent beads are covalently fixed to the surface. In some embodiments, the fluorescent beads are pre-activated to enable chemical attachment to the surface. In some embodiments, the fluorescent beads are pre-activated to enable covalent attachment to the surface.

[0189] In some embodiments, the sequencing system 110 is used to simultaneously image the polynucleotide clusters or communities and fluorescent beads captured thereon in one or more sequencing cycles. In some embodiments, the flow cell image of the flow cell 900 obtained may include signals from fluorescent beads and signals from clusters or communities of polynucleotides. Because the fluorescent beads are fixed to the surface, their positioning relative to the surface remains fixed, and even if the polynucleotides are captured on the surface, they may move relative to the surface. The movement of the polynucleotides in different flow cell images may cause base call errors, thereby requiring image registration before base call. Fixed fluorescent beads can be used as fiducial markers for registering or aligning clusters or communities across different cycles and / or different channels, and eliminate the need for additional fiducial markers (such as optical test targets disclosed herein) outside the flow cell device. In some embodiments, the same fluorescent beads can be used to determine the transformation between two different flow cell images in two different images across cycles and / or channels, and the transformation can be applied to the positioning of clusters or communities to perform image registration of clusters or communities. The transformation may include one or more transformation matrices for some regions or the entire flow cell image. In embodiments where at least some clusters or populations move across cycles and / or channels, the flow cells disclosed herein can advantageously facilitate image registration of flow cell images and allow for more accurate and more reliable base calls following image registration.

[0190] In some embodiments, clusters or colonies and fluorescent beads can be imaged in different cycles. For example, clusters or colonies can be imaged during the bright cycle, while fluorescent beads can be imaged in the dark cycle.

[0191] In some embodiments, clusters or colonies and fluorescent beads can be imaged in the same cycle. For example, clusters, colonies, and fluorescent beads can be imaged simultaneously in the first cycle and the Nth cycle, where N can be any integer greater than 1. Since the fluorescent beads remain fixed to the surface, the change in the positioning of the fluorescent beads between cycles can be used to determine movement that can be attributed to sources external to the sequencing system and the flow cell device, such as movement of the platform, optical elements, etc. The positioning of the clusters or colonies relative to the fluorescent beads can be used to determine movement due to the movement of the clusters or colonies on the surface, so that the clusters or colonies in the first and nth cycles can be aligned / registered without being affected by movement due to external causes of the flow cell device.

[0192] In some embodiments, clusters or colonies and fluorescent beads can be imaged in the same channel of the same or different cycles so that image transformations caused by differences in elements of the sequencing system or other cross-cycle variances can be determined when aligning / registering clusters or colonies in different flow cell images. In some embodiments, clusters or colonies and fluorescent beads can be imaged in different channels of the same or different cycles so that image transformations can account for transformations across channels alone or in combination with transformations across cycles.

[0193] In some embodiments, the flow cell 900 with fluorescent beads can be used alone or in combination with the test targets disclosed herein for image registration. For example, the test target can be used to register images from different channels within the same cycle, and the fluorescent beads can be used to register images from different cycles but the same channel. As another example, a solid-state optical test target can be used to register images from a reference cycle to a template coordinate system, and the fluorescent beads can be used to register images acquired from all channels in cycles other than the reference cycle to the template coordinate system.

[0194] In some embodiments, method 2400 includes operation 2430 of determining a first plurality of transformations of the first flow cell image. Each transformation in the first plurality of transformations may correspond to a sub-tile of the first flow cell image and may be configured to register or align the corresponding sub-tile to a predetermined coordinate system (e.g., a reference coordinate system) or a coordinate system of another flow cell image from a different channel.

[0195] Operation 2430 may be performed by a processor as disclosed herein.Determination of the first plurality of transforms may be based on one or more of: a second plurality of transforms of the second flow cell image; a third plurality of transforms of the first rescue image; and a fourth plurality of transforms of the second rescue image.

[0196] In some embodiments, operation 2430 may include determining each transformation corresponding to one or more selected regions in the sub-tile of the first flow cell image.

[0197] One or more regions can be selected from the sub-tiles of the flow cell image. Each selected region in the one or more selected regions can have various sizes not greater than the corresponding sub-tiles, for example, each selected region can be 128x 128, 96x 96, 64x 64 or other various sizes. For example, the region can be selected as the central pixel of the enclosed sub-tile. For images from cell samples or other various samples unevenly distributed above the FOV, the region of each sub-tile can be selected at different positions in the sub-tile. In some embodiments, the region is selected to ensure that there are at least a threshold number of cells or colonies in the selected region. For example, operation 510 can include an operation to determine the number of cells and / or colonies in the region. The region including the central pixel, for example, 128x 128, can be selected as a default. In response to determining that the central region does not include cells and / or colonies that meet a predetermined threshold, method 500 may include an operation to move the selected region until an area that meets the predetermined threshold is found. The selected region may be located at the same position across different color channels and / or cycles. The predetermined number of colonies or clusters can be 20, 40, 60, 80 or more. The predetermined number of cells may be 1, 2, 5, 8, 10 or more.

[0198] The area of ​​each sub-tile can be selected at different positions of the sub-tile. In some embodiments, based on various criteria, such as maximum signal variance, highest average signal strength or maximum signal strength standard deviation, etc., the area is selected in the selected area of ​​multiple candidate areas in the same sub-tile. The area selected with various criteria (e.g., larger signal variance) may be more likely to contain stronger signals of colonies and / or cells than other areas with lower signal variance, and may be more convenient for image registration than other areas. In some embodiments, some or all possible options for selecting the area in the sub-tile are included as candidate areas. For example, for a 100x 100 area size, a sliding window across all rows and columns of the sub-tile can provide all possible options for selecting a 100x 100 area. In some embodiments, the selected area can be located at different positions of different sub-tiles. For the same sub-tile in different cycles or across different color channels, the selected area can remain the same. In some embodiments, each sub-tile can include one or more selected areas. Two different sub-tiles can include different numbers of selected areas. The size of the selected area can be the same or different. For example, sub-tile 1 may have a single selected region including its center pixel, sub-tile 2 may have 3 selected regions, none of which include any center pixel, and the sizes of the 3 selected regions may be different. Such selection based on a predetermined threshold may also be used for 2D samples with sparse populations or clusters within the sub-tile (e.g., less than 100 populations in a 128 x 128 area) or weak signals within the sub-tile (e.g., less than 10%, 20%, or other percentages of the average signal intensity compared to another area).

[0199] Selecting regions within each subtile can help ensure that the Fourier transform of the selected region contains at least some signal from the community and not just background and / or noise.

[0200] The region may be of various two-dimensional shapes, such as a rectangle, a circle, or a square. As a non-limiting example, the selected region may include one or more central pixels of the sub-tile. Figure 2 A non-limiting example of a selected region 230 is shown. Region 230 may include Figure 2 The plurality of colonies 232 in the flow cell image can be selected. The size of the region can be determined to balance the trade-offs between different factors, including but not limited to computational complexity, time consumption, and accuracy of image registration. For example, selecting a 64x 64 region may be computationally simpler than selecting a 128x 128 region, but may not be as accurate. The colony can be included in a tile or sub-tile of the flow cell image. In some embodiments, the colony can be from one or more selected regions of a sub-tile in the first flow cell image.

[0201] Once a region is selected in the first flow cell image, a corresponding region can be determined in the second flow cell image, the first rescue image, and the second rescue image. The corresponding region can be determined in the second flow cell image, and the first rescue image and the second rescue image can be located at the same coordinates of the corresponding image. The selected region can be used to represent its corresponding sub-tile, but with a smaller size to reduce the computational time and cost of image registration and image registration rescue, while still achieving accurate and reliable registration and / or registration rescue. In some embodiments, one or more selected regions can be selected to cover the entire sub-tile, but there is a tradeoff of increased computational time and cost.

[0202] In some embodiments, operation 2430 may include an operation of determining a corresponding displacement d2(i,j), d3(i,j), or d4(i,j) for each sub-tile of the second flow cell image, the first rescue image, and the second rescue image, respectively. The coordinate (i,j) may indicate that the displacement d2(i,j) is located at the sub-tile at the i-th row and j-th column of the flow cell image, where i may be any number from 1 to the total number of rows, and j may be any number from 1 to the total number of columns, and where d2, d3, or d4 each collectively represent all possible displacements d2(i,j), d3(i,j), or d4(i,j), respectively. The corresponding displacement may have a sub-pixel resolution, for example, a 0.05 or 0.1 pixel resolution. The corresponding displacement d2(i,j), d3(i,j), or d4(i,j) may be 2D or 3D. The corresponding displacements d2d2(i,j), d3(i,j) or d4(i,j), d3, d4 may be determined based on corresponding areas in the second flow cell image, the first rescue image, and the second rescue image.

[0203] Various methods may be used to determine the spatial displacement of the image relative to the reference, or more specifically, the displacement of the same group of communities in one or more selected areas of the second flow cell image, the first rescue image, and the second rescue image relative to the corresponding template image or reference coordinate system. For example, method 2400 may include an operation of determining the corresponding cross-correlation of one or more selected areas of the corresponding sub-tiles of the second flow cell image, the first rescue image, and the second rescue image with the template image. One or more template images are disclosed herein with respect to method 500. For example, one or more template images are 2D and their size may be approximately the same as the size of the corresponding sub-tile. The example flow cell may have 54 template images, each template image corresponding to a sub-tile of the same size as the corresponding sub-tile. One or more template images may include communities from all channels, so that at least some of the same communities in the selected areas of the second flow cell image, the first rescue image, and the second rescue image are included in the template image for alignment and registration. Each 2D displacement that optimizes (e.g., maximizes) the cross-correlation between corresponding sub-tiles containing at least some of the same communities may be determined as a 2D displacement of the corresponding sub-tile. Each sub-tile may have its corresponding 2D displacement. An example flow cell may have 54 displacements, eg, each of d2, d3, and d4 is a matrix with 54 entries.

[0204] As another example, method 2400 may include an operation of determining a spatial displacement (e.g., a 2D displacement) by generating a Fourier transformed image (FTI) of the second flow cell image and one or more template images, and determining a 2D displacement of a corresponding sub-tile in the frequency domain based on the FTI.

[0205] In some embodiments, operation 2430 may include an operation of determining a second plurality of transformations of the second flow cell image, each transformation corresponding to a sub-tile of the second flow cell image. An individual transformation may be based on a corresponding displacement d2 of one or more adjacent sub-tiles thereof. Similarly, operation 2430 may include determining a third plurality of transformations of the first rescue image based on a corresponding displacement d3 of one or more adjacent sub-tiles; and determining a fourth plurality of transformations of the second rescue image based on a corresponding displacement d4 of one or more adjacent sub-tiles.

[0206] Details of determining the transformation of a sub-block or any region of an image based on displacements of neighboring sub-blocks or regions using equations (1) to (3) are disclosed herein with respect to method 500 .

[0207] In some embodiments, operation 2430 may include determining the first plurality of transforms (eg, d) based on the second, third, and fourth plurality of transforms (eg, d2, d3, and d4).

[0208] In some embodiments, each of the plurality of transformations disclosed herein, e.g., the first, second, third, or fourth plurality of transformations, comprises one or more nonlinear transformations. In some embodiments, each of the plurality of transformations comprises one or more linear transformations. In some embodiments, each of the plurality of transformations comprises one or more affine transformations. In some embodiments, each of the transformations disclosed herein is an affine transformation.

[0209] In some embodiments, operation 2430 may include determining each transform T1_i in the first plurality of transforms based on corresponding transforms T2_i, T3_i, and T4_i in the second, third, and fourth pluralities of transforms as follows:

[0210] (T1_i) -1 =(T2_i) -1 ·T4_i·(T3_i) -1

[0211] Where i is from 1 to the total number of sub-tiles, and where T1_i, T2_i, T3_i and T4_i are nxn transformation matrices, where n is an integer. For example, n=3.

[0212] When it may not be possible to directly determine the transformation (i.e., T1_i) using its corresponding displacement, for example, using equations (1)-(3), method 2400 can be used to accurately and reliably indirectly determine such transformations. For example, the first flow cell image may have fewer signal spots, i.e., less than 10% of the total community, so that directly determining the transformation may include errors. On the contrary, the transformation can be indirectly determined based on other images with more signal spots and their relationship with other images. The transformations T2_i, T3_i and T4_i with a high nucleotide portion and therefore a higher diversity can be determined in a similar manner as disclosed herein about method 500, for example, using equations (1)-(3). In other words, when the diversity of sample nucleotide acid molecules in the flow cycle is low, some channels may cause traditional image registration failure due to low community / signal levels caused by low diversity data. The failed image registration of the first flow cell image can be rescued by indirectly deriving the transformation from other images with sufficient signal levels to generate a reliable image registration. Image registration herein may be with respect to any commonly shared coordinate system or image, such as a template image. The method 2400 may be used to rescue image registration that may fail in various regions, and is not limited to the rescue of registration of low diversity data.

[0213] In some embodiments, method 2400 may include one or more operations during one or more reference cycles. In some embodiments, such operations may include acquiring a reference flow cell image by imager 116 in one or more reference flow cycles. Such operations may further include determining the coordinates of a colony in a reference coordinate system, wherein the colony is obtained from the reference flow cell image in one or more reference flow cycles. In some embodiments, method 2400 may include an operation of generating one or more template images in a reference coordinate system by registering the colony to one or more template images using the coordinates of the colony. One or more template images may be used in subsequent operations (e.g., 2430) for determining a transformation of an image (e.g., a first flow cell image) having a low colony / signal level.

[0214] In some embodiments, operation 2430 may include determining the coordinates of a colony in one or more of the following: a first flow cell image, a second flow cell image, a first rescue image, and a second rescue image. A list of colony coordinates may also be used to determine displacement, rather than using an image or an area of ​​an image to determine displacement. The coordinates of a colony may be in a reference coordinate system, or in a commonly shared coordinate system among a first flow cell image, a second flow cell image, a first rescue image, a second rescue image, or a combination thereof. The coordinates of a colony may be two-dimensional (2D) or three-dimensional (3D). The coordinates of a colony may be used to determine a spatial displacement between two images, for example, a 2D displacement, where the same colony will appear in two images, and due to the displacement between the two images, the same colony is optionally located at different coordinates.

[0215] In some embodiments, method 2400 may include an operation of transmitting the first plurality of transformations to a processing unit. For example, the first plurality of transformations may be determined by an FPGA to improve the computational speed relative to a general-purpose CPU, and the transformations (e.g., affine matrices) may be transmitted back to the processing unit for subsequent processing steps. In some embodiments, the processing unit is a central processing unit (CPU). In some embodiments, method 2400 may include an operation of extracting image intensities of colonies from a first flow cell image, for example, by a CPU based on the first plurality of transformations.

[0216] In some embodiments, method 2400 may include an operation of performing base calling using image intensities of a population of the first flow cell image based on the first plurality of transformations. Base calling may be performed after image registration rescue using the first plurality of transformations, such that the accuracy and reliability of base calling may be improved compared to conventional methods, especially in the case of low nucleotide diversity data. For example, after image registration rescue of low diversity data using the methods disclosed herein, index unassignment rate and mismatch rate may be improved. Figure 22-23The improved low diversity index unassignment rate and mismatch rate using the image registration rescue method 2400 are shown. Fig. 22 In the figure, the points above the diagonal indicate the improved index assignment rate. For low-diversity patches (green points), 24 out of 142 patches (about 17%) are improved, and 9 out of these 24 patches have wrong output without registration rescue. Various high-diversity patches also have some improvements compared to the assignment rate without image registration rescue. Fig.23 Green points below the diagonal line are shown, which have improved index mismatch rates. As shown by the points on the diagonal line, the image registration rescue 2400 does not cause negative interference to the high diversity data tiles in terms of index assignment or mismatch rate.

[0217] The unassigned rate can be the fraction of populations that do not match any mapped index sequence and / or the fraction of populations that match two mapped index sequences of two or more different samples. In other words, within the tolerance, the fraction of populations whose index sequence does not match the reference index sequence of a single sample relative to the total number of populations.

[0218] The distribution rate can be the fraction of populations whose index sequence matches the mapped index sequence of a single sample. In other words, the fraction of populations that match the reference index sequence of a single sample relative to the total number of populations within the tolerance.

[0219] The mismatch rate can be the fraction of populations that match (within the tolerance) the reference index sequence of a single sample but have some base mismatches relative to the total number of populations.

[0220] The match rate can be the fraction of populations that match the reference index sequence of a single sample and are error-free relative to the total number of populations.

[0221] In some embodiments, after determining the first plurality of transformations in operation 2430, method 2400 may include an operation of registering the sub-tiles of the first flow cell image using the first plurality of transformations. In some embodiments, registering the sub-tiles of the first flow cell image includes an operation of registering each sub-tile of the first flow cell image to a coordinate system (e.g., a reference coordinate system) using a corresponding transformation of the first plurality of transformations. Such registration is equivalent to registering the first flow cell image to other images in a predetermined coordinate system, such as one or more template images, a second flow cell image, or a community map.

[0222] In certain embodiments, method 2400 includes determining whether the number of colonies in the first flow cell image meets the operation of a predetermined threshold. In response to determining to meet the predetermined threshold, image registration rescue can be turned on. Image registration rescue result can replace any pre-existing image registration result of the first flow cell image. In certain embodiments, a custom predetermined threshold can be run for various sequencing. For example, a predetermined threshold can be the scope of the number of colonies in the flow cell image in a specified area, such as being less than 100, 200, 400, 500, 600 or 800 colonies in a 128 pixel x 128 pixel area of ​​a flow cell image of 5472 pixels x 3648 pixels. In certain embodiments, a predetermined threshold can be an unassigned rate and / or mismatch rate of colonies in a flow cycle. In certain embodiments, a predetermined threshold can be a certain quantity or scope of image quality (such as signal-to-noise ratio or contrast-to-noise ratio).

[0223] In some embodiments, the processor herein includes one or more processing units; one or more integrated circuits; or a combination thereof. In some embodiments, the processor includes: one or more central processing units (CPUs); one or more field programmable gate arrays (FPGAs); or a combination thereof.

[0224] In some embodiments, method 2400 includes the operation of performing, by a processor, one or more pre-processing steps on a flow cell image, eg, a first flow cell image, a second flow cell image, a first rescue image, a second rescue image, or a combination thereof.

[0225] In some embodiments, the operation of performing one or more pre-processing steps can be performed by FPGA. In some embodiments, the data after the operation can be transmitted by FPGA to CPU so that CPU can use such data to perform subsequent operations in method 500.

[0226] In some embodiments, preprocessing steps of one or more flow cell images in a reference cycle may be performed before operation 2430. In some embodiments, preprocessing steps of one or more flow cell images in a reference cycle may be performed after operation 2410 of obtaining a flow cell image. In some embodiments, one or more preprocessing steps of a flow cell image in a reference cycle may be performed before obtaining image intensity, size, shape, or a combination thereof of a colony from a plurality of sub-tiles of the flow cell image in the reference cycle.

[0227] One or more pre-processing steps may include background subtraction. Background subtraction is configured to remove at least some background signals that may interfere with the signal of interest, i.e., the image intensity of the colony. Background signals may be noise caused by multiple sources, including flow cell 112, imager 115, sequencer 114, and other sources. Background subtraction may be adjusted to avoid excessive subtraction.

[0228] One or more pre-processing steps may include image sharpening to optimize the image intensity of the colonies in view of their surroundings in the flow cell image. For example, sharpening may be performed using a Laplacian of Gaussian (LoG) filter.

[0229] One or more pre-processing steps may include intensity offset adjustment, which may remove intensity offset that was not removed during background subtraction.

[0230] One or more pre-processing steps may include color correction to remove interference to one channel from other channels or colors.

[0231] One or more pre-processing steps may include lag and lead corrections configured to correct image intensity within a particular cycle by eliminating intensity bias caused by sequencing of DNA fragments that are out of sync with other fragments due to lagging or leading.

[0232] One or more pre-processing steps may include intensity normalization to normalize image intensities of populations from different channels to be within a predetermined range.

[0233] The one or more pre-processing steps may include: background subtraction; image sharpening; or a combination thereof.

[0234] In some embodiments, method 2400 can be used for image registration rescue without any template image. Method 2400 can determine the transformation between two images in a high diversity flow cycle (e.g., a first rescue image and a second rescue image in a rescue flow cycle), and use such transformation to indirectly determine the transformation of an image in a low diversity flow cycle (e.g., a first flow cell image in a flow cycle) to another image in the same flow cycle (e.g., a second flow cell image in a flow cycle). Eliminating the need for any template image can advantageously save computation time, storage space, and reduce delays in image registration rescue of low diversity images.

[0235] In such embodiments, method 2400 may include operations 2410 and 2420 disclosed above. Fig. 24BAs shown, method 2400 may include operation 2430' instead of 2430 for determining a transformation for image registration rescue. Operation 2430' may be to determine a first plurality of transformations of the first flow cell image based on a plurality of transformations, the plurality of transformations being based on the first rescue image and the second rescue image. The plurality of transformations may be between the first rescue image and the second rescue image without using any template image. Each of the first plurality of transformations may correspond to a sub-tile of the first flow cell image and may be configured to register the sub-tile to one or more template images or the second flow cell image. In some embodiments, the first rescue image and the second rescue image may include signals / communities appearing in both images, so that the displacement between the two rescue images may be determined using the co-occurring signals / communities.

[0236] In some embodiments, the operation 2430' of determining the first plurality of transformations may include, for each pair of corresponding sub-tiles in the first rescue image and the second rescue image, determining a displacement d34 between the pair of sub-tiles. The displacement d34 may be determined using similar operations for determining other displacements d, d2, d3, and d4. The displacement d34 may be a 2D displacement, such as other displacements d2, d3, etc. The displacement d34 may be determined by calculating a cross-correlation of a selected area of ​​the corresponding sub-tile of the first rescue image with a selected area of ​​the corresponding sub-tile of the second rescue image. Similarly, as disclosed herein for d2, d3, d4, the displacement d34 may be determined by using the FTI of the selected area of ​​the corresponding sub-tile of the first rescue image and the FTI of the selected area of ​​the corresponding sub-tile of the second rescue image. For a flow cell having 54 sub-tiles, it may have 54 different displacements d34 corresponding to each of the sub-tiles.

[0237] In some embodiments, operation 2430' may include determining a transformation of a corresponding pair of sub-blocks of the first rescue image and the second rescue image based on the corresponding displacement d34 and other displacements of one or more adjacent sub-blocks. Such determination may be repeated for each corresponding pair of sub-blocks of the first rescue image and the second rescue image, thereby generating a plurality of transformations T34_i, where i is in the range of 1 to the total number of sub-blocks, and where the transformation T34 is from the first rescue image to the second rescue image. The transformation T43 may be from the second rescue image to the first rescue image. Operation 2430' may further include determining a first plurality of transformations based on a plurality of transformations. In some embodiments, operation 2430' may include determining each transformation T12_i in the first plurality of transformations based on a corresponding transformation T34_i in the plurality of transformations as follows: T12_i=T34_i or (T12_i) -1 =T43_i, where i is in the range of 1 to the total number of sub-tiles.

[0238] In some embodiments, multiple transformations (e.g., T3_i, T4_i, T34_i of the rescue cycle) and displacements (e.g., d3, d4, d34) may be pre-calculated and saved after the rescue cycle has been determined and the flow cell image has been acquired so that they can be retrieved when needed to determine T1_i or T12_i to improve the efficiency of image registration rescue.

[0239] Parameter estimation robustness

[0240] In some embodiments, one or more pre-processing steps or other image processing steps in the preliminary analysis, for example, steps involving image intensity, may be affected by low diversity data. In some embodiments, when some flow cell images in a particular flow cycle have low signal levels or low population numbers (e.g., less than 5% or 10% of the total population), one or more pre-processing steps or other image processing steps performed on such flow cell images may be inaccurate or unreliable. As a result, image processing may be erroneous, for example, color correction, intensity normalization, background subtraction, intensity offset correction, etc., and ultimately lead to errors in base calling using the processed images.

[0241] The parameter estimation method of this paper can be used to improve or remove such image processing steps that generate erroneous results. In certain embodiments, such image processing problems can be indirectly solved by using high signal level images from different channels to obtain the same image processing results (e.g., processing parameters). In certain embodiments, such image processing problems can be solved indirectly using images of the same channel but different cycles. Different cycles can be predetermined based on a custom threshold. For example, different cycles can be rescue cycles. Different cycles can include more than one cycle in which data has high nucleotide diversity. Different cycles can include (1) being close to an inaccurate or unreliable cycle with an image processing result and (2) having one or more cycles of high nucleotide diversity data. For example, if the color correction of cycle 60 fails. The color correction parameters from cycles 59, 58 or 57 can be used as its substitute. Wherein, a cycle in which a color correction does not fail can be randomly selected, or the closest cycle 59 (if its color correction does not fail) can be selected.

[0242] In some embodiments, the parameter estimation method of the present invention includes an operation of receiving one or more thresholds for image processing results. For different image processing results, the thresholds may be different. The thresholds can be customized based on various factors, including but not limited to: sequencing chemistry, characteristics of the sample being sequenced, and parameters of the optical system. In some embodiments, the parameter estimation method of the present invention includes an operation of determining whether one or more flow cell images in the first channel of the first flow cycle meet one or more thresholds. In response to determining that at least one of the thresholds is not met, the parameter estimation method of the present invention can initiate an operation of indirectly determining the image processing results of one or more flow cell images using images from channels other than the first channel in the first flow cycle. Alternatively, in response to determining that at least one of the thresholds is not met, the parameter estimation method of the present invention can initiate an operation of indirectly determining the image processing results of one or more flow cell images using images from the first channel in one or more rescue cycles. In some embodiments, the parameter estimation method of the present invention can initiate an operation of indirectly determining the image processing results of one or more flow cell images using images from all channels in one or more rescue cycles. The image processing results can be various results that can be derived from the processing of the flow cell images. For example, the image processing results can be an offset of image intensity, a color correction parameter of different images, and a normalization parameter of image intensity.

[0243] In some embodiments, the parameter estimation method can be turned on and / or off for different cycles or different channels. For example, it can be turned on only for cycles including low diversity data without introducing unnecessary delays when determining image processing results in other cycles with higher diversity data.

[0244] In some embodiments, the parameter estimation method can be executed immediately after the image processing results of the current cycle (e.g., cycle N) are generated, while the next cycle of image acquisition and / or image processing is being performed or has not yet been performed, so that the total sequencing time and preliminary analysis time can be reduced.

[0245] In some embodiments, the parameter estimation method can be performed using any computer system or processor disclosed herein (including CPUs and FPGAs).

[0246] Computer Systems

[0247] Various embodiments of methods 500 and 2400 may be implemented, for example, using one or more computer systems (e.g., Figure 4 For example, one or more computer systems 400 may be used to implement any of the various embodiments discussed herein, as well as combinations and sub-combinations thereof.

[0248] Computer system 400 may include one or more hardware processors 404. Hardware processor 404 may be a central processing unit (CPU), a graphics processing unit (GPU), or a combination thereof. Processor 404 may be connected to a bus or communication infrastructure 406.

[0249] The computer system 400 may also include a user input / output device 403, such as a display, a keyboard, a pointing device, etc. The computer system may communicate with a communication infrastructure 406 via a user input / output interface 402. The user input / output device 403 may be coupled to Figure 1 The user interface 124 in FIG.

[0250] One or more of the processors 404 may be a graphics processing unit (GPU). In one embodiment, a GPU may be a processor that is a dedicated electronic circuit designed to process mathematically intensive applications. A GPU may have a parallel structure that is very efficient for processing large blocks of data in parallel (such as mathematically intensive data common in computer graphics applications, images, videos, vector processing, array processing), as well as cryptography (including brute force cracking), generating encrypted hashes or hash sequences for some blockchain-based applications, solving partial hash reversal problems, and / or generating results for other proof-of-work calculations. With the power of general-purpose computing on a graphics processing unit (GPGPU), a GPU may be particularly useful at least in the image recognition and machine learning embodiments described herein.

[0251] Additionally, one or more of the processors 404 may include a coprocessor or other logic implementation for accelerating cryptographic calculations or other specialized mathematical functions, including a hardware-accelerated cryptographic coprocessor. Such an accelerated processor may further include an instruction set for accelerating using the coprocessor and / or other logic to facilitate such acceleration.

[0252] The computer system 400 may also include data storage devices, such as a main memory (primary memory) 408, for example, a random access memory (RAM). The main memory 408 may include one or more levels of cache. The main memory 408 may store control logic (i.e., computer software) and / or data therein.

[0253] The computer system 400 may also include one or more secondary data storage devices or secondary memories 410. The secondary memories 410 may include, for example, a primary storage drive 412 and / or a removable storage device or drive 414. The primary storage drive 412 may be, for example, a hard disk drive or a solid state drive. The removable storage drive 414 may be a floppy disk drive, a tape drive, an optical disk drive, an optical storage device, a tape backup device, and / or any other storage device / drive.

[0254] Removable storage drive 414 may interact with removable storage unit 418 .

[0255] The removable storage unit 418 may include a computer usable or readable storage device having computer software and / or data stored thereon. The software may include control logic. The software may include instructions that can be executed by the hardware processor 404. The removable storage unit 418 may be a floppy disk, a magnetic tape, an optical disk, a DVD, an optical storage disk, and / or any other computer data storage device. The removable storage drive 414 may read from and / or write to the removable storage unit 418.

[0256] Secondary storage 410 may include other means, devices, components, tools, or other methods for allowing computer system 400 to access computer programs and / or other instructions and / or data. Such means, devices, components, tools, or other methods may include, for example, a removable storage unit 422 and an interface 420. Examples of removable storage unit 422 and interface 420 may include a program cartridge and cartridge interface (such as those found in video game devices), a removable memory chip (such as an EPROM or PROM) and an associated socket, a memory stick and USB port, a memory card and an associated memory card slot, and / or any other removable storage unit and associated interface.

[0257] The computer system 400 may further include a communication or network interface 424. The communication interface 424 may enable the computer system 400 to communicate and interact with any combination of external devices, external networks, external entities, etc. (individually and collectively referenced by reference numeral 428). For example, the communication interface 424 may allow the computer system 400 to communicate with an external or remote device 428 via a communication path 426, which may be wired and / or wireless (or a combination thereof) and may include any combination of a LAN, a WAN, the Internet, etc. Control logic and / or data may be transmitted to and from the computer system 400 via the communication path 426. In some embodiments, the communication path 426 is a connection to the cloud 130, such as Figure 1 The external device or the like indicated by reference numeral 428 may be a device, a network, an entity, or the like in the cloud 130 .

[0258] The computer system 400 may also be any one or any combination of the following: a personal digital assistant (PDA), a desktop workstation, a laptop or notebook computer, a netbook, a tablet computer, a smart phone, a smart watch or other wearable device, an appliance, a part of the Internet of Things (IoT), and / or an embedded system, to name a few non-limiting examples.

[0259] It should be understood that the framework described herein can be implemented as a method, process, apparatus, system or article of manufacture, such as a non-transitory computer-readable medium or device. For illustrative purposes, the present framework can be described in the context of a distributed ledger that is publicly available or at least available to untrusted third parties. As an example of a modern use case is a blockchain-based system. However, it should be understood that the present framework can also be applied to other settings where sensitive or confidential information may need to pass through the hands of untrusted third parties, and this technology is by no means limited to distributed ledger or blockchain use.

[0260] The computer system 400 can be a client or server that accesses or hosts any application and / or data through any delivery paradigm, including but not limited to: remote or distributed cloud computing solutions; local or on-premises software (e.g., "on-premises" cloud-based solutions); "as a service" models (e.g., content as a service (CaaS), digital content as a service (DCaaS), software as a service (SaaS), hosted software as a service (MSaaS), platform as a service (PaaS), desktop as a service (DaaS), framework as a service (FaaS), backend as a service (BaaS), mobile backend as a service (MBaaS), infrastructure as a service (IaaS), database as a service (DBaaS), etc.); and / or a hybrid model including any combination of the foregoing examples or other services or delivery paradigms.

[0261] Any applicable data structures, file formats, and schemas may be derived from standards including, but not limited to, JavaScript Object Notation (JSON), Extensible Markup Language (XML), Yet Another Markup Language (YAML), Extensible Hypertext Markup Language (XHTML), Wireless Markup Language (WML), MessagePack, XML User Interface Language (XUL), or any other functionally similar representations alone or in combination. Alternatively, proprietary data structures, formats, or schemas may be used alone or in combination with known or open standards.

[0262] Any relevant data, files and / or databases may be stored, retrieved, accessed and / or transmitted in a human-readable format (such as digital, textual, graphic or multimedia format, further including various types of markup languages ​​and other possible formats). Alternatively or in combination with the above formats, data, files and / or databases may be stored, retrieved, accessed and / or transmitted in a binary, encoded, compressed and / or encrypted format or any other machine-readable format.

[0263] The interface connections or interconnections between the various systems and layers may employ any number of mechanisms, such as any number of protocols, programming frameworks, layout plans, or application programming interfaces (APIs), including but not limited to Document Object Model (DOM), Discovery Service (DS), NSUserDefaults, Web Services Description Language (WSDL), Message Exchange Pattern (MEP), Web Distributed Data Exchange (WDDX), Web Hypertext Application Technology Working Group (WHATWG) HTML5 web messaging, Representational State Transfer (REST or RESTful web services), Extensible User Interface Protocol (XUP), Simple Object Access Protocol (SOAP), XML Schema Definition (XSD), XML Remote Procedure Call (XML-RPC), or any other open or proprietary mechanism that can achieve similar functions and results.

[0264] Such an interface connection or interconnection can also utilize a uniform resource identifier (URI), which can further include a uniform resource locator (URL) or a uniform resource name (URN). Other forms of uniform and / or unique identifiers, locators or names can be used, used alone or in combination with those forms as described above.

[0265] Any of the above protocols or APIs can be interfaced with or implemented in any programming language (procedural, functional or object-oriented), and can be compiled or interpreted. Non-limiting examples include C, C++, C#, Objective-C, Java, Scala, Clojure, Elixir, Swift, Go, Perl, PHP, Python, Ruby, JavaScript, WebAssembly, or nearly any other language, as well as any other library or pattern in any type of framework, runtime environment, virtual machine, interpreter, stack, engine, or similar mechanism, including but not limited to Node.js, V8, Knockout, jQuery, Dojo, Dijit, OpenUI5, AngularJS, Expressjs, Backbone.js, Ember.js, DHTMLX, Vue, React, Electron, etc., and many other non-limiting examples.

[0266] In some embodiments, a tangible non-transitory device or article of manufacture containing a tangible non-transitory computer usable or readable medium having control logic (software) stored thereon may also be referred to herein as a computer program product or program storage device. This includes, but is not limited to, computer system 400, main memory 408, auxiliary memory 410, and removable storage units 418 and 422, and tangible articles of manufacture embodying any combination of the foregoing. When executed by one or more data processing devices (such as computer system 400), such control logic may cause such data processing devices to operate as described herein.

[0267] Based on the teachings contained in this disclosure, it is obvious to a person skilled in the relevant art how to use different Figure 4 It will be apparent that the data processing devices, computer systems, and / or computer architectures shown can be used to make and use the embodiments of the present disclosure. Specifically, the embodiments can operate with software, hardware, and / or operating system implementations other than those described herein.

[0268] Optical system

[0269] Figure 1 The imager 116 in may include one or more optical systems. Further disclosed herein are optical system design guidelines and high-performance fluorescence imaging methods and systems that provide improved optical resolution and image quality for genomics applications based on fluorescence imaging. The disclosed optical imaging system design provides a larger field of view, increased spatial resolution, improved modulation transfer, contrast-to-noise ratio and image quality, higher spatial sampling frequency, faster transitions between image captures when repositioning the sample plane to capture a series of images (e.g., images of different fields of view), and improved imaging system duty cycle, and thus achieves higher throughput image acquisition and analysis.

[0270] In some cases, improvements in imaging performance, such as for dual-sided (flow cell) imaging applications, can be achieved by using an electro-optic phase plate in conjunction with an objective to compensate for optical aberrations caused by the fluid layer separating the upper (near) and lower (far) inner surfaces of the flow cell. In some cases, this design approach can also compensate for vibrations introduced by, for example, motion-actuated compensators that move in or out of the optical path depending on which surface of the flow cell is being imaged.

[0271] In some cases, improvements in imaging performance, such as for dual-sided (flow cell) imaging applications involving the use of thick flow cell walls (e.g., wall (or coverslip) thickness >700 μm) and fluid channels (e.g., fluid channel height or thickness of 50-200 μm), can be achieved even when using commercially available off-the-shelf objectives by using a tube lens design that corrects for optical aberrations caused by the thick flow cell walls and / or intervening fluid layers in combination with the objectives.

[0272] In some cases, improvements in imaging performance, such as for multi-channel (e.g., two-color or four-color) imaging applications, can be achieved by using multiple tube lenses (one tube lens for each imaging channel), where each tube lens design is optimized for a specific wavelength range used in that imaging channel.

[0273] Example embodiments disclosed herein may include a fluorescence imaging system, the system comprising: a) at least one light source configured to provide excitation light within one or more specified wavelength ranges; b) an objective lens configured to collect fluorescence generated within a specified field of view of a sample plane when the sample plane is exposed to the excitation light, wherein the numerical aperture of the objective lens is at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, or at least 0.9, or a numerical aperture within the range defined by any two of the foregoing; wherein the working distance of the objective lens is at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, at least 1000 μm, or a working distance within the range defined by any two of the foregoing; and wherein the area of ​​the field of view is at least 0.1 mm 2 , at least 0.2mm 2 , at least 0.5mm 2 , at least 0.7mm 2 , at least 1mm 2 , at least 2mm 2 , at least 3mm 2 , at least 5mm 2 or at least 10mm 2 or a field of view falling within the range defined by any two of the above; and c) at least one image sensor, wherein the fluorescence collected by the objective is imaged onto the image sensor, and wherein the pixel size of the image sensor is selected so that the spatial sampling frequency of the fluorescence imaging system is at least twice the optical resolution of the fluorescence imaging system.

[0274] In some embodiments, the numerical aperture may be at least 0.75. In some embodiments, the numerical aperture is at least 1.0. In some embodiments, the working distance is at least 850 μm. In some embodiments, the working distance is at least 1,000 μm. In some embodiments, the area of ​​the field of view may be at least 2.5 mm2. In some embodiments, the area of ​​the field of view may be at least 3 mm2. In some embodiments, the spatial sampling frequency may be at least 2.5 times the optical resolution of the fluorescence imaging system. In some embodiments, the spatial sampling frequency may be at least 3 times the optical resolution of the fluorescence imaging system. In some embodiments, the system may further include an XYZ translation stage, such that the system is configured to acquire a series of two or more fluorescence images in an automated manner, wherein each image in the series is or may be acquired for a different field of view. In some embodiments, the positioning of the sample plane may be adjusted simultaneously in the X direction, the Y direction, and the Z direction to match the positioning of the objective focal plane between acquiring images of different fields of view. In some embodiments, the time required for simultaneous adjustment in the X direction, the Y direction, and the Z direction may be less than 0.3 seconds, less than 0.4 seconds, less than 0.5 seconds, less than 0.7 seconds, or less than 1 second, or a time falling within the range defined by any two of the foregoing. In some embodiments, the system further comprises an autofocus mechanism, the autofocus mechanism being configured to adjust the focal plane positioning before acquiring images of different fields of view if the error signal indicates that the positioning difference between the focal plane and the sample plane in the Z direction is greater than a specified error threshold. In some embodiments, the specified error threshold is 100nm or greater. In some embodiments, the specified error threshold is 50nm or less. In some embodiments, the system comprises three or more image sensors, and wherein the system is configured to image the fluorescence in each wavelength range in three or more wavelength ranges onto different image sensors. In some embodiments, the positioning difference between the focal plane of each image sensor in the three or more image sensors and the sample plane is less than 100nm. In some embodiments, the positioning difference between the focal plane of each image sensor in the three or more image sensors and the sample plane is less than 50nm. In some embodiments, the total time required to reposition the sample plane, adjust the focus (if necessary), and acquire the image is less than 0.4 seconds per field of view. In some embodiments, the total time required to reposition the sample plane, adjust the focus (if necessary), and acquire the image is less than 0.3 seconds per field of view.

[0275] The present invention also discloses a fluorescence imaging system for dual-sided imaging of a flow cell, the fluorescence imaging system comprising: a) an objective lens configured to collect fluorescence generated within a specified field of view of a sample plane in the flow cell; b) at least one tube lens positioned between the objective lens and at least one image sensor, wherein the at least one tube lens is configured to correct an imaging performance metric of a combination of the objective lens, at least two tube lenses, and at least one image sensor when imaging the inner surface of the flow cell, and wherein the wall thickness of the flow cell is at least 700 μm, and the gap between the upper inner surface and the lower inner surface is at least 50 μm; wherein the imaging performance metric is substantially the same for imaging the upper inner surface or the lower inner surface of the flow cell without moving an optical compensator into or out of an optical path between the flow cell and the at least one image sensor, without moving one or more optical elements of the tube lens along the optical path, and without moving one or more optical elements of the tube lens into or out of the optical path.

[0276] In some embodiments, the objective lens may be a commercially available microscope objective lens. In some embodiments, the numerical aperture of the commercially available microscope objective lens may be at least 0.3. In some embodiments, the working distance of the objective lens may be at least 700 μm. In some embodiments, the objective lens may be corrected to compensate for a cover glass thickness (or flow cell wall thickness) of 0.17 mm or a thickness greater than or less than 0.17 mm. In some embodiments, the optical system may be corrected to compensate for the cover glass thickness, the flow cell thickness, or the distance between the desired focal planes. In some embodiments, the correction may be performed by inserting a correction optical device such as a lens or an optical assembly into the optical path of the optical system. In some embodiments, the correction may be performed without inserting a correction optical device such as a lens or an optical assembly into the optical path of the optical system. In some embodiments, the fluorescence imaging system may further include an electro-optical phase plate positioned adjacent to the objective lens and positioned between the objective lens and the tube lens, wherein the electro-optical phase plate may provide correction of optical aberrations caused by a fluid filling the gap between the upper inner surface and the lower inner surface of the flow cell. In some embodiments, at least one tube lens may be a composite lens comprising three or more optical components. In some embodiments, at least one tube lens is a composite lens comprising four optical components, which may include one or more of the following: a first asymmetric convex-convex lens, a second convex-plano lens, a third asymmetric concave-concave lens, and a fourth asymmetric convex-concave lens, which may be present in the order listed above or in any alternative order. In some embodiments, at least one tube lens is configured to correct the imaging performance metric of the combination of the objective lens, at least one tube lens, and at least one image sensor when imaging the inner surface of a flow cell having a wall thickness of at least 1 mm. In some embodiments, at least one tube lens is configured to correct the imaging performance metric of the combination of the objective lens, at least one tube lens, and at least one image sensor when imaging the inner surface of a flow cell having a gap of at least 100 μm. In some embodiments, at least one tube lens is configured to correct the imaging performance metric of the combination of the objective lens, at least one tube lens, and at least one image sensor when imaging the inner surface of a flow cell having a gap of at least 200 μm. In some embodiments, the system comprises a single objective lens, two tube lenses, and two image sensors, and each of the two tube lenses is designed to provide optimal imaging performance at different fluorescence wavelengths. In some embodiments, the system comprises a single objective lens, three tube lenses, and three image sensors, and each of the three tube lenses is designed to provide optimal imaging performance at a different fluorescence wavelength. In some embodiments, the system comprises a single objective lens, four tube lenses, and four image sensors, and each of the four tube lenses is designed to provide optimal imaging performance at a different fluorescence wavelength.In some embodiments, the design of the objective lens or at least one tube lens is configured to optimize the modulation transfer function in the medium to high spatial frequency range. In some embodiments, the imaging performance metric comprises a measurement of the modulation transfer function (MTF), defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, contrast-to-noise ratio (CNR), or any combination thereof at one or more specified spatial frequencies. In some embodiments, the difference in the imaging performance metric for imaging the upper and lower inner surfaces of the flow cell is less than 10%. In some embodiments, the difference in the imaging performance metric for imaging the upper and lower inner surfaces of the flow cell is less than 5%. In some embodiments, the use of at least one tube lens provides at least equivalent or better improvements in imaging performance metrics for dual-sided imaging compared to conventional systems including an objective lens, a motion-actuated compensator, and an image sensor. In some embodiments, the use of at least one tube lens provides at least a 10% improvement in imaging performance metrics for dual-sided imaging compared to conventional systems including an objective lens, a motion-actuated compensator, and an image sensor.

[0277] Disclosed herein is an illumination system for imaging-based solid phase genotyping and sequencing applications, the illumination system comprising: a) a light source; and b) a liquid light guide configured to collect light emitted by the light source and deliver the light to a designated illumination field on a carrier surface comprising tethered biomacromolecules.

[0278] In some embodiments, the illumination system further comprises a condenser lens. In some embodiments, the area of ​​the designated illumination field is at least 2 mm2. In some embodiments, the light delivered to the designated illumination field has uniform intensity across a designated field of view of an imaging system used to acquire an image of the carrier surface. In some embodiments, the area of ​​the designated field of view is at least at least 2 mm2. In some embodiments, the light delivered to the designated illumination field has uniform intensity across the designated field of view when the coefficient of variation (CV) of the light intensity is less than 10%. In some embodiments, the light delivered to the designated illumination field has uniform intensity across the designated field of view when the coefficient of variation (CV) of the light intensity is less than 5%. In some embodiments, the speckle contrast value of the light delivered to the designated illumination field is less than 0.1. In some embodiments, the speckle contrast value of the light delivered to the designated illumination field is less than 0.05.

[0279] Imaging modules and systems: Those skilled in the art will appreciate that, in some cases, the disclosed optical systems, imaging systems or modules may be independent optical systems designed for imaging a sample or substrate surface. In some cases, they may include one or more processors or computers. In some cases, they may include one or more software packages that provide instrument control functions and / or image processing functions. In some cases, in addition to optical components such as light sources (e.g., solid-state lasers, dye lasers, diode lasers, arc lamps, halogen lamps, etc.), lenses, prisms, mirrors, dichroic reflectors, optical filters, optical bandpass filters, apertures, and image sensors (e.g., complementary metal oxide semiconductor (CMOS) image sensors and cameras, charge coupled devices (CCD) image sensors and cameras, etc.), they may also include mechanical and / or optical mechanical components such as XY translation stages, XYZ translation stages, piezoelectric focusing mechanisms, etc. In some cases, they may serve as modules, components, subassemblies, or subsystems of larger systems designed for genomics applications (e.g., genetic testing and / or nucleic acid sequencing applications). For example, in some cases, they can act as modules, components, subassemblies, or subsystems of a larger system, which further includes a light-tight and / or other environmental control housing, a temperature control module, a fluid control module, a fluid dispensing robot, a pick and place robot, one or more processors or computers, one or more local and / or cloud-based software packages (e.g., instrument / system control software packages, image processing software packages, data analysis software packages), data storage modules, data communication modules (e.g., Bluetooth, WiFi, intranet or Internet communication hardware and associated software), a display module, or any combination thereof.

[0280] Methods used for sequencing

[0281] The present disclosure provides a method for sequencing fixed or non-fixed template molecules. The method can be operated in system 100, for example, in a sequencer 114. In some embodiments, the fixed template molecule comprises a plurality of nucleic acid template molecules having a copy of a target sequence of interest. In some embodiments, the nucleic acid template molecule having a copy of a target sequence of interest can be generated by bridge amplification using a linear library molecule. In some embodiments, the fixed template molecule comprises a plurality of nucleic acid template molecules, each nucleic acid template molecule having two or more tandem copies (e.g., concatemer) of a target sequence of interest. In some embodiments, the nucleic acid template molecule comprising a concatemer molecule can be generated by rolling circle amplification of a circularized linear library molecule. In some embodiments, the non-fixed template molecule comprises a circular molecule. In some embodiments, the method for sequencing employs a soluble (e.g., non-fixed) sequencing polymerase or a sequencing polymerase fixed to a carrier.

[0282] In some embodiments, the sequencing reaction uses detectably labeled nucleotide analogs. In some embodiments, the sequencing reaction uses a two-stage sequencing reaction, including binding to a detectably labeled multivalent molecule and incorporation of nucleotide analogs. In some embodiments, the sequencing reaction uses unlabeled nucleotide analogs. In some embodiments, the sequencing reaction uses phosphate-tagged nucleotides.

[0283] In some embodiments, each of the fixed concatemers comprises a tandem repeat unit (e.g., an insertion region) of a sequence of interest and any adapter sequence. For example, the tandem repeat unit comprises: (i) a left universal adapter sequence having a binding sequence for a first surface primer (720) (e.g., a surface pinning primer), (ii) a left universal adapter sequence having a binding sequence for a first sequencing primer (740) (e.g., a forward sequencing primer), (iii) a sequence of interest (710), (iv) a right universal adapter sequence having a binding sequence for a second sequencing primer (750) (e.g., a reverse sequencing primer), (v) a right universal adapter sequence having a binding sequence for a second surface primer (730) (e.g., a surface capture primer), and (vii) a left sample index sequence (760) and / or a right sample index sequence (770). In some embodiments, the tandem repeat unit further comprises a left unique identifier sequence (780) and / or a right unique identifier sequence (790). In some embodiments, the tandem repeat unit further comprises a binding sequence for at least one compaction oligonucleotide. In some embodiments, Figure 7 and 8 Linear library molecules or concatemer molecule units are shown.

[0284] Specifically, Figure 7 is a schematic diagram showing an example linear single-stranded library molecule according to some embodiments. The linear library molecule can have a surface pinning primer binding site (720); an optional left unique identifier sequence (780); a left index sequence (760); a forward sequencing primer binding site (740); an insert region with a sequence of interest (710); a reverse sequencing primer binding site (750); a right index sequence (770); and a surface capture primer binding site (730).

[0285] Figure 8 is a schematic diagram showing an example linear single-stranded library molecule according to some embodiments. The linear library molecule may include: a surface pinning primer binding site (720); a left index sequence (760); a forward sequencing primer binding site (740); an insert region with a sequence of interest (710); a reverse sequencing primer binding site (750); a right index sequence (770); an optional right unique identifier sequence (790); and a surface capture primer binding site (730).

[0286] The fixed concatemer can be collapsed into a compact nucleic acid nanoball by itself. The size and / or shape of the nanoball can be further compacted by including one or more compacting oligonucleotides during the RCA reaction. The increase of the number of tandem repeat units in a given concatemer increases the number of sites along the concatemer for hybridization with a variety of sequencing primers (e.g., sequencing primers with a universal sequence), and the sequencing primers serve as a plurality of starting sites for the sequencing reaction catalyzed by a polymerase. When the sequencing reaction adopts detectably labeled nucleotides and / or detectably labeled multivalent molecules (e.g., with nucleotide units), the signal emitted by the nucleotides or nucleotide units participating in the parallel sequencing reactions along the concatemer produces an increased signal intensity for each concatemer. A plurality of parts of a given concatemer can be sequenced simultaneously. In addition, a plurality of binding complexes can be formed along a specific concatemer molecule, and each binding complex comprises a sequencing polymerase that is combined with a template / primer duplex and combined with a multivalent molecule, wherein the plurality of binding complexes remain stable and do not dissociate, resulting in an increased retention time, which increases signal intensity and reduces imaging time.

[0287] Methods for sequencing using nucleotide analogs

[0288] Embodiments of the present disclosure provide methods for sequencing any immobilized template molecule described herein, the method comprising step (a): contacting a sequencing polymerase with (i) a nucleic acid template molecule and (ii) a nucleic acid sequencing primer, wherein the contact is performed under conditions suitable for binding the sequencing polymerase to the nucleic acid template molecule hybridized to the nucleic acid primer, wherein the nucleic acid template molecule hybridized to the nucleic acid primer forms a nucleic acid duplex. In some embodiments, the sequencing polymerase comprises a recombinant mutant sequencing polymerase that can bind and incorporate nucleotide analogs.

[0289] In some embodiments, in a method for sequencing a template molecule, a sequencing primer comprises a 3' extendable end or a 3' non-extendable end. In some embodiments, a plurality of nucleic acid template molecules comprise amplified template molecules (e.g., clonally amplified template molecules). In some embodiments, a plurality of nucleic acid template molecules comprise a copy of a target sequence of interest. In some embodiments, a plurality of nucleic acid molecules comprise two or more tandem copies of a target sequence of interest (e.g., concatemers). In some embodiments, a plurality of nucleic acid template molecules comprise the same target sequence of interest or different target sequences of interest. In some embodiments, a plurality of nucleic acid primers are in solution or fixed to a carrier. In some embodiments, when a plurality of nucleic acid template molecules and / or a plurality of nucleic acid primers are fixed to a carrier, a plurality of fixed first composite polymerases are produced in combination with a first sequencing polymerase. In some embodiments, a plurality of nucleic acid template molecules and / or nucleic acid primers are fixed to 10 2 -10 15In some embodiments, the combination of multiple template molecules and nucleic acid primers with multiple first sequencing polymerases produces 10 2 -10 15 In some embodiments, the plurality of fixed first composite polymerases on the carrier are fixed to predetermined sites or random sites on the carrier. In some embodiments, the plurality of fixed first composite polymerases are fluidically connected to each other to allow a reagent solution (e.g., an enzyme including a sequencing polymerase, a multivalent molecule, a nucleotide, and / or a divalent cation) to flow onto the carrier, so that the plurality of fixed composite polymerases on the carrier react with the reagent solution in a massively parallel manner.

[0290] In some embodiments, the method for sequencing further comprises step (b): contacting a sequencing polymerase with a plurality of nucleotides under conditions suitable for binding at least one nucleotide to a sequencing polymerase bound to a nucleic acid duplex and suitable for incorporating a nucleotide catalyzed by the polymerase, incorporating the nucleotide catalyzed by the polymerase to extend the sequencing primer by one nucleotide. In some embodiments, the sequencing polymerase is contacted with a plurality of nucleotides in the presence of at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, a plurality of nucleotides comprises at least one nucleotide analog having a chain termination portion at a sugar 2' or 3' location. In some embodiments, the chain termination portion can be removed from the sugar 2' or 3' location to convert the chain termination portion into an OH or H group. In some embodiments, a plurality of nucleotides comprises at least one nucleotide lacking a chain termination portion. In some embodiments, at least one nucleotide is labeled with a detectable reporter gene portion (e.g., a fluorophore) that emits a detectable signal. The detectable reporter gene portion comprises a fluorophore. In some embodiments, the fluorophore is connected to a nucleobase. In some embodiments, the fluorophore is connected to a nucleobase with a joint that can be cut / removed from a base. In some embodiments, at least one nucleotide in a plurality of nucleotides is not labeled with a detectable reporter gene portion. In some embodiments, the specific detectable reporter moiety (e.g., a fluorophore) attached to the nucleotide can correspond to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow detection and identification of the nucleobase. When the incorporated chain-terminating nucleotide is detectably labeled, step (b) further comprises detecting a signal emitted from the incorporated chain-terminating nucleotide. In some embodiments, step (b) further comprises identifying the nucleobase of the incorporated chain-terminating nucleotide.

[0291] In some embodiments, the method for sequencing further comprises step (c): removing the chain terminating moiety from the incorporated chain terminating nucleotide to generate an extendable 3'OH group. In some embodiments, step (c) further comprises removing the detectable label from the incorporated chain terminating nucleotide. In some embodiments, the sequencing polymerase remains bound to the template molecule, which is hybridized to the sequencing primer extended by one nucleobase.

[0292] In some embodiments, the method for sequencing further comprises step (d): repeating steps (b) and (c) at least once.

[0293] Two-stage method for sequencing nucleic acids

[0294] Embodiments of the present disclosure provide a two-stage method for sequencing any immobilized template molecule described herein. In some embodiments, the first stage generally comprises combining a multivalent molecule with a composite polymerase to form a multivalent composite polymerase, and detecting the multivalent composite polymerase.

[0295] In some embodiments, the first stage comprises step (a): contacting a plurality of first sequencing polymerases with (i) a plurality of nucleic acid template molecules and (ii) a plurality of nucleic acid sequencing primers, wherein the contacting is performed under conditions suitable for binding the plurality of first sequencing polymerases to the plurality of nucleic acid template molecules and the plurality of nucleic acid primers, thereby forming a plurality of first composite polymerases, each of which comprises a first sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer. In some embodiments, the first polymerase comprises a recombinant mutant sequencing polymerase.

[0296] In some embodiments, in a method for sequencing a template molecule, a sequencing primer comprises an oligonucleotide having a 3' extendable end or a 3' non-extendable end. In some embodiments, a plurality of nucleic acid template molecules comprise amplified template molecules (e.g., clonally amplified template molecules). In some embodiments, a plurality of nucleic acid template molecules comprise a copy of a target sequence of interest. In some embodiments, a plurality of nucleic acid molecules comprise two or more tandem copies of a target sequence of interest (e.g., concatemers). In some embodiments, the nucleic acid template molecules in a plurality of nucleic acid template molecules comprise the same target sequence of interest or different target sequences of interest. In some embodiments, a plurality of nucleic acid template molecules and / or a plurality of nucleic acid primers are in a solution or fixed to a carrier. In some embodiments, when a plurality of nucleic acid template molecules and / or a plurality of nucleic acid primers are fixed to a carrier, a plurality of fixed first composite polymerases are produced in combination with a first sequencing polymerase. In some embodiments, a plurality of nucleic acid template molecules and / or nucleic acid primers are fixed to 10 2 -10 15In some embodiments, the combination of multiple template molecules and nucleic acid primers with multiple first sequencing polymerases produces 10 2 -10 15 In some embodiments, the plurality of fixed first composite polymerases on the carrier are fixed to predetermined sites or random sites on the carrier. In some embodiments, the plurality of fixed first composite polymerases are fluidically connected to each other to allow a reagent solution (e.g., an enzyme including a sequencing polymerase, a multivalent molecule, a nucleotide, and / or a divalent cation) to flow onto the carrier, so that the plurality of fixed composite polymerases on the carrier react with the reagent solution in a massively parallel manner.

[0297] In some embodiments, the method for sequencing further comprises step (b): contacting the plurality of first composite polymerases with the plurality of multivalent molecules to form a plurality of multivalent composite polymerases (e.g., binding complexes). In some embodiments, a single multivalent molecule in the plurality of multivalent molecules comprises a core connected to a plurality of nucleotide arms, and each nucleotide arm is connected to a nucleotide (e.g., a nucleotide unit) (e.g., Figure 9-13 In some embodiments, the contacting of step (b) is performed under conditions suitable for binding the complementary nucleotide units of the multivalent molecule to at least two of the plurality of first composite polymerases, thereby forming a plurality of multivalent composite polymerases. In some embodiments, the conditions are suitable for inhibiting polymerase-catalytic incorporation of complementary nucleotide units into primers of the plurality of multivalent composite polymerases. In some embodiments, the plurality of multivalent molecules comprises at least one multivalent molecule having a plurality of nucleotide arms (e.g., Figure 9-12 ), each nucleotide arm is connected to a nucleotide analog (e.g., a nucleotide analog unit), wherein the nucleotide analog includes a chain termination portion at the sugar 2' and / or 3' location. In some embodiments, a plurality of multivalent molecules comprises at least one multivalent molecule comprising a plurality of nucleotide arms, each nucleotide arm being attached with a nucleotide unit lacking a chain termination portion. In some embodiments, at least one of the multivalent molecules in the plurality of multivalent molecules is labeled with a detectable reporter gene portion that emits a signal. In some embodiments, the detectable reporter gene portion comprises a fluorophore. In some embodiments, the contacting of step (b) is performed in the presence of at least one non-catalytic cation comprising strontium, barium and / or calcium.

[0298] In some embodiments, the method for sequencing further comprises step (c): detecting a plurality of multivalent composite polymerases. In some embodiments, the detection comprises detecting a signal emitted by a multivalent molecule bound to the composite polymerase, wherein the complementary nucleotide unit of the multivalent molecule binds to the primer but inhibits the incorporation of the complementary nucleotide unit. In some embodiments, the multivalent molecule is labeled with a detectable reporter moiety to allow detection. In some embodiments, the labeled multivalent molecule comprises a fluorophore attached to the core, linker, and / or nucleotide unit of the multivalent molecule.

[0299] In some embodiments, the method for sequencing further comprises step (d): identifying the nucleobases of the complementary nucleotide units bound to the plurality of first composite polymerases, thereby determining the sequence of the template molecule. In some embodiments, the multivalent molecule is labeled with a detectable reporter moiety corresponding to a specific nucleotide unit attached to the nucleotide arm to allow identification of the complementary nucleotide units (e.g., nucleotide bases adenine, guanine, cytosine, thymine, or uracil) bound to the plurality of first composite polymerases.

[0300] In some embodiments, the method for sequencing further comprises step (e): dissociating the plurality of multivalent complex polymerases and removing the plurality of first sequencing polymerases and the multivalent molecules bound thereto, and retaining the plurality of nucleic acid duplexes.

[0301] In some embodiments, the second stage of the two-stage sequencing method generally comprises nucleotide incorporation. In some embodiments, the method for sequencing further comprises step (f): contacting the plurality of retained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, wherein the contacting is performed under conditions suitable for binding the plurality of second sequencing polymerases to the plurality of retained nucleic acid duplexes, thereby forming a plurality of second composite polymerases each comprising a second sequencing polymerase bound to the nucleic acid duplexes. In some embodiments, the second sequencing polymerase comprises a recombinant mutant sequencing polymerase.

[0302] In some embodiments, the plurality of first sequencing polymerases of step (a) have an amino acid sequence that is 100% identical to the amino acid sequence of the plurality of second sequencing polymerases of step (f). In some embodiments, the plurality of first sequencing polymerases of step (a) have an amino acid sequence that is different from the amino acid sequence of the plurality of second sequencing polymerases of step (f).

[0303] In some embodiments, the method for sequencing further comprises step (g): contacting a plurality of second composite polymerases with a plurality of nucleotides, wherein the contact is carried out under conditions suitable for combining complementary nucleotides from a plurality of nucleotides with at least two second composite polymerases in the second composite polymerase, thereby forming a plurality of nucleotide composite polymerases. In some embodiments, the contact of step (g) is carried out under conditions suitable for promoting the catalytic incorporation of the bound complementary nucleotide polymerase into the primer of the nucleotide composite polymerase, thereby extending the sequencing primer by one nucleobase. In some embodiments, the incorporation of nucleotides into the 3' end of the sequencing primer in step (g) comprises a primer extension reaction. In some embodiments, the contact of step (g) is carried out in the presence of at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, a plurality of nucleotides comprises natural nucleotides (e.g., non-analog nucleotides) or nucleotide analogs. In some embodiments, a plurality of nucleotides comprises removable or non-removable 2' and / or 3' chain termination portions. In some embodiments, at least one nucleotide in a plurality of nucleotides is not labeled with a detectable reporter gene portion. In some embodiments, a plurality of nucleotides are unlabeled. In some embodiments, a plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter gene portion. The detectable reporter gene portion comprises a fluorophore. In some embodiments, the fluorophore is linked to a nucleotide base. In some embodiments, the fluorophore is linked to a nucleotide base with a linker that may or may not be cleavable / removable from the base. In some embodiments, a specific detectable reporter gene moiety (e.g., a fluorophore) linked to a nucleotide may correspond to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow detection and identification of the nucleotide base.

[0304] In some embodiments, when the plurality of nucleotides in step (g) are detectably labeled, the method for sequencing further comprises step (h): detecting complementary nucleotides incorporated into the primer of the nucleotide composite polymerase. In some embodiments, the plurality of nucleotides are labeled with a detectable reporter gene moiety to allow detection. In some embodiments, when the plurality of nucleotides in step (g) are not labeled, the detection of step (h) is omitted.

[0305] In some embodiments, when the plurality of nucleotides in step (g) are detectably labeled, the method for sequencing further comprises step (i): identifying the base of the complementary nucleotide incorporated into the primer of the nucleotide composite polymerase. In some embodiments, the identification of the incorporated complementary nucleotide in step (i) can be used to confirm the identity of the complementary nucleotide of the multivalent molecule bound to the plurality of first composite polymerases in step (d). In some embodiments, the identification of step (i) can be used to determine the sequence of the nucleic acid template molecule. In some embodiments, when the plurality of nucleotides in step (g) are not labeled, the identification of step (i) is omitted.

[0306] In some embodiments, the method for sequencing further comprises step (j): removing the chain terminating moiety from the incorporated nucleotides when step (g) is performed by contacting a plurality of second composite polymerases with a plurality of nucleotides comprising at least one nucleotide having a 2' and / or 3' chain terminating moiety.

[0307] In some embodiments, the method for sequencing further comprises step (k): repeating steps (a) to (j) at least once. In some embodiments, the sequence of the nucleic acid template molecule can be determined by detecting and identifying the multivalent molecule that binds to the sequencing polymerase but is not incorporated into the 3' end of the primer at steps (c) and (d). In some embodiments, the sequence of the nucleic acid template molecule can be determined (or confirmed) by detecting and identifying the nucleotide incorporated into the 3' end of the primer at steps (h) and (i).

[0308] In some embodiments, in any method for sequencing a nucleic acid molecule, the combination of a plurality of first composite polymerases and a plurality of multivalent molecules forms at least one affinity complex, the method comprising the following steps: (a) combining a first nucleic acid primer, a first sequencing polymerase, and a first multivalent molecule with a first portion of a concatemer template molecule, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds to the first sequencing polymerase; and (b) combining a second nucleic acid primer, a second sequencing polymerase, and the first multivalent molecule with a second portion of the same concatemer template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to the second sequencing polymerase, wherein the first binding complex and the second binding complex comprising the same multivalent molecule form an affinity complex. In some embodiments, the first sequencing polymerase comprises any wild-type or mutant polymerase described herein. In some embodiments, the second sequencing polymerase comprises any wild-type or mutant polymerase described herein. The concatemer template molecule comprises a tandem repeat sequence of a sequence of interest and at least one universal sequencing primer binding site. The first nucleic acid primer and the second nucleic acid primer can bind to the sequencing primer binding site along the concatemer template molecule. Example multivalent molecules are shown in Figure 9-12 middle.

[0309] In some embodiments, in any method for sequencing a nucleic acid molecule, wherein the method comprises combining a plurality of first composite polymerases with a plurality of multivalent molecules to form at least one affinity complex, the method comprises the following steps: (a) contacting a plurality of sequencing polymerases and a plurality of nucleic acid primers with different portions of a concatemer nucleic acid concatemer molecule to form at least a first composite polymerase and a second composite polymerase on the same concatemer template molecule; (b) contacting the plurality of multivalent molecules with the at least first composite polymerase and the second composite polymerase on the same concatemer template molecule under conditions suitable for combining a single multivalent molecule from the plurality of multivalent molecules with the first composite polymerase and the second composite polymerase, wherein at least a first nucleotide unit of the single multivalent molecule is combined with the first composite polymerase, the first composite polymerase comprising a first primer, the first primer hybridizing to a first portion of the concatemer template molecule, thereby forming a first binding complex (e.g., a first ternary complex), and wherein the At least a second nucleotide unit of the single multivalent molecule is bound to the second composite polymerase, the second composite polymerase comprising a second primer, the second primer hybridizing to the second portion of the concatemer template molecule, thereby forming a second binding complex (e.g., a second ternary complex), wherein the contacting is performed under conditions suitable for inhibiting the polymerase-catalytic incorporation of the bound first and second nucleotide units into the first and second binding complexes, and wherein the first and second binding complexes bound to the same multivalent molecule form an affinity complex; and (c) detecting the first and second binding complexes on the same concatemer template molecule; and (d) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the concatemer template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the concatemer template molecule. In some embodiments, the plurality of sequencing polymerases comprises any wild-type or mutant sequencing polymerase described herein. The concatemer template molecule comprises a tandem repeat sequence of a sequence of interest and at least one universal sequencing primer binding site. A plurality of nucleic acid primers can bind to the sequencing primer binding sites along the concatemer template molecule. Example multivalent molecules are shown in Figure 9-12 middle.

[0310] Sequencing by Binding

[0311] Embodiments of the present disclosure provide methods for sequencing any of the immobilized template molecules described herein, wherein the sequencing method comprises a sequencing by binding (SBB) procedure using unlabeled chain terminating nucleotides. In some embodiments, a sequencing by binding (SBB) method comprises the following steps: (a) sequentially contacting a primed template nucleic acid with at least two separate mixtures under ternary complex stabilizing conditions, wherein each of the at least two separate mixtures comprises a polymerase and nucleotides, whereby the sequential contacting in the primed template nucleic acid results in contact with nucleotide homologs of a first, second, and third base type in the template under the ternary complex stabilizing conditions; (b) examining the at least two separate mixtures to determine whether a ternary complex is formed; and (c) identifying the next correct nucleotide of the primed template nucleic acid molecule, wherein if a ternary complex is detected in step (b), the next correct nucleotide is identified as a homolog of the first, second, or third base type, and wherein the next correct nucleotide is estimated to be a nucleotide homolog of a fourth base type based on the absence of a ternary complex in step (b); (d) after step (b), adding the next correct nucleotide to a primer of the primed template nucleic acid, thereby generating an extended primer; and (e) repeating steps (a) to (d) at least once for the primed template nucleic acid comprising the extended primer. Exemplary sequencing-by-binding methods are described in U.S. Pat. Nos. 10,246,744 and 10,731,141 (the contents of both of which are hereby incorporated by reference in their entireties).

[0312] Method for sequencing using nucleotides labeled with phosphate chains

[0313] Embodiments of the present disclosure provide methods for sequencing using an immobilized sequencing polymerase bound to an unimmobilized template molecule, wherein a sequencing reaction is performed with nucleotides labeled with a phosphate chain. In some embodiments, the sequencing method comprises step (a): providing a carrier on which a plurality of sequencing polymerases are immobilized. In some embodiments, the sequencing polymerase comprises a processive DNA polymerase. In some embodiments, the sequencing polymerase comprises a wild-type or mutant DNA polymerase, including, for example, a Phi29 DNA polymerase. In some embodiments, the carrier comprises a plurality of separate compartments, and the sequencing polymerase is fixed to the bottom of the compartment. In some embodiments, the separate compartments comprise a silica bottom through which light can penetrate. In some embodiments, the separate compartments comprise a silica bottom configured with a nanophotonic confinement structure comprising a hole in a metal coating (e.g., an aluminum coating). In some embodiments, the small aperture of the hole in the metal coating is, for example, about 70 nm. In some embodiments, the height of the nanophotonic confinement structure is about 100 nm. In some embodiments, the nanophotonic confinement structure comprises a zero mode waveguide (ZMW). In some embodiments, the nanophotonic confinement structure contains a liquid.

[0314] In some embodiments, the sequencing method further comprises step (b): contacting a plurality of immobilized sequencing polymerases with a plurality of single-stranded circular nucleic acid template molecules and a plurality of oligonucleotide sequencing primers under conditions suitable for a single immobilized sequencing polymerase to bind to a single-stranded circular template molecule and suitable for a single sequencing primer to hybridize to a single single-stranded circular template molecule, thereby generating a plurality of polymerase / template / primer complexes. In some embodiments, a single sequencing primer hybridizes to a universal sequencing primer binding site on a single-stranded circular template molecule.

[0315] In some embodiments, the sequencing method further comprises step (c): contacting a plurality of polymerase / template / primer complexes with a plurality of nucleotides labeled with phosphate chains, each nucleotide labeled with a phosphate chain comprising an aromatic base, a pentose (e.g., ribose or deoxyribose) and a phosphate chain comprising 3-20 phosphate groups, wherein the terminal phosphate group is connected to a detectable reporter gene portion (e.g., a fluorophore). The first phosphate group, the second phosphate group, and the third phosphate group may be referred to as α, β, and γ phosphate groups. In some embodiments, the specific detectable reporter gene portion connected to the terminal phosphate group corresponds to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow detection and identification of the nucleobase. In some embodiments, under conditions suitable for polymerase-catalyzed nucleotide incorporation, contacting a plurality of polymerase / template / primer complexes with a plurality of nucleotides labeled with phosphate chains. In some embodiments, the sequencing polymerase is capable of binding to nucleotides labeled with complementary phosphate chains and incorporating complementary nucleotides relative to the nucleotides in the template molecule. In some embodiments, the polymerase catalyzed nucleotide incorporation reaction cleaves between the alpha phosphate group and the beta phosphate group, thereby releasing the polyphosphate chain attached to the fluorophore.

[0316] In some embodiments, the sequencing method further comprises step (d): detecting a fluorescent signal emitted by a nucleotide labeled with a phosphate chain, which is bound by a sequencing polymerase and incorporated into the end of a sequencing primer. In some embodiments, step (d) further comprises identifying the nucleotide labeled with a phosphate chain bound by a sequencing polymerase and incorporated into the end of a sequencing primer.

[0317] In some embodiments, the sequencing method further comprises step (d): repeating steps (c)-(d) at least once. In some embodiments, the sequencing method using nucleotides labeled with phosphate chains can be performed according to the methods described in U.S. Patent Nos. 7,170,050; 7,302,146; and / or 7,405,281.

[0318] Sequencing polymerase

[0319] Embodiments of the present disclosure provide methods for sequencing nucleic acid molecules, wherein any sequencing method described herein employs at least one type of sequencing polymerase and a plurality of nucleotides, or employs at least one type of sequencing polymerase and a plurality of nucleotides and a plurality of multivalent molecules. In some embodiments, the sequencing polymerase is capable of incorporating complementary nucleotides relative to the nucleotides in the template molecule. In some embodiments, the sequencing polymerase is capable of binding to complementary nucleotide units of multivalent molecules relative to the nucleotides in the template molecule. In some embodiments, a plurality of sequencing polymerases comprises a recombinant mutant polymerase.

[0320] Examples of suitable polymerases for sequencing with nucleotides and / or multivalent molecules include, but are not limited to, Klenow DNA polymerase; Thermus aquaticus DNA polymerase I (Taq polymerase); KlenTaq polymerase; Candidatus altiarchaeales archaeon; Candidatus Hadarchaeum Yellowstonense; Hadesarchaea archaeon; Euryarchaeota archaeon; Thermoplasmata archaeon; Thermococcus polymerases, such as Thermococcus litoralis, bacteriophage T7 DNA polymerase; human alpha, delta, and epsilon DNA polymerases; bacteriophage polymerases, such as T4, RB69, and phi29 phage DNA polymerases; Pyrococcus furiosus furiosus DNA polymerase (Pfu polymerase); Bacillus subtilis DNA polymerase III; Escherichia coli (E. coli) DNA polymerase III alpha and epsilon; 9 degree N polymerase; reverse transcriptase, such as HIV type M or type O reverse transcriptase; avian myeloblastosis virus reverse transcriptase; Moloney Murine Leukemia Virus (MMLV) reverse transcriptase; or telomerase. Additional non-limiting examples of DNA polymerases include those from various Archaea (e.g., Aeropyrum, Archaeoglobus, Desulfurococcus, Pyrobaculum, Pyrococcus, Pyrolobus, Pyrodictium, Staphylothermus, Stetteria, Sulfolobus, Thermococcus, and Vulcanisaeta, etc., or variants thereof), including such polymerases as are known in the art, such as 9 degrees N, VENT, DEEP VENT, THERMINATOR, Pfu, KOD, Pfx, Tgo, and RB69 polymerases.

[0321] Multivalent molecules

[0322] Embodiments of the present disclosure provide methods for sequencing nucleic acid molecules, wherein any of the sequencing methods described herein employ at least one multivalent molecule. In some embodiments, the multivalent molecule comprises a plurality of nucleotide arms attached to a core and has any configuration, including a starburst, a helter skelter, or a bottlebrush configuration (e.g., Fig. 9 ). The multivalent molecule comprises: (1) a core; and (2) a plurality of nucleotide arms, the plurality of nucleotide arms comprising (i) a core linking portion, (ii) a spacer, the spacer comprising a PEG portion, (iii) a linker, and (iv) a nucleotide unit, wherein the core is linked to the plurality of nucleotide arms, wherein the spacer is connected to the linker, wherein the linker is connected to the nucleotide unit. In some embodiments, the nucleotide unit comprises a base, a sugar, and at least one phosphate group, and the linker is connected to the nucleotide unit via the base. In some embodiments, the linker comprises an aliphatic chain or an oligoethylene glycol chain, wherein both linker chains have 2-6 subunits. In some embodiments, the linker further comprises an aromatic portion. Example nucleotide arms are shown in Fig.13 Example multivalent molecules are shown in Figure 9-12 Example spacers are shown in Fig.14 (top) and example joints are shown in Fig.14 (bottom) and Fig.15 Example nucleotides linked to the linker are shown in Figure 16-19 An example biotinylated nucleotide arm is shown in Fig. 20 middle.

[0323] In some embodiments, the multivalent molecule comprises a core linked to a plurality of nucleotide arms, and wherein the plurality of nucleotide arms have the same type of nucleotide units selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and dUTP.

[0324] In certain embodiments, the multivalent molecule comprises a core connected to a plurality of nucleotide arms, wherein each arm comprises a nucleotide unit. The nucleotide unit comprises an aromatic base, a pentose (e.g., ribose or deoxyribose) and one or more phosphate groups (e.g., 1-10 phosphate groups). A plurality of multivalent molecules can comprise a type of multivalent molecule having a type of nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP and dUTP. A plurality of multivalent molecules can comprise a mixture of any combination of two or more types of multivalent molecules, wherein the independent multivalent molecule in the mixture comprises a nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP and / or dUTP.

[0325] In some embodiments, the nucleotide unit comprises a chain of one, two or three phosphorus atoms, wherein the chain is generally connected to the 5' carbon of the sugar moiety by an ester or phosphoramide bond. In some embodiments, at least one nucleotide unit is a nucleotide analog with a phosphorus chain, wherein the phosphorus atoms are linked together with an intervening O, S, NH, methylene or ethylene. In some embodiments, the phosphorus atoms in the chain include substituted side groups (including O, S or BH3). In some embodiments, the chain includes a phosphate group substituted with an analog, including phosphoramide, phosphorothioate, phosphorodithioate and O-methylphosphoramidite groups.

[0326] In some embodiments, the multivalent molecule comprises a core connected to a plurality of nucleotide arms, and wherein the individual nucleotide arms comprise nucleotide units, which are nucleotide analogs having a chain termination portion (e.g., a blocking portion) at a sugar 2' position, at a sugar 3' position, or at a sugar 2' and 3' position. In some embodiments, the nucleotide unit comprises a chain termination portion (e.g., a blocking portion) at a sugar 2' position, at a sugar 3' position, or at a sugar 2' and 3' position. In some embodiments, the chain termination portion can inhibit the polymerase-catalyzed incorporation of subsequent nucleotide units or free nucleotides in the nascent chain during a primer extension reaction. In some embodiments, the chain termination portion is connected to a 3' sugar position, wherein the sugar comprises a ribose or deoxyribose portion. In some embodiments, the chain termination portion can be removed / cut from the 3' sugar position to produce a nucleotide having a 3'OH sugar group, which can be extended with subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain termination portion comprises an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, an azide group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group or a silyl group. In some embodiments, the chain termination portion can be cut / removed from the nucleotide unit, for example, by reacting the chain termination portion with a chemical agent, a pH change, light or heat. In some embodiments, the chain termination portion alkyl, alkenyl, alkynyl and allyl groups can be cut with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the chain termination portion aryl and benzyl groups can be cut with H2Pd / C. In some embodiments, the chain termination portion amine, amide, ketone, isocyanate, phosphate, thio, disulfide can be cut with a phosphine or thiol group (including β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, the chain-terminating moiety carbonate can be cleaved with MeOH containing potassium carbonate (K2CO3), with pyridine containing triethylamine, or with acetic acid (AcOH) containing Zn. In some embodiments, the chain-terminating moieties urea and silyl can be cleaved with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride.

[0327] In some embodiments, the nucleotide unit comprises a chain terminating portion (e.g., a blocking portion) at the sugar 2' position, at the sugar 3' position, or at the sugar 2' and 3' positions. In some embodiments, the chain terminating portion comprises an azide, an azido, or an azidomethyl. In some embodiments, the chain terminating portion comprises a 3'-O-azido or a 3'-O-azidomethyl. In some embodiments, the chain terminating portion azide, an azido, and an azidomethyl can be cleaved / removed with a phosphine compound. In some embodiments, the phosphine compound comprises a derived trialkylphosphine portion or a derived triarylphosphine portion. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), or bissulfotriphenylphosphine (BS-TPP) or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[0328] In some embodiments, the nucleotide unit comprises a chain terminating portion selected from the group consisting of: 3'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-thiol, 3'-aminomethyl, 3'-ethyl, 3'butyl, 3'-tert-butyl, 3'-fluorenylmethyloxycarbonyl, 3'-tert-butoxycarbonyl, 3'-O-alkylhydroxyamino, 3'-phosphorothioate, and 3-O-benzyl or a derivative thereof.

[0329] In some embodiments, the multivalent molecule comprises a core connected to a plurality of nucleotide arms, wherein the nucleotide arms comprise spacers, joints and nucleotide units, and wherein the core, joints and / or nucleotide units are labeled with detectable reporter gene moieties. In some embodiments, the detectable reporter gene moieties comprise fluorophores. In some embodiments, the specific detectable reporter gene moieties (e.g., fluorophores) connected to the multivalent molecule can correspond to the bases (e.g., dATP, dGTP, dCTP, dTTP or dUTP) of the nucleotide units to allow detection and identification of nucleotide bases.

[0330] In some embodiments, at least one nucleotide arm of the multivalent molecule has a nucleotide unit connected to a detectable reporter gene portion. In some embodiments, the detectable reporter gene portion is connected to a nucleotide base. In some embodiments, the detectable reporter gene portion comprises a fluorophore. In some embodiments, the specific detectable reporter gene portion (e.g., a fluorophore) connected to the multivalent molecule can correspond to the base (e.g., dATP, dGTP, dCTP, dTTP or dUTP) of the nucleotide unit to allow detection and identification of the nucleotide base.

[0331] In some embodiments, the core of the multivalent molecule comprises an avidin-like or streptavidin-like portion and the core-linked portion comprises biotin. In some embodiments, the core comprises a streptavidin-type or avidin-type portion (including avidin protein), and any derivatives, analogs, and other non-natural forms of avidin that can be bound to at least one biotin portion. Other forms of avidin moieties include natural and recombinant avidin and streptavidin and derived molecules, such as non-glycosylated avidin and truncated streptavidin. For example, avidin moieties include deglycosylated forms of avidin, bacterial streptavidin produced by Streptomyces (Streptomyces) (e.g., Streptomyces avidinii) and derived forms, such as N-acyl avidin, such as N-acetyl, N-phthaloyl, and N-succinyl avidin, and commercially available products EXTRAVIDIN, CAPTAVIDIN, NEUTRAVIDIN, and NEUTRALIITEAVIDIN.

[0332] In some embodiments, any of the methods described herein for sequencing nucleic acid molecules may include forming a binding complex, wherein the binding complex comprises (i) a polymerase, a nucleic acid template molecule duplexed with a primer, and nucleotides, or the binding complex comprises (ii) a polymerase, a nucleic acid template molecule duplexed with a primer, and a nucleotide unit of a multivalent molecule. In some embodiments, the residence time of the binding complex is greater than about 0.1 seconds, 0.2 seconds, 0.3 seconds, 0.4 seconds, 0.5 seconds, 0.6 seconds, 0.7 seconds, 0.8 seconds, 0.9 seconds, or 1 second. The residence time of the binding complex is greater than about 0.1-0.25 seconds, or about 0.25-0.5 seconds, or about 0.5-0.75 seconds, or about 0.75-1 seconds, or about 1-2 seconds, or about 2-3 seconds, or about 3-4 seconds, or about 4-5 seconds, and / or wherein the method is or can be performed at a temperature of at or above 15°C, at or above 20°C, at or above 25°C, at or above 35°C, at or above 37°C, at or above 42°C, at or above 55°C, at or above 60°C, or at or above 72°C, or at or above 80°C, or within a range defined by any of the foregoing. The binding complex (e.g., ternary complex) remains stable until subjected to conditions that result in dissociation of the interaction between any of the polymerase, template molecule, primer, and / or nucleotide units or nucleotides. For example, the dissociation conditions include contacting the binding complex with any one of a detergent, EDTA and / or water or any combination thereof. In some embodiments, the disclosure provides the method, wherein the binding complex is deposited onto a surface, or attached or hybridized to the surface, and the surface shows a contrast-to-noise ratio greater than 20 in the detection step. In some embodiments, the disclosure provides the method, wherein the contacting is performed under conditions that stabilize the binding complex when the nucleotide or nucleotide unit is complementary to the next base of the template nucleic acid, and destabilize the binding complex when the nucleotide or nucleotide unit is not complementary to the next base of the template nucleic acid.

[0333] Fig. 9 Schematic diagrams of multivalent molecules with "starburst" or "helter-skelter" configurations are shown (left panel (class I)); schematic diagrams of multivalent molecules with dendrimer configurations (middle panel (class II)); and schematic diagrams of multiple multivalent molecules formed by reacting streptavidin with 4-arm or 8-arm PEG-NHS with biotin and dNTPs (right panel (class III)). The nucleotide unit is designated as 'N', biotin is designated as 'B', and streptavidin is designated as 'SA'.

[0334] Fig.10 is a schematic diagram of an example multivalent molecule comprising a universal core linked to multiple nucleotide arms according to some embodiments.

[0335] Fig.11 is a schematic diagram of an example multivalent molecule comprising a dendrimer core linked to a plurality of nucleotide arms according to some embodiments.

[0336] Fig.12 A schematic diagram of an example multivalent molecule comprising a core linked to a plurality of nucleotide arms is shown, wherein the nucleotide arms comprise biotin, a spacer, a linker, and a nucleotide unit, according to some embodiments.

[0337] Fig.13 is a schematic diagram of an example nucleotide arm comprising a core linking moiety, a spacer, a linker, and a nucleotide unit according to some embodiments.

[0338] Fig.14 Shown are the chemical structures of example spacers according to some embodiments (top), as well as the chemical structures of various example linkers, including an 11-atom linker, a 16-atom linker, a 23-atom linker, and an N3 linker (bottom).

[0339] Fig.15 Shown are the chemical structures of various example linkers (including linkers 1-9) according to some embodiments.

[0340] Fig.16 Shown are chemical structures of various example linkers joined / connected to nucleotide units according to some embodiments.

[0341] Fig.17 Shown are chemical structures of various example linkers joined / connected to nucleotide units according to some embodiments.

[0342] Fig.18 Shown are chemical structures of various example linkers joined / connected to nucleotide units according to some embodiments.

[0343] Fig.19 Shown are chemical structures of various example linkers joined / connected to nucleotide units according to some embodiments.

[0344] Fig. 20 The chemical structure of an example biotinylated nucleotide arm is shown. In this example, the nucleotide unit is connected to the linker via propargylamine attachment at the 5 position of a pyrimidine base or at the 7 position of a purine base, according to some embodiments.

[0345] Fig. 27 A schematic diagram of one embodiment of a low binding solid support of the present disclosure is shown, wherein the support comprises alternating layers of a glass substrate and a hydrophilic coating covalently or non-covalently adhered to the glass, and the support further comprises chemically reactive functional groups that serve as attachment sites for oligonucleotide primers.

[0346] Compacted oligonucleotides

[0347] The compaction oligonucleotide comprises a single-stranded linear oligonucleotide and a compaction oligonucleotide, the single-stranded linear oligonucleotide having a 5' region that can be hybridized with the first part of the concatemer molecule, and the compaction oligonucleotide having a 3' region that can be hybridized with the second part of the concatemer molecule (e.g., identical concatemer molecule). In certain embodiments, the hybridization of the compaction oligonucleotide and the individual concatemer molecule causes the concatemer molecule to collapse or fold into a DNA nanoball, which is more compact in shape and size compared to the non-collapsed DNA molecule. The point image of the DNA nanoball can be represented as a Gaussian spot, and the size can be measured as a half-maximum full width (FWHM). The smaller point size as indicated by the smaller FWHM is generally associated with the improved image of the point. In certain embodiments, the FWHM of the DNA nanoball point can be about 10um or less. The DNA nanoball can be a compact nucleic acid structure, which has a smaller half-maximum full width (FWHM) compared to the concatemer that is not collapsed / folded into the DNA nanoball.

[0348] In some embodiments, the compacted oligonucleotide comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The compacted oligonucleotide can be any length, including 20-150 nucleotides, or 30-100 nucleotides, or 40-80 nucleotides in length.

[0349] In some embodiments, the compacted oligonucleotide comprises a 5' region and a 3' region, and optionally an intermediate region between the 5' region and the 3' region. The intermediate region can be any length, for example, a length of about 2-20 nucleotides. The intermediate region comprises a homopolymer with continuous identical bases (e.g., AAA, GGG, CCC, TTT or UUU). The intermediate region comprises a non-homopolymer sequence.

[0350] The 5' region of the compacted oligonucleotide can be fully complementary or partially complementary to the first portion of the concatemer molecule along its length. The 3' region of the compacted oligonucleotide can be fully complementary or partially complementary to the second portion of the concatemer molecule along its length. The 5' region of the compacted oligonucleotide can hybridize with the first universal sequence portion of the concatemer molecule. The 3' region of the compacted oligonucleotide can hybridize with the second universal sequence portion of the concatemer molecule. The 5' and 3' regions of the compacted oligonucleotide can hybridize with the concatemer to pull the distal portion of the concatemer together, thereby compacting the concatemer to form a DNA nanoball.

[0351] The 5' region of the compacted oligonucleotide may have the same sequence as the 3' region. The 5' region of the compacted oligonucleotide may have a different sequence than the 3' region. The 3' region of the compacted oligonucleotide may have the opposite sequence than the 5' region.

[0352] In some embodiments, sequence data can be obtained by nanopore sequencing, which comprises sequencing nucleic acids by translocating the nucleic acids across a membrane (such as through a hole), and wherein sequence reading or base calling is performed by measuring one or more signals (such as impedance, current, voltage, or capacitance) during the translocation event. In some embodiments, the identity of the nucleotide can be determined by a unique electrical feature, such as the timing, duration, degree, or linearity of a current block, impedance change, voltage change, or capacitance change. Sequencing nucleic acids by translocation across a membrane and / or through a hole does not exclude alternative detection methods, such as optical, chemical, biochemical, fluorescent, luminescent, magnetic, electromagnetic, acoustic, or electroacoustic detection.

[0353] Supports and low non-specific coatings

[0354] In some embodiments, Figure 1 The flow cell 112 in can include a carrier, for example, a solid phase carrier as disclosed herein. Embodiments of the present disclosure provide paired sequencing compositions and methods, which use a carrier containing multiple oligonucleotide surface primers fixed thereon. In certain embodiments, the carrier is passivated with a low non-specific binding coating. The surface coating described herein exhibits very low non-specific binding to reagents (such as dyes, nucleotides, enzymes, and nucleic acid primers) that are commonly used for nucleic acid capture, amplification, and sequencing workflows. Compared with conventional surface coatings, the surface coating exhibits low background fluorescence signals or high contrast to noise (CNR) ratios.

[0355] The low nonspecific binding coating comprises one or more layers ( Fig. 27). In some embodiments, a variety of surface primers are fixed to the low non-specific binding coating. In some embodiments, at least one surface primer is embedded in the low non-specific binding coating. The low non-specific binding coating enables improved nucleic acid hybridization and amplification performance. Typically, the carrier comprises a substrate (or carrier structure), one or more layers (e.g., silane layers, polymer films) of a covalently or non-covalently attached low binding chemical modification layer, and one or more surface primers that can be used to tether single-stranded nucleic acid library molecules to the carrier's covalently or non-covalently attached. In some embodiments, the coating's deployment (e.g., the chemical composition of one or more layers, the coupling chemistry for cross-linking one or more layers to the carrier and / or cross-linking each other, and the total number of layers) can vary so that proteins, nucleic acid molecules, and other hybridization and amplification reaction components are minimized or reduced relative to comparable monolayers. The deployment of the coating described herein can vary so that relative to comparable monolayers, non-specific hybridization on the coating is minimized or reduced. The deployment of the coating can vary so that relative to comparable monolayers, non-specific amplification on the coating is minimized or reduced. The deployment of the coating can vary so that the specific amplification rate and / or yield on the coating is maximized. In some cases disclosed herein, amplification levels suitable for detection are achieved in no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 cycles of amplification.

[0356] The support structure comprising one or more chemically modified layers (e.g., a layer of a low non-specific binding polymer) can be independent or can be integrated into another structure or component. For example, in some embodiments, the support structure can comprise one or more surfaces within an integrated or assembled microfluidic flow cell. The support structure can comprise one or more surfaces within a microplate format (e.g., the bottom surface of a hole in a microplate). In some embodiments, the support structure comprises the inner surface of a capillary (e.g., the lumen surface). In some embodiments, the support structure comprises the inner surface of a capillary etched into a planar chip (e.g., the lumen surface).

[0357] The attachment chemistry for grafting the first chemically modified layer to the surface of the carrier will generally depend on both the material of the manufacturing surface and the chemical properties of the layer. In some embodiments, the first layer can be covalently attached to the surface. In some embodiments, the first layer can be non-covalently attached to the carrier (e.g., adsorbed to the carrier) by non-covalent interactions (such as electrostatic interactions, hydrogen bonding, or van der Waals interactions) between the molecular components of the carrier and the first layer. In either case, the carrier can be treated before the connection or deposition of the first layer. Any of the surface preparation techniques known to those skilled in the art can be used to clean or treat the surface. For example, a glass or silicon surface can be pickled using a Piranha solution (a mixture of sulfuric acid (H2SO4) and hydrogen peroxide (H2O2), alkaline treatment in KOH and NaOH, and / or cleaned using an oxygen plasma treatment method.

[0358] Silane chemistry constitutes a non-limiting method for covalently modifying silanol groups on glass or silicon surfaces to attach more reactive functional groups (such as amines or carboxyls), which can then be used to couple linker molecules (e.g., linear hydrocarbon molecules of various lengths, such as C6, C12, C18 hydrocarbons or linear polyethylene glycol (PEG) molecules) or layer molecules (e.g., branched PEG molecules or other polymers) to the surface. Examples of suitable silanes that can be used to produce any of the disclosed low binding coatings include, but are not limited to: (3-aminopropyl) trimethoxysilane (APTMS), (3-aminopropyl) triethoxysilane (APTES), any of a variety of PEG-silanes (e.g., containing molecular weights of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silanes (i.e., containing free amino functional groups), maleimide-PEG silanes, biotin-PEG silanes, and the like.

[0359] Any of a variety of molecules known to those skilled in the art (including but not limited to amino acids, peptides, nucleotides, oligonucleotides, other monomers or polymers, or combinations thereof) can be used to produce one or more chemically modified layers on a support, wherein the selection of components used can be varied to alter one or more properties of the layer (e.g., surface density of functional groups and / or tethered oligonucleotide primers, hydrophilicity / hydrophobicity of the layer, or three-dimensional properties of the layer (i.e., "thickness")). Examples of polymers that can be used to produce one or more layers of low nonspecific binding material in any disclosed coating include, but are not limited to, polyethylene glycol (PEG) of various molecular weights and branched structures, streptavidin, polyacrylamide, polyesters, dextran, polylysine and polylysine copolymers, or any combination thereof. Examples of conjugation chemistries that can be used to graft one or more layers of material (e.g., polymer layers) to a surface and / or cross-link layers to each other include, but are not limited to: biotin-streptavidin interaction (or variants thereof), his tag-Ni / NTA conjugation chemistry, methoxy ether conjugation chemistry, carboxylate conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiols, epoxies, azides, hydrazides, alkynes, isocyanates, and silanes.

[0360] The low non-specific binding surface coating can be applied uniformly across the carrier. Alternatively, the surface coating can be patterned so that the chemically modified layer is confined to one or more discrete regions of the carrier. For example, photolithography can be used to pattern the coating to produce an ordered array or random pattern of chemically modified regions on the carrier. Alternatively or in combination, contact printing and / or inkjet printing techniques can be used, for example, to pattern the coating. In some embodiments, the ordered array or random pattern of chemically modified regions may comprise at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 or more discrete regions.

[0361] In certain embodiments, the low non-specific binding coating comprises a hydrophilic polymer that is non-specifically adsorbed or covalently grafted to a carrier. Typically, poly(ethylene glycol) (PEG, also referred to as polyethylene oxide (PEO) or polyoxyethylene) or other hydrophilic polymers with different molecular weights and end groups connected to the carrier using, for example, silane chemistry are used to perform passivation. End groups away from the surface may include, but are not limited to, biotin, methoxy ethers, carboxylates, amines, NHS esters, maleimides, and disilanes. In certain embodiments, two or more layers of a hydrophilic polymer (e.g., a linear polymer, a branched polymer, or a multi-branched polymer) may be deposited on the surface. In certain embodiments, two or more layers may be covalently coupled to each other or internally cross-linked to improve the stability of the resulting coating. In certain embodiments, surface primers (or other biomolecules, e.g., enzymes or antibodies) with different nucleotide sequences and / or base modifications may be tethered to the resulting layer with various surface densities. In certain embodiments, for example, both surface functional group density and surface primer concentration may vary to obtain a desired surface primer density range. Additionally, the surface primer density can be controlled by diluting the surface primer with other molecules carrying the same functional group. For example, the surface primer labeled with an amine can be diluted with polyethylene glycol labeled with an amine in the reaction with the surface coated with NHS-ester to reduce the final primer density. The surface primer with a joint of different lengths between the hybridization zone and the surface connection functional group can also be applied to control the surface density. The example of a suitable joint includes poly-T and poly-A chains (for example, 0 to 20 bases), PEG joints (for example, 3 to 20 monomer units) and carbon chains (for example, C6, C12, C18, etc.) at the 5' end of the primer. In order to measure the primer density, the primer labeled with a fluorescent mode can be tethered to the surface, and then the fluorescence reading is compared with the fluorescence reading of the dye solution of known concentration.

[0362] In some embodiments, the low nonspecific binding coating comprises a functionalized polymer coating covalently bound to at least a portion of the support via a chemical group on the support, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating and the functionalized polymer coating on the primer. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM).

[0363] In order to scale the primer surface density and add additional dimensions to the hydrophilic or amphiphilic coating, a carrier containing a multilayer coating of PEG and other hydrophilic polymers has been developed. By using hydrophilic and amphiphilic surface layering methods (including but not limited to polymer / copolymer materials described below), the primer loading density on the carrier can be significantly increased. The traditional PEG coating method uses a monolayer primer deposition, which is generally reported for single molecule applications, but does not produce high copy numbers for nucleic acid amplification applications. As described herein, "layering" can be achieved using traditional cross-linking methods with any compatible polymer or monomer subunit, so that a surface containing two or more highly cross-linked layers can be sequentially constructed. Examples of suitable polymers include but are not limited to streptavidin, polyacrylamide, polyester, dextran, polylysine, and copolymers of polylysine and PEG. In certain embodiments, different layers can be connected to each other by any of a variety of conjugation reactions (including but not limited to: biotin-streptavidin binding, azide-alkyne click reaction, amine-NHS ester reaction, thiol-maleimide reaction, and ionic interactions between positively charged polymers and negatively charged polymers). In some embodiments, high primer density materials can be constructed in solution and then layered onto a surface in multiple steps.

[0364] Examples of materials from which the support structure may be made include, but are not limited to, glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic support structures are contemplated.

[0365] The support structure can be presented in any of a variety of geometric shapes and sizes known to those skilled in the art, and can include any of a variety of materials known to those skilled in the art. For example, the support structure can be locally planar (e.g., a surface comprising a microscope slide or a microscope slide). In general, the support structure can be cylindrical (e.g., an inner surface comprising a capillary or a capillary), spherical (e.g., an outer surface comprising a non-porous bead) or irregular (e.g., an outer surface comprising an irregularly shaped non-porous bead or particle). In certain embodiments, the surface of the support structure for nucleic acid hybridization and amplification can be a solid non-porous surface. In certain embodiments, the surface of the support structure for nucleic acid hybridization and amplification can be porous, so that the coating described herein penetrates the porous surface, and the nucleic acid hybridization and amplification reactions performed thereon can occur in the hole.

[0366] The support structure comprising one or more chemically modified layers (e.g., a layer of a low non-specific binding polymer) can be independent or can be integrated into another structure or component. For example, the support structure can comprise one or more surfaces within an integrated or assembled microfluidic flow cell. The support structure can comprise one or more surfaces within a microwell plate format (e.g., the bottom surface of a hole in a microwell plate). In some embodiments, the support structure comprises the inner surface of a capillary (e.g., the lumen surface). In some embodiments, the support structure comprises the inner surface of a capillary etched into a planar chip (e.g., the lumen surface).

[0367] As noted, low non-specific binding carriers of the present disclosure show non-specific binding of the reduction of other components of proteins, nucleic acids and hybridization and / or amplification preparations for solid phase nucleic acid amplification. The degree of non-specific binding shown by a given carrier surface can be assessed in a qualitative manner or in a quantitative manner. For example, under a set of standardized conditions, the surface is exposed to a fluorescent dye (for example, cyanine (such as Cy3 or Cy5, etc.), fluorescein, coumarin, rhodamine, etc. or other dye disclosed herein), through fluorescently labeled nucleotides, through fluorescently labeled oligonucleotides and / or through fluorescently labeled proteins (for example, polymerase), then the specified flushing scheme and fluorescence imaging can be used as a qualitative tool to compare the non-specific binding on the carrier comprising different surface preparations. In some embodiments, in some aspects, the surface is exposed to fluorescent dyes, fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g., polymerases) under a standardized set of conditions, and then a specified wash regimen and fluorescence imaging can be used as a quantitative tool to compare nonspecific binding on carriers containing different surface preparations - provided that care has been taken to ensure that the fluorescence imaging is linearly related (or predictably related) to the number of fluorophores on the carrier surface and that suitable calibration standards are used (e.g., under conditions where signal saturation and / or self-quenching of fluorophores is not a problem). In some embodiments, other techniques known to those skilled in the art (e.g., radioisotope labeling and counting methods) can be used to quantitatively assess the degree of nonspecific binding exhibited by different carrier surface preparations of the present disclosure.

[0368] Some surfaces disclosed herein exhibit a ratio of specific binding to non-specific binding of a fluorophore (such as Cy3) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value encompassed by ranges herein. Some surfaces disclosed herein exhibit a ratio of specific fluorescence to non-specific fluorescence of a fluorophore (such as Cy3) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value encompassed by ranges herein.

[0369] The degree of non-specific binding exhibited by the disclosed low binding carrier can be assessed using a standardized protocol for the following: contact the surface with a labeled protein (e.g., bovine serum albumin (BSA), streptavidin, DNA polymerase, reverse transcriptase, helicase, single-stranded binding protein (SSB), etc., or any combination thereof), labeled nucleotides, labeled oligonucleotides, etc., under a standardized set of incubation and flushing conditions, followed by detection of the amount of labeling remaining on the surface and comparing the signal obtained therefrom with an appropriate calibration standard. In some embodiments, the labeling can include a fluorescent label. In some embodiments, the labeling can include a radioactive isotope. In some embodiments, the labeling can include any other detectable labeling known to those skilled in the art. In some embodiments, the degree of non-specific binding exhibited by a given carrier surface preparation can therefore be assessed based on the number of non-specifically bound protein molecules (or nucleic acid molecules or other molecules) / unit area. In some embodiments, the low binding carrier of the present disclosure may exhibit the following non-specific protein binding (or non-specific binding of other specified molecules (e.g., cyanine (such as Cy3 or Cy5, etc.), fluorescein, coumarin, rhodamine, etc. or other dyes disclosed herein)): less than 0.001 molecules / μm 2 , less than 0.01 molecule / μm 2 , less than 0.1 molecule / μm 2 , less than 0.25 molecules / μm 2 , less than 0.5 molecules / μm 2 , less than 1 molecule / μm 2 , less than 10 molecules / μm 2 , less than 100 molecules / μm 2 or less than 1,000 molecules / μm 2 Those skilled in the art will recognize that a given support surface of the present disclosure may exhibit a density anywhere within this range (e.g., less than 86 molecules / μm2 ) non-specific binding. For example, after contact with 1 μM Cy3-labeled streptavidin (GE Amersham) in phosphate buffered saline (PBS) buffer for 15 minutes, followed by rinsing with deionized water three times, some of the modified surfaces disclosed herein exhibited less than 0.5 molecules / μm 2 Some of the modified surfaces disclosed herein exhibit less than 0.25 molecules / μm 2In a separate nonspecific binding assay, 1 μM labeled Cy3 SA (ThermoFisher), 1 μM Cy5 SA dye (ThermoFisher), 10 μM aminoallyl-dUTP-ATTO-647N (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rhol 1 (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rhol 1 (Jena Biosciences), 10 μM 7-propargylamino-7-deaza-dGTP-Cy5 (Jena Biosciences), and 10 μM 7-propargylamino-7-deaza-dGTP-Cy3 (Jena Biosciences) were incubated on low-binding coated supports in a 384-well plate format at 37°C for 15 minutes. Each well is rinsed 2-3 times with 50ul deionized RNase / DNase-free water, and rinsed 2-3 times with 25mMACES buffer (pH 7.4).Use Cy3, AF555 or Cy5 filter set (according to the dye test performed) as specified by the manufacturer with 800 PMT gain setting and 50-100 μ m resolution on GE Typhoon instrument to image 384 well plates. For higher resolution imaging, images were collected on an Olympus IX83 microscope (e.g., an inverted fluorescence microscope) (Olympus Corp., Center Valley, Pa.) with a total internal reflection fluorescence (TIRF) objective (100×, 1.5 NA, Olympus), a CCD camera (e.g., an Olympus EM-CCD monochrome camera, an Olympus XM-10 monochrome camera, or an Olympus DP80 color and monochrome camera), an illumination source (e.g., an Olympus 100 W mercury lamp, an Olympus 75 W xenon lamp, or an Olympus U-HGLGPS fluorescence light source), and an excitation wavelength of 532 nm or 635 nm. Dichroic mirrors were purchased from Semrock (IDEX Health & Science, LLC, Rochester, NY), such as 405 nm, 488 nm, 532 nm, or 633 nm dichroic mirrors / beam splitters, and the bandpass filter was selected to be a 532LP or 645LP integrated with the appropriate excitation wavelength. Some modified surfaces disclosed herein exhibit less than 0.25 molecules / μm 2 In some embodiments, the coated support is immersed in a buffer (e.g., 25 mM CAES, pH 7.4) while acquiring an image.

[0370] In some embodiments, the surfaces disclosed herein exhibit a ratio of specific binding to non-specific binding of a fluorophore (such as Cy3) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate values ​​encompassed by ranges herein. In some embodiments, the surfaces disclosed herein exhibit a ratio of specific fluorescence signal to non-specific fluorescence signal of a fluorophore (such as Cy3) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate values ​​encompassed by ranges herein.

[0371] Low background surfaces consistent with the disclosure herein can exhibit a ratio of specific dye attachment (e.g., Cy3 attachment) to nonspecific dye adsorption (e.g., Cy3 dye adsorption) of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50 attached specific dye molecules / nonspecifically adsorbed molecules. Similarly, low background surfaces consistent with the disclosure herein that have been attached to fluorophores (e.g., Cy3) can exhibit a ratio of specific fluorescent signals (e.g., generated from Cy3-labeled oligonucleotides attached to the surface) to nonspecifically adsorbed dye fluorescent signals of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50:1 when subjected to excitation energy.

[0372] In some embodiments, the degree of hydrophilicity (or "wetting" with aqueous solutions) of the disclosed carrier surface can be evaluated, for example, by measuring the water contact angle, wherein a small drop of water is placed on the surface and its contact angle with the surface is measured using, for example, an optical tensiometer. In some embodiments, a static contact angle can be determined. In some embodiments, an advancing or receding contact angle can be determined. In some embodiments, the water contact angle for a hydrophilic low binding carrier surface disclosed herein can be in the range of about 0 degrees to about 30 degrees. In some embodiments, the water contact angle for a hydrophilic low binding carrier surface disclosed herein can be no more than 50 degrees, 40 degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees or 1 degree. In many cases, the contact angle does not exceed 40 degrees. Those skilled in the art will recognize that a given hydrophilic low binding carrier surface of the present disclosure can exhibit a water contact angle with a value anywhere within this range.

[0373] In some embodiments, the hydrophilic surfaces disclosed herein help reduce wash times for bioassays, which is generally due to reduced non-specific binding of biomolecules to low binding surfaces. In some embodiments, a sufficient wash step can be performed in less than 60 seconds, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 15 seconds, 10 seconds, or less than 10 seconds. For example, a sufficient wash step can be performed in less than 30 seconds.

[0374] Some low binding surfaces disclosed herein exhibit significant improvements in stability or durability to prolonged exposure to solvents and elevated temperatures or to repeated cycles of solvent exposure or changes in temperature. For example, the stability of the disclosed surfaces can be tested by fluorescently labeling functional groups on the surface or tethered biomolecules (e.g., oligonucleotide primers) on the surface and monitoring the fluorescent signal before, during, and after prolonged exposure to solvents and elevated temperatures or repeated cycles of solvent exposure or changes in temperature. In some embodiments, the degree of change in fluorescence used to assess the quality of a surface can be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% (or any combination of these percentages as measured over these time periods) over a period of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 100 hours of exposure to a solvent and / or elevated temperature. In some embodiments, the degree of change in fluorescence used to assess the quality of a surface can be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20% or 25% (or any combination of these percentages as measured over this range of cycles) over 5 cycles, 10 cycles, 20 cycles, 30 cycles, 40 cycles, 50 cycles, 60 cycles, 70 cycles, 80 cycles, 90 cycles, 100 cycles, 200 cycles, 300 cycles, 400 cycles, 500 cycles, 600 cycles, 700 cycles, 800 cycles, 900 cycles or 1,000 cycles.

[0375] In some embodiments, the surfaces disclosed herein can exhibit a high ratio of specific signal to nonspecific signal or other background. For example, when used for nucleic acid amplification, some surfaces can exhibit an amplified signal that is at least 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 75 times, 100 times, or more than 100 times the signal of the adjacent unpopulated region of the surface. Similarly, some surfaces exhibit an amplified signal that is at least 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 75 times, 100 times, or more than 100 times the signal of the adjacent amplified nucleic acid population region of the surface.

[0376] In some embodiments, when used in nucleic acid hybridization or amplification applications to generate populations of hybridized or clonally amplified nucleic acid molecules (e.g., that have been directly or indirectly labeled with a fluorophore), fluorescent images of the disclosed low background surfaces exhibit a contrast to noise ratio (CNR) of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 210, 220, 230, 240, 250, or greater.

[0377] One or more types of primers can be connected or tethered to the carrier surface. In some embodiments, one or more types of adapters or primers can include a spacer sequence, an adapter sequence for hybridizing to a target library nucleic acid sequence connected to the adapter, a forward amplification primer, a reverse amplification primer, a sequencing primer, and / or a molecular barcode sequence, or any combination thereof. In some embodiments, 1 primer or adapter sequence can be tethered to at least one layer of the surface. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different primers or adapter sequences can be tethered to at least one layer of the surface.

[0378] In some embodiments, the length of the tethered adapter and / or primer sequence can be in the range of about 10 nucleotides to about 100 nucleotides. In some embodiments, the length of the tethered adapter and / or primer sequence can be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides. In some embodiments, the length of the tethered adapter and / or primer sequence can be at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, or at most 10 nucleotides. Any of the lower and upper limit values ​​described in this paragraph can be combined to form a range included in the present disclosure, for example, in some embodiments, the length of the tethered adapter and / or primer sequence can be in the range of about 20 nucleotides to about 80 nucleotides. One skilled in the art will recognize that the length of the tethered adaptor and / or primer sequence can have any value within this range, for example, about 24 nucleotides.

[0379] In some embodiments, the resulting surface density of primers (eg, capture primers) on the surface of a low binding support of the present disclosure can be between about 100 primer molecules / μm 2 To about 100,000 primer molecules / μm 2 In some embodiments, the resulting surface density of primers on the surface of the low binding support of the present disclosure can be about 1,000 primer molecules / μm 2 To about 1,000,000 primer molecules / μm 2 In some embodiments, the surface density of the primers can be at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 molecules / μm 2 In some embodiments, the surface density of primers may be at most 1,000,000, at most 100,000, at most 10,000, or at most 1,000 molecules / μm 2 Any of the lower and upper values ​​described in this paragraph can be combined to form a range included in the present disclosure. For example, in some embodiments, the surface density of primers can be about 10,000 molecules / μm. 2 to about 100,000 molecules / μm 2 Those skilled in the art will recognize that the surface density of primer molecules can have any value within this range, for example, about 455,000 molecules / μm 2In some embodiments, the surface density of target library nucleic acid sequences that initially hybridize to adapter or primer sequences on the support surface can be less than or equal to the surface density indicated by the surface density of tethered primers. In some embodiments, the surface density of clonally amplified target library nucleic acid sequences that hybridize to adapter or primer sequences on the support surface can span the same range as the range indicated by the surface density of tethered primers.

[0380] The local densities listed above do not exclude variations in density across the surface, such that a surface may contain oligonucleotides at a density of, for example, 500,000 / μm 2 region, while also comprising at least a second region having a significantly different local density.

[0381] In some embodiments, the performance of nucleic acid hybridization and / or amplification reactions using the disclosed reaction formulations and low-binding carriers can be evaluated using fluorescent imaging techniques, wherein the contrast-to-noise ratio (CNR) of the image provides a key metric in evaluating amplification-specific and non-specific binding on the carrier. CNR is generally defined as: CNR = (signal-background) / noise. The background term is generally considered to be the signal measured for the gap region around a specific feature (diffraction-limited spot (DLS)) in a specified region of interest (ROI). Although the signal-to-noise ratio (SNR) is generally considered to be a benchmark for overall signal quality, it can be demonstrated that in applications requiring rapid image capture (e.g., sequencing applications where cycle time must be minimized), improved CNR can provide significant advantages over SNR as a benchmark for signal quality, as shown in the examples below. At high CNRs, even with moderate improvements in CNR, the imaging time required to achieve accurate differentiation (and therefore accurate base calls in the case of sequencing applications) can be greatly reduced. Improved CNR in imaging data with respect to imaging integration time provides a method for more accurately detecting features (such as clonally amplified nucleic acid colonies on the surface of a carrier).

[0382] In most ensemble-based sequencing methods, the background term is usually measured as the signal associated with the 'gap' region. 间隙 ), the “intrastitial” background (B 内质 ) is present in the area occupied by the amplified DNA colonies. The combination of these two background signals determines the achievable CNR and subsequently directly affects the optical instrument requirements, architecture cost, reagent cost, run time, cost / genome, and ultimately the accuracy and data quality of cycle array-based sequencing applications. 间隙Background signals are generated from a variety of sources; some examples include: autofluorescence from consumable flow cells, nonspecific adsorption of detection molecules that produce false fluorescent signals that may obscure signals from the ROI, and the presence of nonspecific DNA amplification products (e.g., nonspecific DNA amplification products generated from primer dimers). In typical next-generation sequencing (NGS) applications, this background signal in the current field of view (FOV) is averaged and subtracted over time. The signals generated from individual DNA colonies (i.e., (signal)-B(gap) in the FOV) generate distinguishable features that can be classified. In some embodiments, endoplasmic background (B(endoplasmic)) can contribute confounding fluorescent signals that are not specific to the target of interest, but are present in the same ROI, thus making averaging and subtraction much more difficult.

[0383] Nucleic acid amplification on low-binding coated carriers described herein can reduce B (gap) background signals by reducing nonspecific binding, which can lead to improvements in specific nucleic acid amplification and can lead to reductions in nonspecific amplification (which can affect background signals generated from both gaps and endoplasmic regions). In some embodiments, the disclosed low-binding coated carriers (optionally used in combination with the disclosed hybridization and / or amplification reaction preparations) can improve CNR by 2-fold, 5-fold, 10-fold, 100-fold, 250-fold, 500-fold, or 1000-fold compared to improvements achieved using conventional carriers and hybridization, amplification, and / or sequencing protocols. Although described herein in the context of using fluorescence imaging as a readout or detection mode, the same principles also apply to using the disclosed low-binding coated carriers and nucleic acid hybridization and amplification preparations for other detection modes (including both optical and non-optical detection modes).

[0384] The headings provided herein are not limitations of the various embodiments of the disclosure, which can be understood by referring to the specification as a whole.

[0385] Unless otherwise defined, the technology and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art. In general, the terms related to the technology of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production and hybridization described herein are well-known and commonly used terms in the art. The technology and procedures described herein are usually performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, see Sambrook et al., " Molecular Cloning: Laboratory Manual (Molecular Cloning:A Laboratory Manual)" (3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) 2000). See also Ausubel et al., " Molecular Biology Experiment Guide (Current Protocols in Molecular Biology) ", Greene Publishing Associates (Greene Publishing Associates) (1992). The nomenclature used in combination with laboratory procedures and techniques described herein is well known and commonly used in the art.

[0386] Unless otherwise required by context herein, singular terms shall include pluralities and plural terms shall include the singular. The singular forms "a / an" and "the" and any word used in the singular include plural referents unless expressly and unequivocally limited to one referent.

[0387] It should be understood that the use of alternative terms (eg, "or") is taken to mean one or both of the alternatives, or any combination thereof.

[0388] As used herein, the term "and / or" will be deemed to mean that each of the specified features or components is explicitly disclosed with or without the other. For example, the term "and / or" used in phrases such as "A and / or B" herein is intended to include: "A and B"; "A or B"; "A" (A alone); and "B" (B alone). In a similar manner, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: "A, B, and C"; "A, B, or C"; "A or C"; "A or B"; "B or C"; "A and B"; "B and C"; "A and C"; "A" (A alone); "B" (B alone); and "C" (C alone).

[0389] As used herein and in the appended claims, the terms "comprising," "including," "having," and "containing," and grammatical variations thereof, as used herein, are intended to be non-limiting, such that one or more items in a list are not exclusive of other items that can be substituted or added to the listed items. It should be understood that whenever an embodiment is described herein with the language "comprising," other similar embodiments described with "consisting of" and / or "consisting essentially of" are also provided.

[0390] As used herein, the terms "about", "approximately" and "substantially" refer to values ​​or compositions within an acceptable error range for a particular value or composition as determined by a person of ordinary skill in the art, which will depend in part on how to measure or determine the value or composition, i.e., the limitations of the measurement system. For example, according to practice in the art, "about", "approximately" or "substantially" may mean within one or more than one standard deviation. Alternatively, "about" or "approximately" may mean a range of up to 10% (i.e., ± 10%) or more, depending on the limitations of the measurement system. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. In addition, particularly for biological systems or methods, the term may mean a value of up to an order of magnitude or up to 5 times. When a particular value or composition is provided in the present disclosure, unless otherwise stated, the meaning of "about", "approximately", "substantially" should be assumed to be within an acceptable error range for the particular value or composition. In addition, in the case of providing a range and / or sub-range of a value, the range and / or sub-range may include the endpoints of the range and / or sub-range.

[0391] The term "colony" as used herein refers to a nucleic acid library molecule that can be cloned in a solution or on a carrier to generate an amplicon that can serve as a template molecule for sequencing. In certain embodiments, a linear library molecule can be cyclized to generate a cyclized library molecule, and the cyclized library molecule can be cloned in a solution or on a carrier to generate a concatemer. In certain embodiments, a concatemer can serve as a nucleic acid template molecule that can be sequenced. A concatemer is sometimes referred to as a colony. In certain embodiments, a colony includes a nucleotide chain.

[0392] The terms "peptide", "polypeptide" and "protein" and other related terms used herein are used interchangeably and refer to polymers of amino acids and are not limited to any specific length. Polypeptides may contain natural and non-natural amino acids. Polypeptides include recombinant or chemically synthesized forms. Polypeptides also include precursor molecules that have not yet undergone post-translational modifications, such as proteolytic cleavage, cleavage due to ribosome skipping, hydroxylation, methylation, lipidation, acetylation, SUMOylation, ubiquitination, glycosylation, phosphorylation and / or disulfide bond formation. These terms encompass natural and artificial proteins, protein fragments and polypeptide analogs (such as mutant proteins, variants, chimeric proteins and fusion proteins) of protein sequences, as well as proteins that are covalently or non-covalently modified after translation or otherwise.

[0393] As used herein, the term "polymerase" and its variants include any enzyme that can catalyze nucleotides (including analogs thereof) to polymerize into nucleic acid chains. Generally, but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Generally, the polymerase includes one or more active sites at which nucleotide binding and / or nucleotide polymerization catalysis can occur. In some embodiments, the polymerase includes other enzymatic activities, such as 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, the polymerase has strand displacement activity. The polymerase may include, but is not limited to, naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fused or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives or fragments thereof (e.g., catalytically active fragments) that retain the ability to catalyze nucleotide polymerization. In some embodiments, the polymerase may be separated from a cell or produced using recombinant DNA technology or chemical synthesis methods. In some embodiments, the polymerase may be expressed in prokaryotes, eukaryotes, viruses or bacteriophage organisms. In some embodiments, the polymerase may be a protein or fragment thereof that is post-translationally modified. The polymerase may be derived from a prokaryotic organism, a eukaryotic organism, a virus or a bacteriophage. The polymerase comprises a DNA-guided DNA polymerase and an RNA-guided DNA polymerase.

[0394] As used herein, the term "fidelity" refers to the accuracy of DNA polymerization by a template-dependent DNA polymerase. The fidelity of a DNA polymerase is usually measured by the error rate (the frequency of incorporation of inaccurate nucleotides (i.e., nucleotides that are not complementary to the template nucleotides)). The accuracy or fidelity of DNA polymerization is maintained by the polymerase activity and 3'-5' exonuclease activity of the DNA polymerase.

[0395] As used herein, the term "binding complex" refers to a complex formed by binding together free nucleotides or nucleotide units of a nucleic acid duplex, a polymerase, and a multivalent molecule, wherein the nucleic acid duplex comprises a nucleic acid template molecule hybridized with a nucleic acid primer. In the binding complex, the free nucleotides or nucleotide units may or may not bind to the 3' end of the nucleic acid primer at a location relative to the complementary nucleotide in the nucleic acid template molecule. A "ternary complex" is an example of a binding complex formed by binding together free nucleotides or nucleotide units of a nucleic acid duplex, a polymerase, and a multivalent molecule, wherein the free nucleotides or nucleotide units bind to the 3' end of the nucleic acid primer (as part of the nucleic acid duplex) at a location relative to the complementary nucleotide in the nucleic acid template molecule.

[0396] The term "residence time" and related terms refer to the length of time that a binding complex remains stable without any component dissociation, wherein the components of the binding complex include nucleic acid templates and nucleic acid primers, polymerases, nucleotide units of multivalent molecules or free (e.g., unconjugated) nucleotides. Nucleotide units or free nucleotides can be complementary or non-complementary to the nucleotide residues in the template molecule. Nucleotide units or free nucleotides can be combined with the 3' end of the nucleic acid primer at a location relative to the complementary nucleotide residues in the nucleic acid template molecule. The residence time indicates the stability of the binding complex and the intensity of the binding interaction. The residence time can be measured by observing the start and / or duration of the binding complex (such as by observing the signal of the labeled component from the binding complex). For example, labeled nucleotides or labeled reagents comprising one or more nucleotides can be present in the binding complex, so that the signal from the label is allowed to be detected during the residence time of the binding complex. An example label is a fluorescent label. The binding complex (e.g., a ternary complex) remains stable before being subjected to the conditions of the dissociation of the interaction between any one of the polymerase, template molecule, primer and / or nucleotide units or nucleotides. For example, the dissociation conditions comprise contacting the bound complex with any one of a detergent, EDTA, and / or water, or any combination thereof.

[0397] The terms "nucleic acid", "polynucleotide" and "oligonucleotide" and other related terms used herein are used interchangeably and refer to polymers of nucleotides and are not limited to any specific length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids and non-natural nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids include polymers of nucleotides, wherein nucleotides include natural or non-natural bases and / or sugars. Nucleic acids include naturally occurring internucleoside bonds, such as phosphodiester bonds. Nucleic acids include non-natural internucleoside bonds, including thiophosphates, sulfur-containing phosphates, or peptide nucleic acid (PNA) bonds. In some embodiments, nucleic acids include a mixture of one type of polynucleotide or two or more different types of polynucleotides.

[0398] The term "primer" and related terms used herein refer to natural or synthetic oligonucleotides that can hybridize with DNA and / or RNA polynucleotide templates to form duplex molecules. Primers can have any length, but generally range from 4 to 50 nucleotides. Typical primers include 5' ends and 3' ends. The 3' end of a primer can include a 3'OH portion that serves as a nucleotide polymerization initiation site in a polymerase-mediated primer extension reaction. Alternatively, the 3' end of a primer can lack a 3'OH portion or can include a terminal 3' blocking group that inhibits nucleotide polymerization in a polymerase-mediated reaction. Any one nucleotide along the length of the primer or more than one nucleotide can be labeled with a detectable reporter gene portion. Primers can be in solution (e.g., soluble primers) or can be fixed to a carrier (e.g., capture primers).

[0399] The terms "template nucleic acid", "template polynucleotide", "target nucleic acid", "target polynucleotide", "template strand" and other variants refer to a nucleic acid strand that serves as a base nucleic acid molecule for generating a complementary nucleic acid strand. The template nucleic acid may be single-stranded or double-stranded, or the template nucleic acid may have a single-stranded or double-stranded portion. The sequence of the template nucleic acid may be partially or completely complementary to the sequence of the complementary strand. The template nucleic acid may be obtained from a naturally occurring source, a recombinant form, or chemically synthesized to include any type of nucleic acid analog. The template nucleic acid may be linear, circular, or in other forms. The template nucleic acid may include an insertion region having an insertion sequence also referred to as a sequence of interest. The template nucleic acid may also include at least one adapter sequence. The template nucleic acid may be a concatemer having two or tandem copies of a sequence of interest and at least one adapter sequence. The insertion region may be isolated in any form, including chromosomes, genomes, organelles (e.g., mitochondria, chloroplasts, or ribosomes), recombinant molecules, cloned, amplified cDNA, RNA (e.g., pre-mRNA or mRNA), oligonucleotides, whole genomic DNA obtained from freshly frozen paraffin-embedded tissue, needle biopsies, cell-free circulating DNA, or any type of nucleic acid library. Insertion regions can be isolated from any source, including from organisms such as prokaryotes, eukaryotes (e.g., humans, plants, and animals), fungi, viruses, cells, tissues, normal or diseased cells or tissues, body fluids, including blood, urine, serum, lymph, tumors, saliva, anal and vaginal secretions, amniotic fluid samples, sweat, semen, environmental samples, culture samples, or synthetic nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. Insertion regions can be isolated from any organ, including the head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestine, bladder, prostate, testis, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin, heart, larynx, or other organs. Template nucleic acids can be subjected to nucleic acid analysis (including sequencing and composition analysis).

[0400] When used to refer to nucleic acid molecules, the term "hybridize" or "hybridizing" or "hybridization" or other related terms refer to hydrogen bonding between two different nucleic acids to form a duplex nucleic acid. Hybridization also includes hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridizing molecule with a duplex region. Hybridization can include Watson-Crick or Hoogstein binding to form a duplex double-stranded nucleic acid or a double-stranded region within a nucleic acid molecule. Two different regions of a double-stranded nucleic acid or a single nucleic acid can be fully complementary or partially complementary. Complementary nucleic acid chains do not need to hybridize to each other across their entire length. Complementary base pairing can be standard AT or CG base pairing or can be other forms of base pairing interactions. Duplex nucleic acids can include mismatched base-paired nucleotides.

[0401] The term "nucleotide" and related terms refer to molecules comprising an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose) and at least one phosphate group. Regular or non-regular nucleotides are consistent with the use of the term. In certain embodiments, phosphates comprise monophosphates, diphosphates or triphosphates, or corresponding phosphate analogs. In certain embodiments, nucleotides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate groups. The term "nucleoside" refers to a molecule comprising an aromatic base and a sugar.

[0402] Nucleotides (and nucleosides) typically contain heterocyclic bases, including substituted or unsubstituted nitrogen-containing parent heteroaromatic rings (typically present in nucleic acids), including naturally occurring, substituted, modified or engineered variants or analogs thereof. The base of a nucleotide (or nucleoside) is capable of forming Watson-Crick and / or Hoostein hydrogen bonds with an appropriate complementary base. Example bases include, but are not limited to, purines and pyrimidines, such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethyleneadenine, N 6 -Δ 2 -Isopentenyl adenine (6iA), N 6 -Δ 2 -Isopentenyl-2-methylthioadenine (2ms6iA), N 6 -methyladenine, guanine (G), isoguanine, N 2 -dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O 6-methylguanine; 7-deaza-purines, such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines, such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O 4 -methylthymine, uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; agarosin; inosine; hydroxymethylcytosine; 5-methylcytosine; base (Y); and methylated, glycosylated and acylated base moieties; etc. Additional example bases can be found in Fasman, 1989, "Practical Handbook of Biochemistry and Molecular Biology", pp. 385-394, CRC Press, Boca Raton, Fla.

[0403] Nucleotides (and nucleosides) typically contain sugar moieties such as carbocyclic moieties (Ferraro and Gotor 2000 Chem. Rev. 100:4319-48), acyclic moieties (Martinez et al. 1999 Nucleic Acids Research 27:1271-1274; Martinez et al. 1997 Bioorganic & Medicinal Chemistry Letters 7:3013-3016), and other sugar moieties (Joeng et al. 1993 J. Med. Chem. 36:2627-2638; Kim et al. 1993 J. Med. Chem. 36:30-7; Eschenmosser et al. 1994 J. Med. Chem. 36:31-32; Kim et al. 1994 J. Med. Chem. 36:32-33; Eschenmosser et al. 1994 J. Med. Chem. 36:33-34; Kim et al. 1994 J. Med. Chem. 36:33-33; Eschenmosser ... 1999 Science 284:2118-2124; and U.S. Pat. No. 5,558,991). The sugar moiety includes: ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-thiolribosyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-thiolribosyl; 3'-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

[0404] In some embodiments, the nucleotide comprises a chain of one, two or three phosphorus atoms, wherein the chain is generally connected to the 5' carbon of the sugar moiety by an ester or phosphoramide bond. In some embodiments, the nucleotide is an analog with a phosphorus chain, wherein the phosphorus atoms are linked together with an intervening O, S, NH, methylene or ethylene. In some embodiments, the phosphorus atoms in the chain include substituted side groups (including O, S or BH3). In some embodiments, the chain includes a phosphate group substituted with an analog, including phosphoramide, phosphorothioate, phosphorodithioate and O-methylphosphoramidite groups.

[0405] When used in reference to nucleic acids, the terms "extend," "extending," "extension," and other variants refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation comprises polymerization of one or more nucleotides into the terminal 3'OH end of a nucleic acid chain, resulting in extension of the nucleic acid chain. Nucleotide incorporation can be performed with natural nucleotides and / or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent manner. Any suitable method for extending nucleic acid molecules can be used, including primer extension catalyzed by a DNA polymerase or an RNA polymerase.

[0406] The term "reporter moiety", "reporter moieties" or related terms refers to a compound that produces or causes a detectable signal. The reporter moiety is sometimes referred to as a "label". Any suitable reporter moiety can be used, including luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophore, radioisotope, electrochemistry, mass spectrometry, Raman, hapten, affinity tag, atom or enzyme. The reporter moiety produces a detectable signal caused by a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzymatic activity or adjacent event). The adjacent event includes two reporter moieties approaching, associating or binding to each other. It is well known to those skilled in the art that the reporter moiety is selected so that each reporter moiety absorbs excitation radiation and / or emits fluorescence at a wavelength that can be distinguished from other reporter moieties to allow monitoring of the presence of different reporter moieties in the same reaction or in different reactions. Two or more different reporter moieties with spectrally distinct emission curves or with minimal overlapping spectral emission curves can be selected. The reporter moiety can be linked (eg, operably linked) to a nucleotide, nucleoside, nucleic acid, enzyme (eg, polymerase or reverse transcriptase), or carrier (eg, surface).

[0407] The reporter gene portion (or label) comprises a fluorescent marker or fluorophore. Example fluorescent moieties that can serve as fluorescent markers or fluorophores include, but are not limited to, fluorescein and fluorescein derivatives (such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein semicarbazide, carbohydrazinomethylthioacetamidofluorescein), rhodamine and rhodamine derivatives (such as TRITC, TMR, lissamine rhodamine (lissamine rhodamine), Texas Red, Rhodamine B, Rhodamine 6G, Rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazide, Texas Red sulfonyl chloride, Texas Red hydrazide), coumarin and coumarin derivatives (such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide), BODIPY and derivatives (such as BODIPY FL C3-SE, BODIPY 530 / 550C3, BODIPY 530 / 550C3-SE, BODIPY 530 / 550C3 hydrazide, BODIPY 493 / 503C3 hydrazide, BODIPY FLC3 hydrazide, BODIPY FL IA, BODIPY 530 / 551IA, Br-BODIPY 493 / 503), Cascade Blue and derivatives (such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide), Fluorescent Yellow and derivatives (such as Fluorescent Yellow iodoacetamide, Fluorescent Yellow CH), Cyanine and derivatives (such as indolium-based cyanine dyes, benzindolium-based cyanine dyes, pyridinium-based cyanine dyes, thiazolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy3, Cy5), lanthanide chelates and derivatives (such as BCPDA, TBP, TMT, BHHCT, BCOT, europium chelates, terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour dyes), JOE and its derivatives, Oregon Green (Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, stilbene, DEG dyes, NR dyes, near infrared dyes, and other fluorescent moieties known in the art, such as Haugland, Molecular Probes Handbook, 6th ed. (Eugene, Oreg.);A fluorescent moiety as described in Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed., Plenum Press New York (1999) or Hermanson, Bioconjugate Techniques, 2nd ed., or a derivative thereof;or any combination thereof. Cyanine dyes may exist in sulfonated or non-sulfonated form and consist of two indolenine, benzindolium, pyridinium, thiazolium and / or quinolinium groups separated by a polymethine bridge between the two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3 (which may contain 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium or 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which may include 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene )penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium or 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolyl-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate) and Cy7 (which may contain 1-(5-carboxypentyl) 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium or 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate), where "Cy" stands for 'cyanine' and the first number indicates the number of carbon atoms between the two indolenine groups. Cy2 is an oxazole derivative rather than an indolenine, and the benzo-derived Cy3.5, Cy5.5 and Cy7.5 are exceptions to this rule. ;

[0408] In some embodiments, the reporter moiety may be a FRET pair, so that multiple classifications may be performed with a single excitation and imaging step. As used herein, FRET may comprise excitation exchange (Forster) transfer or electron exchange (Dexter) transfer.

[0409] The terms "linked", "engaged", "attached" and variations thereof include any type of fusion, binding, adhesion or association between any combination of compounds or molecules that are sufficiently stable to withstand use in a particular procedure. The procedure may include, but is not limited to: transient binding of nucleotides; nucleotide incorporation; deblocking; washing; removal; flow; detection; imaging and / or identification. Such bonds may include, for example, covalent bonding, ionic bonding, hydrogen bonding, dipole-dipole bonding, hydrophilic bonding, hydrophobic bonding or affinity bonding, bonds or associations involving van der Waals forces, mechanical bonding, and the like. In some embodiments, such bonds occur within a molecule, such as connecting the ends of a single-stranded or double-stranded linear nucleic acid molecule together to form a circular molecule. In some embodiments, such bonds may occur between combinations of different molecules or between a molecule and a non-molecule, including, but not limited to, bonds between a nucleic acid molecule and a solid surface; bonds between a protein and a detectable reporter gene portion; bonds between a nucleotide and a detectable reporter gene portion; and the like. Some examples of bonds can be found, for example, in Hermanson, G., "Bioconjugation Technology", 2nd edition (2008); Aslam, M., Dent, A., "Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences", London: Macmillan (1998); Aslam, M., Dent, A., "Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences", London: Macmillan (1998).

[0410] As used herein, the terms "operably connected" and "operably joined" or related terms refer to the juxtaposition of components. The juxtaposed components can be covalently linked together. For example, two nucleic acid components can be enzymatically linked together, wherein the bond joining the two components together comprises a phosphodiester bond. A first nucleic acid component and a second nucleic acid component can be linked together, wherein the first nucleic acid component can confer a function to the second nucleic acid component. For example, the bond between the primer binding sequence and the sequence of interest forms a nucleic acid library molecule having a portion that can bind to a primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or a nucleic acid sequence of interest) can be linked to a vector, wherein the bond allows the transgenic sequence contained in the vector to be expressed or function. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects transgenic expression. In some embodiments, the vector comprises at least one host cell regulatory sequence, comprising a promoter sequence, an enhancer, a transcription and / or translation initiation sequence, a transcription and / or translation termination sequence, a polypeptide secretion signal sequence, and the like. In some embodiments, the host cell regulatory sequence controls the expression of the level, timing, and / or position of the transgene.

[0411] The term "adapter" and related terms refer to an oligonucleotide that can be operably connected (attached) to a target polynucleotide, wherein the adaptor confers function to the co-engaged adaptor-target molecule. The adaptor comprises DNA, RNA, chimeric DNA / RNA or its analogs. The adaptor may include at least one ribonucleoside residue. The adaptor may be single-stranded, double-stranded or have a single-stranded and / or double-stranded portion. The adaptor may be configured into a linear form, a stem-loop form, a hairpin form or a Y-shaped form. The adaptor may be of any length, including 4-100 nucleotides or longer. The adaptor may have a blunt end, an overhang end or a combination of the two. The overhang end includes a 5' overhang end and a 3' overhang end. The 5' end of a single-stranded adaptor or one chain of a double-stranded adaptor may have a 5' phosphate group or lack a 5' phosphate group. The adaptor may include a 5' tail (e.g., a tailed adaptor) that is not hybridized with a target polynucleotide, or the adaptor may be tailless. The adapter may include a sequence complementary to at least a portion of a primer, such as an amplification primer, a sequencing primer, or a capture primer (e.g., a soluble or immobilized capture primer). The adapter may include a random sequence or a degenerate sequence. The adapter may include at least one inosine residue. The adapter may include at least one phosphorothioate, phosphorothiol, and / or phosphoramide bond. The adapter may include a barcode sequence that can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. The adapter may include a unique identifier sequence (e.g., a unique molecular index, UMI; or a unique molecular tag) that can be used to uniquely identify the nucleic acid molecule to which the adapter is attached. In some embodiments, the unique identifier sequence can be used to increase error correction and accuracy, reduce false positive variant call rates, and / or increase the sensitivity of variant detection. The adapter may include at least one restriction enzyme recognition sequence, including any one or any combination of two or more selected from the group consisting of: type I, type II, type III, type IV, type Hs, or type IIB.

[0412] The terms "universal sequence," "universal adapter sequence," and related terms refer to a sequence in a nucleic acid molecule that is shared between two or more polynucleotide molecules. For example, an adapter having the same universal sequence can be joined to multiple polynucleotides such that the population of co-joined molecules carries the same universal adapter sequence. Examples of universal adapter sequences include amplification primer sequences, sequencing primer sequences, or capture primer sequences (e.g., soluble or carrier-immobilized capture primers).

[0413] In some embodiments, the carrier is solid, semi-solid, or a combination of the two. In some embodiments, the carrier is porous, semi-porous, non-porous, or any combination of porosity. In some embodiments, the carrier can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the carrier can be cylindrical, such as comprising a capillary or the inner surface of a capillary.

[0414] In some embodiments, the surface of the carrier can be substantially smooth. In some embodiments, the carrier can have a regular or irregular texture, including bumps, etchings, holes, three-dimensional scaffolds, or any combination thereof.

[0415] In some embodiments, the support comprises beads having any shape, including spheres, hemispheres, cylinders, barrels, rings, disks, rods, cones, triangles, cubes, polygons, tubes, or wires.

[0416] The support may be made of any material including, but not limited to, glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

[0417] In certain embodiments, the surface of the carrier is coated with one or more compounds to produce a passivation layer on the carrier. In certain embo...

Claims

1. A computer-implemented method for image registration in a preliminary analysis, the method comprising: determining, by a processor, coordinates of a colony in a reference coordinate system, wherein the colony is obtained from an image of the flow cell during one or more reference cycles; generating, by the processor, the one or more template images in the reference coordinate system by registering the colony to one or more template images using the coordinates of the colony; acquiring, by the processor, an image of the flow cell in a loop; as well as A plurality of transformations of the flow cell image are determined, by the processor, based on the one or more template images, each of the plurality of transformations corresponding to a sub-patch of the flow cell image and configured to register the sub-patch of the flow cell image to the one or more template images.

2. The computer-implemented method of any preceding claim, further comprising: An image intensity, size, shape, or a combination thereof of the colonies is obtained from a plurality of sub-tiles of the flow cell image in the one or more reference cycles.

3. The computer-implemented method of any of the preceding claims, wherein determining the coordinates of the colony in the reference coordinate system is based on the flow cell image in the one or more reference cycles and one or more fiducial markers external to the flow cell image in the cycle.

4. The computer-implemented method of any preceding claim, wherein the processor comprises: one or more processing units; one or more integrated circuits; or Its combination.

5. The computer-implemented method of any one of the preceding claims, wherein the processor comprises: one or more central processing units (CPUs); One or more field programmable gate arrays (FPGAs); or a combination thereof.

6. The computer-implemented method of any one of the preceding claims, wherein the processor comprises one or more field programmable gate arrays (FPGAs).

7. The computer-implemented method of any preceding claim, further comprising: performing, by the processor, one or more pre-processing steps on the flow cell images of the one or more reference cycles, the flow cell images in the cycles, or both, the one or more pre-processing steps comprising: Background subtraction; Image sharpening; or Its combination.

8. The computer-implemented method of any preceding claim, further comprising: performing, by the processor, one or more pre-processing steps on the flow cell images of the one or more reference cycles, the flow cell images in the cycles, or both, the one or more pre-processing steps comprising: background subtraction; Image sharpening; Intensity offset adjustment; Color correction; Intensity normalization; Phasing and prephasing correction; or Its combination.

9. The computer-implemented method of any preceding claim, further comprising: performing, by the processor, one or more subsequent steps on the flow cell images of the one or more reference cycles, the flow cell images in the cycle, or both, the one or more subsequent steps comprising: Color correction; Intensity extraction; or Its combination.

10. The computer-implemented method of any preceding claim, further comprising: performing, by the processor, one or more subsequent steps on the flow cell images of the one or more reference cycles, the flow cell images in the cycle, or both, the one or more subsequent steps comprising: Background subtraction; Image sharpening; Color correction; Intensity offset adjustment; Strength extraction; Intensity Normalization Falling behind and leading ahead correction; or Its combination.

11. The computer-implemented method of any preceding claim, further comprising: The plurality of transforms are used by the processor to register a sub-tile of the flow cell image to the one or more template images.

12. The computer-implemented method of any one of the preceding claims, wherein the one or more reference cycles include a cycle among the first five cycles, and the cycle is different from the one or more reference cycles.

13. The computer-implemented method of any one of the preceding claims, wherein the flow cell images are from 1 channel, or 2, 3 or 4 different channels.

14. The computer-implemented method of any preceding claim, wherein some or all of the populations are included in the flow cell image.

15. The computer-implemented method of any preceding claim, wherein some or all of the populations are included in the sub-tile of the flow cell image.

16. The computer-implemented method of any preceding claim, wherein some or all of the colonies are included in selected areas in the sub-tile of the flow cell image.

17. The computer-implemented method of any preceding claim, wherein one or more of the selected regions are located within each of the sub-tiles.

18. The computer-implemented method of any one of the preceding claims, wherein determining the plurality of transformations comprises: Each transformation corresponding to one or more selected regions in the sub-tile of the flow cell image is determined.

19. The computer-implemented method of any preceding claim, wherein the one or more selected areas in the sub-tile include some or all of the communities.

20. The computer-implemented method of any preceding claim, wherein the one or more selected regions include one or more center pixels of the sub-tile.

21. The computer-implemented method of any preceding claim, wherein the plurality of transformations comprises one or more affine transformations.

22. The computer-implemented method of any preceding claim, wherein each of the plurality of transformations comprises an affine transformation.

23. The computer-implemented method of any preceding claim, further comprising One or more transformed images of the one or more template images are generated by the processor.

24. The computer-implemented method of any preceding claim, further comprising One or more Fourier Transformed Images (FTI) of the one or more template images are generated by the processor.

25. The computer-implemented method of any preceding claim, wherein determining the plurality of transformations comprises: For each transform in the plurality of transforms, A corresponding cross-correlation of the selected region of the sub-tile with the one or more template images is determined.

26. The computer-implemented method of any preceding claim, wherein determining the plurality of transformations comprises: For each transform in the plurality of transforms, A corresponding cross-correlation of the sub-block with the FTI of the one or more template images is determined.

27. The computer-implemented method of any one of the preceding claims, wherein determining the plurality of transformations comprises: For each transform in the plurality of transforms, A corresponding cross-correlation of the selected region of the sub-tile with the FTI of the one or more template images is determined.

28. The computer-implemented method of any preceding claim, wherein determining the plurality of transformations comprises: For each transform in the plurality of transforms, A corresponding cross-correlation of the selected region with a corresponding FTI of the one or more template images is determined.

29. The computer-implemented method of any preceding claim, wherein determining the plurality of transformations comprises: For each transform in the plurality of transforms, A corresponding cross-correlation of the selected region with a corresponding FTI of the one or more template images is determined using Fourier transform (FT) or fast Fourier transform (FFT).

30. The computer-implemented method of any preceding claim, wherein determining the plurality of transformations comprises: For each sub-tile, The displacement d is determined with sub-pixel resolution based on the corresponding cross-correlations.

31. The computer-implemented method of any one of the preceding claims, wherein the displacement d is two-dimensional (2D).

32. The computer-implemented method of any preceding claim, determining the plurality of transformations comprising: For each transform in the plurality of transforms, The transformation of the sub-block is determined based on a displacement of one or more neighboring sub-blocks of the sub-block.

33. The computer-implemented method of any preceding claim, wherein the one or more adjacent sub-tiles comprises 3, 4, 5, 6, 7, 8, or 9 adjacent sub-tiles.

34. The computer-implemented method of any preceding claim, wherein the transformation of the flow cell image is linear.

35. The computer-implemented method of any preceding claim, wherein the transformation of the flow cell image is non-linear.

36. The computer-implemented method of any preceding claim, wherein each transform of the plurality of transforms is linear.

37. The computer-implemented method of any preceding claim, wherein each of the plurality of transformations is an affine transformation.

38. The computer-implemented method of any preceding claim, wherein the one or more template images are 2D and are approximately the same size as the sub-tiles.

39. The computer-implemented method of any preceding claim, wherein the size of the selected region is approximately 128 x 128 or approximately 64 x 64.

40. The computer-implemented method of any preceding claim, further comprising: The plurality of transforms are transmitted by the processor to a processing unit.

41. The computer-implemented method of any one of the preceding claims, wherein the processing unit is a central processing unit (CPU).

42. The computer-implemented method of any preceding claim, further comprising: Image intensities of a plurality of colonies are extracted, by the processing unit, from the sub-tile of the flow cell image based on the transmitted plurality of transforms.

43. The computer-implemented method of any preceding claim, further comprising: Base calling is performed by the processing unit based on the transmitted plurality of transformations using image intensities of the sub-tiles of the flow cell image.

44. The computer-implemented method of any preceding claim, further comprising: receiving, by the processor, the flow cell image from an optical system during the one or more reference cycles; and The flow cell image is received, by the processor, from the optical system in the cycle, wherein the cycle is subsequent to the one or more reference cycles.

45. The computer-implemented method of any preceding claim, wherein determining the plurality of transformations comprises: Each transformation corresponding to a plurality of selected regions in the sub-tile of the flow cell image is determined.

46. ​​The computer-implemented method of any preceding claim, wherein determining the plurality of transformations comprises: Each transformation corresponding to 3, 4, 5, 6, 7, 8 or 9 selected regions in the sub-tile of the flow cell image is determined.

47. The computer-implemented method of any preceding claim, wherein determining the plurality of transformations comprises: For each transform in the plurality of transforms, A corresponding cross-correlation of each selected region within the sub-tile with the one or more template images is determined.

48. The computer-implemented method of any preceding claim, wherein determining the plurality of transformations comprises: For each transform in the plurality of transforms, A corresponding cross-correlation of each selected region of the sub-tile with the FTI of the one or more template images is determined.

49. The computer-implemented method of any preceding claim, wherein determining the plurality of transformations comprises: For each transform in the plurality of transforms, A corresponding cross-correlation of each selected region of the sub-tile with a corresponding Fourier transformed image (FTI) of the one or more template images is determined.

50. The computer-implemented method of any preceding claim, wherein determining the plurality of transformations comprises: For each transform in the plurality of transforms, Using a Fourier Transform (FT) or a Fast Fourier Transform (FFT), a corresponding cross-correlation of each selected region of the sub-tile with a corresponding Fourier Transformed Image (FTI) of the one or more template images is determined.

51. The computer-implemented method of any preceding claim, wherein determining the plurality of transformations comprises: For each sub-tile, A plurality of shifts d are determined with sub-pixel resolution, each shift being based on the corresponding cross-correlation.

52. The computer-implemented method of any preceding claim, wherein the displacement d is two-dimensional (2D).

53. The computer-implemented method of any preceding claim, determining the plurality of transformations comprising: For each transform in the plurality of transforms, The transform of the sub-tile is determined based on the plurality of displacements.

54. The method according to any one of the preceding claims, further comprising: A plurality of nucleic acid template molecules immobilized on a carrier are provided, wherein each nucleic acid template molecule comprises an insertion sequence.

55. The method according to any one of the preceding claims, further comprising: The flow cell image is generated by a sequencing system through one or more cycles of sequencing reaction on the plurality of nucleic acid template molecules immobilized on the carrier.

56. The method according to any one of the preceding claims, further comprising: The flow cell image is generated by the sequencing system by performing one or more cycles of sequencing reaction on the cell sample immobilized on the carrier, wherein the flow cell image is generated from two or more different z positions along the axial axis.

57. The method of any one of the preceding claims, wherein performing the one or more cycles of the sequencing reaction comprises: A plurality of nucleotide template molecules are contacted with a plurality of nucleotide reagents comprising a mixture of different types of nucleotide bases A, G, C, and T / U.

58. The method of any of the preceding claims, wherein the individual nucleotide reagents comprise a different detectable color label corresponding to each different type of nucleotide base.

59. The method of any one of the preceding claims, wherein performing the one or more cycles of the sequencing reaction comprises: The plurality of nucleotide template molecules are contacted with a mixture of a plurality of sequencing primers, a plurality of polymerases, and different types of avidites.

60. The method of any of the preceding claims, wherein an individual affimer in the mixture comprises a core to which are attached a plurality of nucleotide arms, and each arm of the individual affimer comprises the same type of nucleotide base.

61. The method of any one of the preceding claims, wherein performing the one or more cycles of the sequencing reaction comprises: In each of the one or more cycles, optical color signals emitted from nucleotide reagents bound to the plurality of template molecules are imaged by an optical system.

62. The method of any one of the preceding claims, wherein performing the one or more cycles of the sequencing reaction comprises: In each of the one or more cycles, the flow cell image is acquired by an optical system, the flow cell image comprising optical color signals emitted from nucleotide reagents bound to the plurality of template molecules.

63. A method according to any of the preceding claims, wherein the flow cell image comprises optical signals emitted from nucleotide reagents that bind to an unbalanced diversity of nucleotide bases A, G, C, and T / U in the plurality of nucleic acid template molecules immobilized on the support during the one or more cycles.

64. A method according to any of the preceding claims, wherein the plurality of populations comprises an unbalanced diversity of nucleotide bases A, G, C, and T / U, and wherein the unbalanced diversity comprises: (1) the percentage of the number of one or more types of nucleotide bases relative to (2) the total number of nucleotide bases, and wherein in the cycle N, the percentage is less than 20%, 15%, 10% or 5%.

65. The method of any of the preceding claims, wherein the plurality of populations correspond to the plurality of nucleotide template molecules.

66. The method according to any one of the preceding claims, further comprising: A cell sample is provided, wherein the cell sample has a plurality of concatemer molecules immobilized on a carrier, wherein each concatemer molecule corresponds to a target RNA of the cell sample.

67. The method according to any one of the preceding claims, further comprising: The sequencing system generates a flow cell image by performing one or more cycles of sequencing reaction on the plurality of concatemer molecules immobilized on the carrier.

68. The method of any one of the preceding claims, wherein performing the one or more cycles of the sequencing reaction comprises: The plurality of concatemer molecules are contacted with a plurality of nucleotide reagents comprising a mixture of different types of nucleotide bases A, G, C, and T / U.

69. The method of any one of the preceding claims, wherein performing the one or more cycles of the sequencing reaction comprises: The plurality of concatemer molecules are contacted with a mixture of a plurality of sequencing primers, a plurality of polymerases, and different types of affimers.

70. The method of any of the preceding claims, wherein the individual affimers in the mixture comprise a core to which are attached a plurality of nucleotide arms, and each arm of the individual affimers comprises the same type of nucleotide base.

71. The method of any one of the preceding claims, wherein performing the one or more cycles of the sequencing reaction comprises: In each of the one or more cycles, optical color signals emitted from nucleotide reagents bound to the plurality of concatemer molecules are imaged by an optical system.

72. The method of any one of the preceding claims, wherein performing the one or more cycles of the sequencing reaction comprises: In each of the one or more cycles, the flow cell image is acquired by an optical system, the flow cell image comprising optical color signals emitted from nucleotide reagents bound to the plurality of concatemer molecules.

73. A computer-implemented system for image registration in a preliminary analysis, the system comprising: one or more hardware processors; One or more data storage devices storing instructions executable by the one or more hardware processors to cause the one or more hardware processors to perform operations comprising: determining coordinates of a colony in a reference coordinate system, wherein the colony is obtained from an image of the flow cell during one or more reference cycles; generating the one or more template images in the reference coordinate system by registering the colony to one or more template images using the coordinates of the colony; Flow cell images are acquired in cycles; as well as A plurality of transformations of the flow cell image are determined based on the one or more template images, each of the plurality of transformations corresponding to a sub-patch of the flow cell image and configured to register the sub-patch of the flow cell image to the one or more template images.

74. A computer-implemented system for image registration in a preliminary analysis, the system comprising: one or more hardware processors; One or more data storage devices, wherein the one or more data storage devices store instructions, wherein the instructions are executable by the one or more hardware processors to cause the one or more hardware processors to perform operations, wherein the operations include any one of claims 1 to 72.

75. One or more non-transitory computer storage media encoded with instructions that, when executed by one or more hardware processors, cause the one or more hardware processors to perform operations for image registration in a preliminary analysis, the operations comprising: determining coordinates of a colony in a reference coordinate system, wherein the colony is obtained from an image of the flow cell during one or more reference cycles; generating the one or more template images in the reference coordinate system by registering the colony to one or more template images using the coordinates of the colony; Flow cell images are acquired in cycles; as well as A plurality of transformations of the flow cell image are determined based on the one or more template images, each of the plurality of transformations corresponding to a sub-patch of the flow cell image and configured to register the sub-patch of the flow cell image to the one or more template images.

76. One or more non-transitory computer storage media encoded with instructions that, when executed by one or more hardware processors, cause the one or more processors to perform operations for image registration in a preliminary analysis, the operations comprising any of claims 1 to 72.

77. A method for image registration rescue in preliminary analysis, the method comprising: obtaining, by a processor, a first flow cell image from a first channel in a first flow cycle and a second flow cell image from a second channel in the first flow cycle, wherein the number of colonies in the first flow cell image is less than 1%, 2%, 5%, 10%, or 15% of the total number of colonies in flow cell images acquired in all channels in the first flow cycle; obtaining, by the processor, a first rescue image from the first channel in a rescue flow cycle and obtaining a second rescue image from the second channel in the rescue flow cycle; A first plurality of transformations of the first flow cell image is determined, by the processor, based on: a second plurality of transformations of the second flow cell image; a third plurality of transformations of the first rescue image; as well as a fourth plurality of transformations of the second rescue image, Each transform of the first plurality of transforms corresponds to a sub-patch of the first flow cell image and is configured to register the sub-patch to one or more template images or the second flow cell image.

78. A method for image registration rescue in preliminary analysis, the method comprising: obtaining, by a processor, a first flow cell image from a first channel in a first flow cycle and a second flow cell image from a second channel in said first flow cycle; obtaining, by the processor, a first rescue image from the first channel in a rescue flow cycle and obtaining a second rescue image from the second channel in the rescue flow cycle; A first plurality of transformations of the first flow cell image is determined, by the processor, based on: a second plurality of transformations of the second flow cell image; a third plurality of transformations of the first rescue image; and a fourth plurality of transformations of the second rescue image, Wherein the first plurality of transformations are configured to register the first flow cell image to one or more template images or the second flow cell image.

79. A method for image registration rescue in preliminary analysis, the method comprising: obtaining, by a processor, a first flow cell image from a first channel in a first flow cycle and a second flow cell image from a second channel in said first flow cycle; obtaining, by the processor, a first rescue image from the first channel in a rescue flow cycle and obtaining a second rescue image from the second channel in the rescue flow cycle; determining, by the processor, that the first flow cell image from the first channel in the first flow cycle has satisfied a predetermined threshold for image registration rescue; A first plurality of transformations of the first flow cell image is determined, by the processor, based on: a second plurality of transformations of the second flow cell image; a third plurality of transformations of the first rescue image; as well as a fourth plurality of transformations of the second rescue image, Wherein the first plurality of transformations are configured to register the first flow cell image to one or more template images or the second flow cell image.

80. A method for image registration rescue in preliminary analysis, the method comprising: obtaining, by a processor, a first flow cell image from a first channel in a first flow cycle and a second flow cell image from a second channel in said first flow cycle; obtaining, by the processor, a first rescue image from the first channel in a rescue flow cycle and obtaining a second rescue image from the second channel in the rescue flow cycle; In response to determining that the first flow cell image from the first channel in the first flow cycle has satisfied a predetermined threshold for image registration rescue, determining, by the processor, a first plurality of transformations of the first flow cell image based on: a second plurality of transformations of the second flow cell image; a third plurality of transformations of the first rescue image; as well as a fourth plurality of transformations of the second rescue image, Wherein the first plurality of transformations are configured to register the first flow cell image to one or more template images or the second flow cell image.

81. A method for image registration rescue in preliminary analysis, the method comprising: obtaining, by a processor, a first flow cell image from a first channel in a first flow cycle and a second flow cell image from a second channel in said first flow cycle; obtaining, by the processor, a first rescue image from the first channel in a rescue flow cycle and obtaining a second rescue image from the second channel in the rescue flow cycle; A first plurality of transformations of the first flow cell image is determined, by the processor, based on: a plurality of transformations between the first rescue image and the second rescue image, Each transform of the first plurality of transforms corresponds to a sub-patch of the first flow cell image and is configured to register the sub-patch to one or more template images or the second flow cell image.

82. A method according to any of the preceding claims, wherein the one or more template images are located in a reference coordinate system.

83. A method according to any of the preceding claims, wherein the reference coordinate system is determined in one or more reference cycles.

84. The method of any preceding claim, wherein the one or more reference cycles are completed prior to the first flow cycle and the rescue flow cycle.

85. A method according to any of the preceding claims, wherein the number of colonies in the first flow cell image is less than 1%, 2%, 5%, 10% or 15% of the total number of colonies in the flow cell images acquired in all channels in the first flow cycle.

86. A method according to any of the preceding claims, wherein the number of colonies in the second flow cell image exceeds 1%, 2%, 5%, 10% or 15% of the total number of colonies in the flow cell images acquired in all of the channels in the first flow cycle.

87. The method according to any one of the preceding claims, further comprising: The processor determines whether the number of colonies in the first flow cell image satisfies a predetermined threshold.

88. The method of any of the preceding claims, wherein the number of colonies in each of the first rescue image and the second rescue image exceeds 1%, 2%, 5%, 10% or 15% of the total number of colonies in the flow cell images acquired in all channels in the rescue flow cycle.

89. The method of any preceding claim, wherein the first flow cycle is different from the rescue flow cycle.

90. The method of any preceding claim, wherein the rescue flow cycle is completed prior to the first flow cycle.

91. The method of any preceding claim, wherein the rescue flow cycle is completed immediately prior to the first flow cycle.

92. The method of any of the preceding claims, wherein the first flow cell image and the second flow cell image are flow cell images of a sample fixed on a carrier, and wherein the first rescue image and the second rescue image are rescue images of the sample fixed on the carrier.

93. A method according to any of the preceding claims, wherein the sample comprises nucleotide acid template molecules.

94. The method of any of the preceding claims, wherein the first flow cell image and the second flow cell image are flow cell images of a sample immobilized on a carrier, and wherein the first rescue image and the second rescue image are rescue images of a test object.

95. The method of any preceding claim, wherein the first rescue image and the second rescue image are rescue images of the test object in the absence of the sample immobilized on the carrier.

96. The method of any of the preceding claims, wherein the test target emits a fluorescent signal detectable in all channels during the rescue flow cycle.

97. The method of any of the preceding claims, wherein the first flow cycle is among cycle 1 to cycle 30 of a sequencing run.

98. The method of any of the preceding claims, wherein the first flow cycle is within cycle 2 to cycle 200 of a sequencing run.

99. The method of any of the preceding claims, wherein the first flow cycle is between cycle 2 and the last cycle of a sequencing run.

100. The method according to any one of the preceding claims, further comprising: Providing a plurality of nucleic acid template molecules immobilized on the carrier, wherein each nucleic acid template molecule comprises: a first insertion sequence and a first sample index sequence, wherein the first sample index sequence comprises a first universal sample index sequence, and the first universal sample index identifies a sample source of the insertion sequence; and Before performing one or more cycles of sequencing reaction on the insert sequence, the sequencing system performs one or more cycles of sequencing reaction on the first sample index sequence to generate a flow cell image including the first flow cell image and the second flow cell image in the first flow cycle.

101. The method according to any one of the preceding claims, further comprising: Providing a plurality of nucleic acid template molecules immobilized on the carrier, wherein each nucleic acid template molecule comprises: a first insertion sequence and a first sample index sequence, wherein the first sample index sequence comprises a first universal sample index sequence, and the first universal sample index identifies a sample source of the insertion sequence; and Before performing one or more cycles of sequencing reaction on the first sample index sequence, the sequencing system performs one or more cycles of sequencing reaction on the first insert sequence to generate a flow cell image including the first flow cell image and the second flow cell image in the first flow cycle.

102. The method of any preceding claim, wherein determining the first plurality of transformations of the first flow cell image comprises: Coordinates of colonies in one or more of: the first flow cell image, the second flow cell image, the first rescue image, and the second rescue image are determined.

103. A method according to any of the preceding claims, wherein the coordinates of the colony are located within a reference coordinate system.

104. The method of any preceding claim, wherein the processor comprises: one or more processing units; one or more integrated circuits; or Its combination.

105. The method of any preceding claim, wherein the processor comprises: one or more central processing units (CPUs); One or more field programmable gate arrays (FPGAs); or a combination thereof.

106. A method according to any of the preceding claims, wherein the processor comprises one or more field programmable gate arrays (FPGAs).

107. The method according to any one of the preceding claims, further comprising: The processor performs one or more pre-processing steps on the first flow cell image, the second flow cell image, the first rescue image, the second rescue image, or a combination thereof, the one or more pre-processing steps comprising: Background subtraction; Image sharpening; or Its combination.

108. The method according to any one of the preceding claims, further comprising: The processor performs one or more pre-processing steps on the first flow cell image, the second flow cell image, the first rescue image, the second rescue image, or a combination thereof, the one or more pre-processing steps comprising: Background subtraction; Image sharpening; Intensity offset adjustment; Color correction; Intensity normalization; Falling behind and leading ahead correction; or Its combination.

109. The method according to any one of the preceding claims, further comprising: performing, by the processor, one or more subsequent steps on the first flow cell image, the second flow cell image, the first rescue image, the second rescue image, or a combination thereof, the one or more subsequent steps comprising: Color correction; Intensity extraction; or Its combination.

110. The method according to any one of the preceding claims, further comprising: performing, by the processor, one or more subsequent steps on the first flow cell image, the second flow cell image, the first rescue image, the second rescue image, or a combination thereof, the one or more subsequent steps comprising: Background subtraction; Image sharpening; Color correction; Intensity offset adjustment; Strength extraction; Intensity Normalization Falling behind and leading ahead correction; or Its combination.

111. The method according to any one of the preceding claims, further comprising: The first plurality of transforms are used by the processor to register a sub-tile of the first flow cell image to the one or more template images or the second flow cell image.

112. The method according to any one of the preceding claims, further comprising: Each sub-tile of the first flow cell image is registered, by the processor, to the one or more template images or the second flow cell image using a corresponding transform of the first plurality of transforms.

113. A method according to any of the preceding claims, wherein the first channel and the second channel are different.

114. The method of any preceding claim, wherein determining the first plurality of transformations comprises: Each transformation corresponding to one or more selected regions of a sub-tile of the first flow cell image is determined.

115. A method according to any of the preceding claims, wherein the one or more selected regions include one or more central pixels of the sub-tile.

116. A method according to any of the preceding claims, wherein the first plurality of transformations comprises one or more non-linear transformations.

117. A method according to any of the preceding claims, wherein the first plurality of transformations comprises one or more linear transformations.

118. A method according to any of the preceding claims, wherein the first plurality of transformations comprises one or more affine transformations.

119. A method according to any of the preceding claims, wherein each transform in the first plurality of transforms comprises an affine transform.

120. The method according to any one of the preceding claims, further comprising: One or more Fourier transformed images (FTIs) of at least a portion of the first flow cell image, the second flow cell image, the first rescue image, the second rescue image, the one or more template images, or a combination thereof are generated by the processor.

121. The method of any preceding claim, wherein determining the first plurality of transformations comprises: For each transform in the first plurality of transforms, A corresponding cross-correlation of the one or more selected regions of corresponding sub-tiles of the second flow cell image, the first rescue image, the second rescue image, or a combination thereof with the one or more template images is determined.

122. The method of any preceding claim, wherein determining the first plurality of transformations comprises: For each transform in the first plurality of transforms, A corresponding cross-correlation of the one or more selected regions of the corresponding sub-block of the first rescue image with the second rescue image is determined.

123. The method of any preceding claim, wherein determining the first plurality of transformations comprises: For each transform in the first plurality of transforms, A corresponding cross-correlation of the one or more selected regions of the corresponding sub-block of the first rescue image with the FTI of the second rescue image is determined.

124. The method of any preceding claim, wherein determining the first plurality of transformations comprises: For each transform in the first plurality of transforms, A corresponding cross-correlation of the one or more selected regions of the first rescue image with a corresponding FTI of the second rescue image is determined using a Fourier Transform (FT) or a Fast Fourier Transform (FFT).

125. The method of any preceding claim, wherein determining the first plurality of transformations comprises: For each sub-block in the first rescue image or the second rescue image, A corresponding displacement d34 is determined with sub-pixel resolution based on the corresponding cross-correlations.

126. The method of any preceding claim, wherein determining the first plurality of transformations comprises: For each transform in the first plurality of transforms and the corresponding sub-tile, determining each of the plurality of transformations between the first rescue image and the second rescue image based on a corresponding displacement d34 of one or more adjacent sub-blocks of the first rescue image and the second rescue image; and The first plurality of transforms is determined based on the plurality of transforms.

127. The method of any preceding claim, wherein determining the first plurality of transforms based on the plurality of transforms comprises: Based on a corresponding transform T34_i in the plurality of transforms, each transform T12_i in the first plurality of transforms is determined as follows: T12_i=T34_i.

128. The method of any preceding claim, wherein determining the first plurality of transformations comprises: For each transform in the first plurality of transforms, A corresponding cross-correlation of the one or more selected regions of corresponding sub-tiles of the second flow cell image, the first rescue image, the second rescue image, or a combination thereof with the FTI of the one or more template images is determined.

129. The method of any preceding claim, wherein determining the first plurality of transformations comprises: For each transform in the first plurality of transforms, A corresponding cross-correlation of the one or more selected regions of the second flow cell image, the first rescue image, the second rescue image, or a combination thereof with a corresponding FTI of the one or more template images is determined using a Fourier Transform (FT) or a Fast Fourier Transform (FFT).

130. The method of any preceding claim, wherein determining the first plurality of transformations comprises: For each sub-tile in the second flow cell image, the first rescue image, and the second rescue image, Based on the corresponding cross-correlations, a corresponding displacement d2, d3 or d4 is determined, optionally with sub-pixel resolution.

131. A method according to any of the preceding claims, wherein the corresponding displacement d2, d3, d4 or d34 is two-dimensional (2D).

132. The method of any preceding claim, wherein determining the first plurality of transformations comprises: For each transform in the first plurality of transforms, determining the second plurality of transformations of the sub-tile of the second flow cell image based on corresponding displacements d2 of one or more neighboring sub-tiles of the sub-tile of the second flow cell image; determining the third plurality of transformations of the sub-block of the first rescue image based on corresponding displacements d3 of one or more neighboring sub-blocks of the sub-block of the first rescue image; determining the fourth plurality of transformations of the sub-block of the second rescue image based on corresponding displacements d4 of one or more neighboring sub-blocks of the sub-block of the second rescue image; as well as The first plurality of transforms is determined based on the second plurality of transforms, the third plurality of transforms, and the fourth plurality of transforms.

133. The method of any preceding claim, wherein determining the first plurality of transforms based on the second plurality of transforms, the third plurality of transforms, and the fourth plurality of transforms comprises: Based on the corresponding transforms T2_i, T3_i and T4_i in the second, third and fourth pluralities of transforms, each transform T1_i in the first plurality of transforms is determined as follows: (T1_i) -1 =(T2_i) -1 ·T4_i·(T3_i) -1 。 134. A method according to any of the preceding claims, wherein the one or more adjacent sub-tiles include at least 3, 4, 5, 6, 7, 8 or 9 adjacent sub-tiles.

135. A method according to any of the preceding claims, wherein the one or more template images are 2D and have a size that is approximately the same as the size of the sub-tile.

136. The method of any of the preceding claims, wherein the size of each of the one or more selected areas is approximately 128 x 128 or approximately 64 x 64.

137. The method further comprises: The first plurality of transforms is transmitted, by the processor, to a processing unit.

138. A method according to any of the preceding claims, wherein the processing unit is a central processing unit (CPU).

139. The method according to any one of the preceding claims, further comprising: Image intensities of colonies are extracted, by the processor, from the first flow cell image based on the first plurality of transformations.

140. The method of any preceding claim, further comprising: Base calls are made, by the processor, based on the first plurality of transformations using the image intensities of the populations of the first flow cell image.

141. The method of any of the preceding claims, wherein the carrier comprises a flow cell.

142. A method according to any of the preceding claims, wherein the support comprises a flow cell with beads.

143. The method of any preceding claim, wherein the beaded flow cell comprises a surface coated with fluorescent beads chemically immobilized to the surface.

144. A method according to any of the preceding claims, wherein the fluorescent beads are randomly distributed on the surface.

145. A method according to any of the preceding claims, wherein the fluorescent beads are distributed on the surface in a repeating pattern.

146. A method according to any of the preceding claims, wherein the fluorescent beads comprise one, two, three, four, five or six different types of beads that emit different colors in response to light stimulation.

147. The method of any of the preceding claims, wherein the fluorescent beads fluoresce at one or more wavelengths in response to the light excitation.

148. A method according to any of the preceding claims, wherein the fluorescent beads emit fluorescence of multiple wavelengths in response to the light excitation, each wavelength in the multiple wavelengths corresponding to a different channel among all of the channels.

149. A method according to any of the preceding claims, wherein the test target comprises a coating of a spatially repeated predetermined geometric shape or pattern.

150. A method according to any of the preceding claims, wherein the predetermined geometric pattern or shape is repeated in one or two dimensions.

151. The method of any preceding claim, wherein the test object lacks a flow cell and a liquid.

152. A method according to any of the preceding claims, wherein the test target comprises one or more substrates having a predetermined refractive index.

153. The method of any preceding claim, wherein the test target comprises a top substrate having a predetermined refractive index.

154. The method of any preceding claim, wherein the test target comprises a base substrate.

155. The method of any of the preceding claims, wherein at least a portion of the first substrate or the second substrate comprises the coating having the predetermined geometric pattern or shape.

156. A method according to any of the preceding claims, wherein the thickness of the first substrate is configured to simulate the presence of a first hypothetical flow cell.

157. The method of any of the preceding claims, wherein the thickness of the top substrate is configured to permit imaging of a bottom surface of the first channel of the hypothetical first flow cell.

158. The method of any of the preceding claims, wherein the predetermined geometry or pattern of the coating comprises optically opaque portions and transparent portions.

159. The method of any preceding claim, further comprising: acquiring, by the optical system, a reference flow cell image during one or more reference flow cycles; determining, by a processor, coordinates of colonies in a reference coordinate system, wherein the colonies are obtained from a reference flow cell image during one or more reference flow cycles; and The one or more template images are generated by the processor in the reference coordinate system by registering the colony to one or more template images using the coordinates of the colony.

160. A computer-implemented system for image registration rescue in a preliminary analysis, the system comprising: one or more hardware processors; One or more data storage devices storing instructions that, when executed by the one or more hardware processors, cause the one or more hardware processors to perform operations comprising: obtaining, by a processor, a first flow cell image from a first channel in a first flow cycle and a second flow cell image from a second channel in the first flow cycle, wherein the number of colonies in the first flow cell image is less than 1%, 2%, 5%, 10%, or 15% of the total number of colonies in flow cell images acquired in all channels in the first flow cycle; obtaining, by the processor, a first rescue image from the first channel in a rescue flow cycle and obtaining a second rescue image from the second channel in the rescue flow cycle; A first plurality of transformations of the first flow cell image is determined, by the processor, based on: a second plurality of transformations of the second flow cell image; a third plurality of transformations of the first rescue image; as well as a fourth plurality of transformations of the second rescue image, Each transform of the first plurality of transforms corresponds to a sub-patch of the first flow cell image and is configured to register the sub-patch to one or more template images or the second flow cell image.

161. A computer-implemented system for image registration rescue in a preliminary analysis, the system comprising: one or more hardware processors; One or more data storage devices storing instructions which, when executed by the one or more hardware processors, cause the one or more hardware processors to perform operations comprising any one of claims 63 to 140.

162. One or more non-transitory computer storage media encoded with instructions that, when executed by one or more hardware processors, cause the one or more hardware processors to perform operations for image registration rescue in a preliminary analysis, the operations comprising: obtaining, by a processor, a first flow cell image from a first channel in a first flow cycle and a second flow cell image from a second channel in the first flow cycle, wherein the number of colonies in the first flow cell image is less than 1%, 2%, 5%, 10%, or 15% of the total number of colonies in flow cell images acquired in all channels in the first flow cycle; obtaining, by the processor, a first rescue image from the first channel in a rescue flow cycle and obtaining a second rescue image from the second channel in the rescue flow cycle; A first plurality of transformations of the first flow cell image is determined, by the processor, based on: a second plurality of transformations of the second flow cell image; a third plurality of transformations of the first rescue image; as well as a fourth plurality of transformations of the second rescue image, Each transform of the first plurality of transforms corresponds to a sub-patch of the first flow cell image and is configured to register the sub-patch to one or more template images or the second flow cell image.

163. One or more non-transitory computer storage media encoded with instructions that, when executed by one or more hardware processors, cause the one or more hardware processors to perform operations for image registration in a preliminary analysis, the operations comprising any of claims 82 to 159.

164. A computer-implemented system for image registration rescue in a preliminary analysis, the system comprising: one or more hardware processors; One or more data storage devices storing instructions that, when executed by the one or more hardware processors, cause the one or more hardware processors to perform operations comprising: obtaining, by a processor, a first flow cell image from a first channel in a first flow cycle and a second flow cell image from a second channel in said first flow cycle; obtaining, by the processor, a first rescue image from the first channel in a rescue flow cycle and obtaining a second rescue image from the second channel in the rescue flow cycle; A first plurality of transformations of the first flow cell image is determined, by the processor, based on: a second plurality of transformations of the second flow cell image; a third plurality of transformations of the first rescue image; and a fourth plurality of transformations of the second rescue image, Wherein the first plurality of transformations are configured to register the first flow cell image to one or more template images or the second flow cell image.

165. A computer-implemented system for image registration rescue in a preliminary analysis, the system comprising: one or more hardware processors; One or more data storage devices storing instructions which, when executed by the one or more hardware processors, cause the one or more hardware processors to perform operations comprising any one of claims 82 to 159.

166. One or more non-transitory computer storage media encoded with instructions that, when executed by one or more hardware processors, cause the one or more hardware processors to perform operations for image registration rescue in a preliminary analysis, the operations comprising: obtaining, by a processor, a first flow cell image from a first channel in a first flow cycle and a second flow cell image from a second channel in said first flow cycle; obtaining, by the processor, a first rescue image from the first channel in a rescue flow cycle and obtaining a second rescue image from the second channel in the rescue flow cycle; A first plurality of transformations of the first flow cell image is determined, by the processor, based on: a second plurality of transformations of the second flow cell image; a third plurality of transformations of the first rescue image; and a fourth plurality of transformations of the second rescue image, Wherein the first plurality of transformations are configured to register the first flow cell image to one or more template images or the second flow cell image.

167. One or more non-transitory computer storage media encoded with instructions that, when executed by one or more hardware processors, cause the one or more hardware processors to perform operations for image registration in a preliminary analysis, the operations comprising any of claims 82 to 159.

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