TIME-BASED CLUSTERING IMAGING OF CONTIGUOUSLY PRESERVED GENOMIC DNA FRAGMENTS AMPLIFIED FROM GENOMIC DNA

Abstract

The present invention relates to a method comprising: generating a series of time-based clustering images for a plurality of contiguous conserved library fragments from a genome sample, characterized in that each time-based clustering image in the series is generated sequentially by: introducing, into a flow cell, a respective sample that includes some of the contiguous conserved library fragments, wherein some of the contiguous conserved library fragments are attached to a solid support or are attached to each other; initiating the release of some of the contiguous conserved library fragments from the solid support or from each other; amplifying some of the contiguous conserved library fragments to generate a plurality of respective template strands; having the respective template strands; and taking images of the respective template strands.

Description

TIME-BASED CLUSTERING IMAGING OF CONTIGUOUSLY PRESERVED GENOMIC DNA FRAGMENTS AMPLIFIED Cross-reference to related request This application claims the benefit of U.S. provisional patent application serial number 62 / 942,563, filed on December 2, 2019, the contents of which are incorporated by reference in the present description in their entirety. Background There are a variety of methods and applications for which generating a library of fragmented and labeled deoxyribonucleic acid (DNA) molecules from double-stranded DNA (dsDNA) molecules is desirable. Frequently, the purpose is to generate smaller DNA molecules (e.g., DNA fragments) from larger dsDNA molecules for use as templates in DNA sequencing reactions. The templates can enable the acquisition of short read lengths. Short sequence reads are typically overlapped with several other short sequence reads to provide redundant coverage over different portions of the longer overall sequence. During data analysis, the overlapping sequences of numerous reads can be used to pool information from longer sequences. As such, the overlapping short sequence reads can be aligned to reconstruct the longer nucleic acid sequences.In some cases, pre-sequencing steps (such as barcoding of particular nucleic acid molecules) can be used to simplify data analysis. Introduction A first aspect described herein is a method comprising generating a series of time-based clustering images for a plurality of contiguously conserved library fragments from a genome sample, wherein each time-based clustering image in the series is generated sequentially by: introducing, into a flow cell, a respective sample that includes some of the contiguously conserved library fragments, wherein some of the contiguously conserved library fragments are attached to a solid support or are attached to each other; initiating the release of some of the contiguously conserved library fragments from the solid support or from each other; and amplifying some of the contiguously conserved library fragments to generate a plurality of strands MA / a / ZUZI / u ι zu io respective templates; dye the respective template strings; and take pictures of the respective template strings. A second aspect described herein is a method comprising preparing a mixture that includes a plurality of contiguously conserved library fragments from a genome sample, the plurality of contiguously conserved library fragments being attached to solid supports or attached to each other; diluting the mixture to generate a predetermined number of dilution samples to be introduced into a flow cell; and generating a time-based clustering image for at least one of the contiguously conserved library fragments by: introducing a first of the dilution samples including some of the contiguously conserved library fragments into the flow cell; initiating the release of some of the contiguously conserved library fragments from the solid support or from each other; amplifying some of the contiguously conserved library fragments to generate a plurality of template strings;dye the plurality of template strings; and take images of the plurality of template strings.; A third aspect described herein is a system comprising a flow cell receptacle; a fluidic control system including fluid supply for delivering a dilution sample and a dye to a flow cell positioned in the flow cell receptacle; an illumination system positioned to illuminate the flow cell positioned in the flow cell receptacle; a detection system positioned to capture an image of the flow cell positioned in the flow cell receptacle; and a controller in operational communication with the fluidic control system, the illumination system, and the detection system; the controller for: causing the supply fluids to introduce the dilution sample into the flow cell positioned in the flow cell receptacle;to cause the delivery fluids to introduce the dye into the flow cell positioned in the flow cell receptacle after template strings are generated in the flow cell positioned in the flow cell receptacle from contiguously conserved gene library fragments present in the dilution sample; to cause the illumination system to illuminate the dyed template strings in the flow cell positioned in the flow cell receptacle; and to cause the detection system to capture the image of the dyed template strings, illuminated in the flow cell positioned in the flow cell receptacle. It should be understood that any of the features of the first method and / or the second method and / or system described herein may be combined with each other in any desirable manner and / or configuration and / or with any of the examples described herein to achieve the benefits as described herein, including, for example, identifying a particular group of template strings using a resolved clustering image. Brief description of the figures The characteristics of the examples in this description will become clear with reference to the detailed description and figures that follow, in which similar reference numbers correspond to similar, though perhaps not identical, components. For the sake of brevity, reference numbers or characteristics that have a function described above may or may not be described in connection with other figures in which they appear. Figure 1 is a schematic illustration of an example of a method for elaborating an example of a complex that includes contiguously preserved gene library fragments attached to a solid support; Figure 2A to Figure 2C are schematic illustrations that together illustrate another example of a method for making another example of a complex that includes contiguously preserved gene library fragments attached to a solid support; Figure 3 is a schematic illustration of the ligation and digestion method from Figure 2A to Figure 20, which takes place in a single, single-vessel reaction; Figure 4 is a schematic illustration of an example of a portion of a gene library preparation process that generates contiguously conserved gene library fragments; Figure 5A is a top view of an example of a flow cell; Figure 5B is an enlarged and partially cropped view of an example of a flow channel of the flow cell; Figure 5C is an enlarged and partially cropped view of another example of a flow channel of the flow cell; Figures 6A to 6D are schematic views of several stages in a method for generating time-based clustering image series; Figure 7 is a schematic illustration of images (on the left side of Figure 7) taken after performing cluster generation for different samples, and resolved cluster images (on the right side of Figure 7) generated for each of the samples; Figure 8 is a schematic illustration of an example of a portion of a gene library preparation process that takes place in a flow cell using the contiguously conserved gene library fragments linked together from Figure 4; Figure 9A to Figure 9C are schematic views of several stages in a method for generating a series of time-based clustering images; Figure 10 is an originally colored screenshot, reproduced in black and white, of an Integrative Genomics Viewer (IGV) browser depicting the results for an AT-rich region of the INTS4P1 gene when a non-transferred strand was removed using thermal denaturation and when a non-transferred strand was removed using T7 exonuclease conditions; and Figure 11 is a graph depicting fluorescence (y-axis, fluorescence units) versus size (x-axis, base pairs) of PCR-amplified library fragments generated by the methods shown in Figure 2A to Figure 2C and in Figure 3. Detailed description Library fragments are deoxyribonucleic acid (DNA) fragments of similar size (e.g., < 1000 bp) from a larger or longer DNA fragment. Library fragments can be pooled together in sequencing data if a common origin compartment can be identified from which the long DNA fragment originates. Compartmentalization of different long DNA fragments may be desirable to achieve subhaploid genome content within each compartment for synthetic long reads. Synthetic long reads (or linked short reads) are enabled when a plurality of short fragments can be pooled together based on the identification of the compartment from which the long DNA fragment originated. Compartmentalization is achieved physically, for example, using wells, globules, droplets, or other physical compartments. All these compartmentalization approaches share the principle of using a barcode sequence, or index sequence, to identify the compartment in which the long DNA fragment originated. The barcode sequence attached to each of the shorter fragments can be unique to a particular long DNA fragment and can therefore help to label different compartments during gene library preparation. It is this barcode sequence that is used to group short reads together to form synthetic long reads, based on the assumption that the short reads all originate from the same compartment. The illustrative methods described herein achieve the compartmentalization of different library fragments without requiring a unique barcode sequence. The method uses contiguous library fragments and imaging to create a series of time-based clustering images. Each time-based clustering image can be used to identify a sample (compartment) from which a particular set of template strings was generated. Furthermore, each sequenced read can be clustered using the time-based clustering images. This clustering allows for linking reads, enabling the reconstitution of a long DNA fragment. Definitions The terms used in this description shall be understood to have their ordinary meaning in the relevant art unless otherwise specified. Several terms used in this description and their meanings are set out below. As used in this description, the singular terms “a,” “an,” and “the” refer to both singular and plural unless the context clearly indicates otherwise. The term “comprising,” as used in this description, is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional unlisted stages of the method or elements. References throughout this specification to “an example,” “another example,” and so on, mean that a particular element (e.g., feature, structure, composition, and / or configuration) described in connection with the example is included in at least one example described herein and may or may not be present in other examples. Furthermore, it is understood that the elements described for any example may be combined in any suitable manner across the various examples unless the context clearly dictates otherwise. IVIA / a / ZUZΊ / UI ¿UI o The terms “substantially” and “approximately” used throughout this specification, including the claims, are used to describe and account for small fluctuations, such as those due to variations in processing. For example, the terms may refer to less than or equal to ±5% of a stated value, such as less than or equal to ±2% of a stated value, such as less than or equal to ±1% of a stated value, such as less than or equal to ±0.5% of a stated value, such as less than or equal to ±0.2% of a stated value, such as less than or equal to ±0.1% of a stated value, such as less than or equal to ±0.05% of a stated value. Adapter: A linear oligonucleotide sequence that can be fused to a nucleic acid molecule, for example, by ligation or tagging. Suitable adapter lengths can range from approximately 10 nucleotides to approximately 100 nucleotides, or from approximately 12 nucleotides to approximately 60 nucleotides, or from approximately 15 nucleotides to approximately 50 nucleotides. The adapter can include any combination of nucleotides and / or nucleic acids. In some examples, the adapter may include a sequence that is complementary to at least a portion of a primer, for example, a primer that includes a universal nucleotide sequence (such as a P5 or P7 sequence).As an example, the adapter at one end of a fragment includes a sequence that is complementary to at least a portion of a first flow cell primer, and the adapter at the other end of the fragment includes a sequence that is identical to at least a portion of a second flow cell primer. The complementary adapter can hybridize to the first flow cell primer, and the identical adapter serves as a template for its complementary copy, which can hybridize to the second flow cell primer during clustering. In some examples, the adapter may include a sequencing primer sequence or a sequencing binding site. Combinations of different adapters can be incorporated into a nucleic acid molecule, such as a DNA fragment. Capture site: A portion of a flow cell surface that has been physically modified and / or chemically modified to enable complex localization. For example, the capture site might include a chemical capture agent (i.e., a material, molecule, or fragment capable of binding to, retaining, or linking a target molecule (e.g., a complex)). Another example of a chemical capture agent is a member of a receptor-ligand binding pair (e.g., avidin, streptavidin, biotin, lectin, carbohydrate, nucleic acid-binding protein, epitope, antibody, etc.) capable of binding to the target molecule (or a binding entity coupled to the target molecule). Yet another example of a chemical capture agent is a chemical reagent capable of forming an electrostatic interaction, a hydrogen bond, or a covalent bond (e.g., thiol-disulfide exchange, click chemistry, Diels-Alder, etc.) with the complex. Complex: A carrier, such as a solid support, and sequencing-ready nucleic acid fragments coupled to the carrier. The carrier may also include one member of a binding pair, the other member of which is part of the capture site. Fragment: A portion or piece of genetic material (e.g., DNA, RNA, etc.). Contiguous library fragments are smaller pieces of the larger nucleic acid sample that has been fragmented, where the smaller fragments are held together in some way (e.g., by a globule, with a transposome, etc.). Nucleic acid molecule or sample: A polymeric form of nucleotides of any length, and may include ribonucleotides, deoxyribonucleotides, their analogues, or mixtures thereof. The term may refer to single-stranded or double-stranded polynucleotides. A nucleic acid molecule (or strand) “template” (or strand) can refer to a sequence to be analyzed. The nucleotides in a nucleic acid sample may include naturally occurring nucleic acids and their functional analogues. Examples of functional analogues are those capable of hybridizing to a nucleic acid in a sequence-specific manner or capable of being used as a template for replicating a particular nucleotide sequence. Naturally occurring nucleotides generally have a backbone containing phosphodiester bonds. An analogue structure may have an alternate backbone linkage that includes any of a variety known to the technique. Naturally occurring nucleotides generally have either a deoxyribose sugar (e.g., found in DNA) or a ribose sugar (e.g., found in RNA). An analogue structure may have an alternate sugar entity that includes any of a variety known to the technique. Nucleotides may include native or non-native bases.Native DNA can include one or more adenine, thymine, cytosine, and / or guanine, and native RNA can include one or more adenine, uracil, cytosine, and / or guanine. Any non-native base can be used, such as a blocked nucleic acid (LNA) and a bridged nucleic acid (BNA). Primer. A nucleic acid molecule that can hybridize to a target sequence, such as an adapter bound to a contiguous conserved library fragment. For example, an amplification primer can serve as a starting point for template amplification and cluster generation. Alternatively, a synthesized nucleic acid strand (template) may include a site to which a primer (e.g., a sequencing primer) can hybridize to initiate the synthesis of a new strand that is complementary to the synthesized nucleic acid strand (template). Any primer may include any combination of nucleotides or nucleotide analogues. In some embodiments, the primer is a single-stranded oligonucleotide or polynucleotide. The primer length can be any number of bases and may include a variety of naturally occurring and / or non-naturally occurring nucleotides.In one example, the sequencing primer is a short string, ranging from 10 to 60 bases, or from 20 to 40 bases. Sequencing-ready nucleic acid fragments. A portion (e.g., a contiguous conserved library fragment) of genetic material that has adapters at its 3' and 5' ends. In the sequencing-ready nucleic acid fragment, each adapter includes a known universal sequence (e.g., complementary to at least a portion of a primer in a flow cell) and a sequencing primer sequence. A sequencing-ready nucleic acid fragment can be joined by inserting transposons attached to the surface of a solid support (e.g., a globule) or immobilized directly via a linker pair or other cleavable linker. Solid carrier: The small body is made of a rigid or semi-rigid material that has a characteristic shape, for example, as a sphere, oval, microsphere, or other recognized particle shape, whether it has regular or irregular dimensions. The solid carrier may have a sequencing library attached to it. Illustrative materials that are useful for the solid carrier include, but are not limited to, glass; plastic, such as acrylic, polystyrene or a copolymer of styrene and another material, polypropylene, polyethylene, polybutylene, polyurethane, or polytetrafluoroethylene (Teflon® from The Chemours Co.); polysaccharides or crosslinked polysaccharides such as agarose or sepharose; nylon; nitrocellulose; resin; silica or silica-based materials, including silicon and modified silicon; carbon fiber; metal; inorganic glass; fiber optic bundles; or a variety of other polymers.Illustrative solid supports include controlled-pore glass globules, paramagnetic globules, sol toria, SEPHAROSE® globules (cross-linked gobled form of agarose, available from Cytivia), nanocrystals, and others known in the art as described, for example, in the Microsphere Detection Guide by Bangs Laboratories, Fishers Ind. ινΐΛ / a / zuzi / ui ¿uio Transposome: A complex formed between an integrating enzyme (e.g., an integrase or a transposase) and a transferable strand, a non-transferable strand, or both transferable and non-transferable strands. Contiguously preserved gene library fragments In the examples described herein, the library fragments introduced into the flow cell are contiguous library fragments. Contiguous library fragments are smaller pieces of a larger nucleic acid sample that has been fragmented, where the smaller pieces are physically held together in some way. In some examples described herein, contiguity can be maintained by using a solid support during library preparation. In other examples described herein, contiguity can be maintained by creating fragments that are joined together during initial library preparation via linked transposomes, introducing the joined fragments into the flow cell, and then completing library preparation in the flow cell. Figure 1 represents an example of a method for forming a complex 10 that includes sequencing-ready nucleic acid fragments 12 that include fragments 14 of the larger nucleic acid sample, the contiguity of which is preserved on a solid support 16. In an illustrative method for forming complex 10 shown in Figure 1, an adapter sequence 18 is attached to the solid support 16 via a member 20 of a binding pair. In one example, this adapter sequence 18 may include a first sequencing primer sequence (e.g., a sequencing primer sequence of read 1) and a first sequence (P5') that is complementary to at least a portion of one of the amplification primers (e.g., P5) in the flow cell (shown in Figures 5A and 5B). The adapter sequence 18 is attached to member 20 of the binding pair (e.g., biotin) in such a way that it can bind to the surface of the solid support 16, which includes the other member (e.g., avidin, streptavidin, etc.) of the binding pair. As illustrated in Figure 1, a transposome complex 24 can also be attached to the solid support 16. Before loading the transposome complex 24 onto the solid support 16, a partial Y-adapter 25' can be mixed with a transposase enzyme 32 (which, although not shown, may include two Tn5 molecules) to form an example of the transposome complex 24. The partial Y-adapter 25' can include two mosaic end sequences Mi, M2 that hybridize to each other. One of the MA / a / ZUZI / u ι zu io mosaic end sequences Mi have a free end that is capable of binding to fragmented DNA strands during labeling and is therefore similar to the transferred strand 26 in Figure 2A to Figure 2C. The other mosaic end sequence M2 can be bound to a second sequencing primer sequence (e.g., a sequencing primer sequence of reading 2) and a second sequence (P7) that has the same sequence as at least a portion of another amplification primer (P7) in the flow cell, so that its copy is complementary (e.g., P7j to amplification primer (P7)). In this example, the other mosaic end sequence M2, the second sequencing primer sequence, and the second sequence make up adapter sequence 22.The adapter sequences 22 are not attached to the fragmented DNA strands during labeling and are therefore similar to the non-transferred strands 28 in Figures 2A to 2C. Loading the transposome complex 24 onto the solid support 16 may involve mixing the transposome complex 24 with the solid support 16 and exposing the mixture to conditions suitable for ligating the mosaic end Mi of the partial Y-adapter 25' to the 3' end of the adapter sequence 18. As shown in Figure 1, individual transposome complexes 24 may be attached to each of the adapter sequences 18 on the solid support 16. In this illustrative method for forming complex 10, a labeling process can then be performed. A fluid (e.g., a labeling regulator) containing the longer nucleic acid sample 30 (e.g., DNA) can be added to the solid support 16, which has the adapter sequence 18 and the transposome complexes 24 attached to it. As the sample comes into contact with the transposome complexes 24, the longer nucleic acid sample is labeled. During this labeling example, the sample 30 is fragmented into 14,14' fragments, and each of the 14,14' fragments is labeled, at its 5' end, with the free end of the Mi mosaic end of the partial Y-adapter 25'. As shown in Figure 1, labeling the longer nucleic acid sample 30 results in a plurality of bridged molecules between the transposome complexes 24. The bridged molecules wrap around the solid support 16. The transposome complexes 24 and the adapter sequences 18 maintain the contiguity of the nucleic acid sample 30 as bridged molecules, and therefore the bridged molecules are the contiguously conserved library fragments 14, 14'. The transposase enzyme can then be removed by treatment with sodium dodecyl sulfate (SDS), heat digestion, or proteinase K. Removal of transposase enzymes IVIA / a / ZUZΊ / UI ¿UI or leaves the conserved gene library fragments contiguously 14, 14' coupled to the solid support 16. To complete the fragments ready for sequencing, an additional extension and ligation (denoted by the stars in Figure 1) is performed to ensure that fragments 14 and 14' are joined to sequences 22. The resulting complex 10 is shown in Figure 1. Each contiguous 14,14' conserved library fragment is part of a respective 12,12' sequencing-ready nucleic acid fragment, each of which also includes the respective adapter sequences 18 and 22 attached to each end. Adapter sequence 18 is initially attached to the solid support 16 and includes the first sequencing primer sequence and the first sequence complementary to one of the flow cell primers. Adapter sequence 18 attaches to member 20 of a joining pair. Adapter sequence 22 is from the partial 25' Y-adapter and includes the second sequence identical to another flow cell primer and the second sequencing primer sequence. Since each 14,14' sequencing-ready nucleic acid fragment includes adapters suitable for bridging and sequencing amplification, PCR amplification is not performed.Therefore, these 12,12' fragments are ready for sequencing. Furthermore, because the contiguous 14,14' conserved library fragments are from the same longer nucleic acid sample 30, the contiguous 14,14' contiguous library fragments may be suitable for the linked long read applications described herein. Another illustrative method for forming another illustrative complex 10' (Figure 2C) is illustrated in Figure 2A to Figure 2C. In this illustrative method, the 18' adapter sequence is attached to the solid support 16 via a member 20 of a binding pair. In one example, this 18' adapter sequence may include a hybridizable sequence H, a first sequencing primer sequence (e.g., a sequencing primer sequence for read 1), and a first sequence (e.g., P5) that is identical to at least a portion of one of the amplification primers (e.g., P5) in the flow cell, such that its copy is complementary (e.g., P5j of the amplification primer (P5)). This 18' adapter sequence is attached to member 20 of the binding pair (e.g., biotin) in such a way that it can bind to the surface of the solid support 16 (which includes the other member (e.g., avidin, streptavidin, etc.) of the binding pair). MA / a / ZUZ 1 / UI ¿UI o As illustrated in Figure 2A, a transposome complex 24 can also be attached to the solid support 16. Before loading the transposome complex 24 onto the solid support 16, an L-adapter 21 can be mixed with a transposase enzyme 32 (e.g., including two Tn5 molecules) to form an example of the transposome complex 24. The L-adapter 21 can include two mosaic end sequences Mi, M2 that hybridize to each other. One of the mosaic end sequences Mi is a transferred strand 26 that is added to one end of each fragment 14, 14' during a ligation process that takes place after a tagging process. The other mosaic end sequence M2 is part of a non-transferred strand 28 that is removed after the ligation and tagging processes.This M2 mosaic end sequence is linked to a complementary hybridizable sequence HC, which is complementary to the hybridizable sequence H of the 18' adapter sequence attached to the solid support 16. The complementary hybridizable sequence HC allows the L-adapter 21 to hybridize to the 18' adapter sequence. Therefore, the complementary hybridizable sequence HC allows the transposome complex 24 to load onto the solid support. Loading the transposome complex 24 onto the solid support 16 may involve mixing the transposome complex 24 with the solid support 16 and exposing the mixture to conditions suitable for hybridization of the complementary hybridizable sequence HC of the L-adapter 21 to the hybridizable sequence H of the adapter sequence 18'. As shown in Figure 2A, the individual transposome complex 24 may be attached to each of the adapter sequences 18' on the solid support 16. In this illustrative method for forming the 10' complex, a labeling process is then performed. A fluid (e.g., a labeling regulator) containing the longer nucleic acid sample (e.g., DNA) 30 can be added to the solid support 16 that has the transposome complex 24 loaded onto it. As the sample 30 comes into contact with the transposome complexes 24 attached to the solid support, the longer nucleic acid sample 30 is labeled. During this labeling example, the sample 30 is fragmented into 14,14' fragments, and each of the 14,14' fragments is labeled, at its 5' end, with the mosaic end sequence Mi of the L-adapter 21. As shown in Figure 2A, labeling the longer nucleic acid sample 30 results in a plurality of bridged molecules between adjacent transposome complexes 24 and therefore adjacent adapter sequences 18'. The bridged molecules wrap around the solid support 16. The transposome complexes 24 and adapter sequences 18' maintain the contiguity of the nucleic acid sample 30 as bridged molecules, and therefore the bridged molecules are the contiguous conserved library fragments 14, 14'. IVIA / a / ¿U¿Ί / UI ¿UI o The transposase 32 enzyme can then be removed by treatment with sodium dodecyl sulfate (SDS) or by heat digestion or proteinase K. Ligation can then be performed to join the free mosaic Mi end sequences to their respective 18' adapter sequences. In Figure 2A, the stars represent where ligation takes place. In one example, ligation can be initiated by introducing a regulator containing a suitable ligase and heating to approximately the appropriate temperature for a time sufficient to initiate enzymatic activity. Examples of suitable ligase enzymes include E. coli DNA ligase, T7 ligase, etc. In one example, the regulator containing E. coli DNA ligase could also include nicotinamide adenine dinucleotide (NAD+). In this example, heating to approximately 16°C for approximately 15 minutes initiates enzymatic activity. The resulting structure is shown in Figure 2B. The untransferred chain 28 can then be removed from the L-adapter 21. In this example, each of the untransferred chains 28 is removed using any suitable 5'-3' exonuclease enzyme 23, such as exonuclease T7. In one example, the removal of the untransferred chain can be initiated by introducing a regulator containing the 5'-3' exonuclease enzyme 23 and waiting for a predetermined time. The 5'-3' exonuclease enzyme 23 is capable of digesting the untransferred chain 28 at room temperature (e.g., from approximately 22 °C to approximately 25 °C), and therefore no additional heating is required. The digested untransferred chains 28 can then be removed by washing. Using an exonuclease enzyme to remove untransferred strands 28 may be more desirable than heat denaturation. The 5'-3' exonuclease enzyme 23 efficiently digests untransferred strands 28, producing a cleaner template (than when using heat denaturation) for hybridization to a subsequently attached adapter sequence (see reference numbers 22 in Figure 2C). This can improve the yield of the gene library. Additionally, removing untransferred strands 28 with the 5'-3' exonuclease enzyme 23 can improve the library coverage of adenine (A) and thymine (T)-rich regions, losses of which have been observed after heat denaturation of untransferred strands 28 (see Figure 11). Furthermore, digestion with the exonuclease enzyme does not increase the overall process time for preparing the gene library. With reference now to Figure 2C, this example of the method for forming complex 10' involves introducing a partial Y-adapter 25. The partial Y-adapter 25 includes a mosaic end sequence M3 that is complementary to the mosaic end sequence Mi and an adapter sequence 22. The adapter sequence 22 may include a second sequencing primer sequence (e.g., a sequencing primer sequence for read 2) and a second sequence (P7j) that is complementary to another of the amplification primers (P7) in the flow cell. As shown in Figure 2C, the mosaic end sequence M3 of the partial Y-adapter 25 hybridizes with the mosaic end sequence Mi of the transferred strand 26 (now linked to the adapter sequence 18j) and thus links the partial Y-adapter 25 to the solid support 16. In the examples described herein, adapter sequences 18, 18', and / or 22 may also include a sequencing sample index or a barcode sequence. These sequences may be used as backups or alternatives to the compartmentalization methods described herein. To generate fragments ready for sequencing, additional extension and ligation are performed to ensure that fragments 14 and 14' are joined to the M3 mosaic end sequence and therefore to sequences 22. The resulting complex 10' is shown in Figure 2C. Yet another illustrative method for forming the 10' complex (shown in Figure 2C) is partially represented in Figure 3. In this illustrative method, ligation of the transferred chain 26 and digestion of the non-transferred chain 28 are performed as part of a single-vessel protocol. In this illustrative method, the adapter sequence 18' is joined to the solid support 16 through a member 20 of a joining pair. The adapter sequence 18', as described with reference to Figure 2A, can be used. This sequence includes the hybridizable sequence H, the first sequencing primer sequence (e.g., a sequencing primer sequence of read 1), and the first sequence (P5) that is identical to at least a portion of one of the amplification primers (e.g., P5) in the flow cell, such that its copy is complementary (e.g., P5j to the amplification primer (P5)). Furthermore, in this illustrative method, the transposome complex 24 is loaded onto the solid support 16 as described with reference to Figure 2A. Briefly, the complementary hybridizable sequence HC of the adapter-L 21 of the transposome complex 24 is hybridized to the hybridizable sequence H of the adapter sequence 18'. In this illustrative method for forming the 10' complex (Figure 2C), the labeling is done as described with reference to Figure 2A. The ligation of the transferred strand 26 and the digestion of the non-transferred strand 28 can then be performed together. This is illustrated schematically in Figure 3. For the ligase and exonuclease enzymes to function synergistically, the reagent formulation introduced onto the labeled solid support includes a regulator, the ligase enzyme, the 5'-3' exonuclease enzyme 23 (e.g., exonuclease T7), and any other components required by the respective enzymes (e.g., a cofactor such as NAD+). In one example, ligation and digestion can be initiated by introducing the reagent formulation and heating to approximately 25 °C for about 15 minutes to start enzymatic activity. Incorporating the ligase and exonuclease enzyme into the same reagent formulation can decrease the protocol time (e.g., by approximately 10 minutes compared to the illustrative method shown in Figure 2A to Figure 2C) and also reduces the number of washing steps. This example of the method then continues with the introduction of the partial Y-adapter 25, as described with reference to Figure 2C. To generate sequencing-ready fragments and the 10' end complex, further extension and ligation are performed to ensure that fragments 14 and 14' join to the mosaic end sequence M3 and thus to sequences 22. The methods described with reference to Figure 1, Figure 2A-Figure 2C, and Figure 3 for making the 10,10' complexes provide a few examples, but it should be understood that other methods can be used as long as the nucleic acid fragments ready for sequencing 12,12' are attached to the solid support 16. In other examples described in this description, contiguity information can be preserved by performing a portion of the library preparation outside the flow cell, and performing a portion of the library preparation on the flow cell. ινΐΛ / a / zuz i / ui zu io In this example, the gene library preparation can be initiated outside the flow cell using labeling, as shown schematically in Figure 4. In the example shown, a fluid (e.g., a labeling regulator) containing the longest nucleic acid sample 30 (e.g., double-stranded DNA) can be mixed with 24' transposome complexes. In the example shown in Figure 4, each 24' transposome complex is a dimer, comprising two transposase enzymes (collectively shown as “32” in Figure 4), a 26' transferred strand, and a 28' non-transferred strand. In other examples, different transposome complexes can be used, for example, one comprising the 32 transposose enzymes and the 26' transferred strand, and another comprising the 32 transposose enzymes and the 28' non-transferred strand. In this example, the 26' transferred strands are adapters that are added to one end of each 14,14' fragment during the labeling process. Specifically, each 26' transferred strand is an adapter that includes a first sequencing primer sequence (e.g., a sequencing primer sequence for read 1) and a first sequence (P5j) that is complementary to at least part of one of the amplification primers (e.g., P5) in the flow cell. In this example, the 28' non-transferred strands are adapters that are not incorporated into the 14,14' fragment during labeling, but rather can be subsequently ligated to the other end of each 14,14' fragment. As shown in Figure 4, the 28' non-transferred strands can be joined to the 26' transferred strand through at least partial base pairing during labeling. In this example, the 28' non-transferred strand is an adapter that includes a second sequencing primer sequence (e.g., a sequencing primer sequence from read 2) and a second sequence (P7) that is identical to at least a portion of one of the amplification primers (e.g., P7) in the flow cell, such that its copy is complementary (e.g., P7j of the amplification primer (P7)). As shown in Figure 4, within the fluid, the 24' transposomes fragment the longer nucleic acid sample 30 into 14,14' fragments and ligate the 26' transferred strands to the 5' end of each 14,14' fragment. In one example, the 26' transferred strand is incorporated into the 5' end of each 14,14' fragment of the longer nucleic acid sample 30 by unilateral transposition. The 28' non-transferred strands can then attach to the 26' transferred strand via base pairing. This illustrative labeling process maintains the contiguity of the longest nucleic acid sample 30 because the generated fragments 14,14' (and any of the transferred strands 26' and non-transferred strands 28' bound directly or indirectly to these) remain bound together through transposase 32. The contiguously conserved library fragments bound 14,14' are referred to in the present description as bound fragments 34. As mentioned in this description, the joined fragments 34 can be introduced into the flow cell, where further processing can be performed to complete the preparation of the gene library. This is shown schematically in Figure 8, which will be further described with reference to the methods described herein. Flow cell The methods described herein may use a flow cell 36, an example of which is illustrated in Figure 5A. The flow cell 36 includes a substrate 38 that at least partially defines a flow lane or channel 40. Substrate 38 can be a single layer / material. Examples of suitable single-layer substrates include epoxysiloxane, glass, modified or functionalized glass, plastics (including acrylics, polystyrene and styrene copolymers and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene (such as Chemours' TEFLON®), cyclic olefin / cycloolefin (COP) polymers (such as Zeon's ZEONOR®), polyimides, etc.), nylon (polyamides), ceramics / ceramic oxides, silica, fused silica or silica-based materials, aluminum silicate, silicon and modified silicon (e.g., boron-doped p+ silicon), silicon nitride (Si3N4), silicon oxide (SiO2), tantalum pentoxide (TaS0) or other tantalum oxide(s) (TaOx), hafnium oxide (HaO2), carbon, metals, inorganic glasses, or Similarly. Substrate 38 can also be a multilayer structure.Examples of multilayer structures include glass or silicon with a coating layer of tantalum oxide or another ceramic oxide on the surface. Other examples include an underlying substrate (e.g., glass or silicon) with a patterned resin on top. Still other examples of multilayer substrates include a silicon-on-insulator (SOI) substrate. In one example, substrate 38 can have a diameter ranging from approximately 2 mm to approximately 300 mm, or be a rectangular sheet or panel with its largest dimension up to approximately 10 feet (3 meters). In another example, substrate 38 is a wafer with a diameter ranging from approximately 200 mm to approximately 300 mm. In yet another example, substrate 38 is a template with a width ranging from approximately 0.1 mm to approximately 10 mm. While illustrative dimensions have been provided, it should be understood that substrate 38 of any suitable dimensions can be used. For another example, a panel, which is a rectangular support, can be used, having a surface area larger than a 300 mm round wafer. In the example illustrated in Figure 5A, flow cell 36 includes flow channels 40. Although several flow channels 40 are shown, it should be understood that any number of channels 40 can be included in flow cell 36 (e.g., a single channel 40, four channels 40, etc.). Each flow channel 40 is a defined area between two joined components (e.g., substrate 36 and a lid, or two substrates 36) that can have fluids (e.g., those described herein) introduced into and removed from them. Each flow channel 40 can be isolated from the other flow channel 40 so that fluid introduced into any particular flow channel 40 does not flow into any adjacent flow channel 40. Some examples of fluids introduced into flow channels 40 can introduce reaction components (e.g., contiguously conserved gene library fragments 14, 14' (e.g., on the solid support 16 or joined together), polymerases, sequencing primers, nucleotides, etc.), washing solutions, softening agents, etc. The flow channel 40 can be defined on the substrate 38 using any suitable technique, which depends, in part, on the material or materials of the substrate 38. In one example, the flow channel 40 is etched into a glass substrate 38. In another example, the flow channel 40 can be modeled in a resin of a multilayer substrate 38 using photolithography, nanoimprint lithography, etc. In yet another example, a separate material (not shown) can be applied to the substrate 38 such that the separate material defines the walls of the flow channel 40 and the substrate 38 defines the bottom of the flow channel 40. In one example, flow channel 40 has a straight configuration. The length and width of flow channel 40 may be smaller than the length and width of substrate 38, respectively, such that the portion of the substrate surface surrounding flow channel 40 is available for bonding to a cap (not shown) or another substrate 38. In some cases, the width of each flow channel 40 may be at least approximately 1 mm, at least approximately 2.5 mm, at least approximately 5 mm, at least approximately 7 mm, at least approximately 10 mm, or greater. In some cases, the length of each flow channel 40 may be at least approximately 10 mm, at least approximately 25 mm, at least approximately 50 mm, at least approximately 100 mm, or greater. The width and / or length of each flow channel 40 may be greater than, less than, or between the values ​​specified above.In another example, the flow channel 40 is square (e.g., 10 mm x 10 mm). IVIA / a / ¿U¿Ί / UI ¿UI o The depth of each flow channel 40 can be as small as one monolayer thickness when micro-contact printing, spraying, or dye jetting is used to deposit a separate material that defines the flow channel walls. The depth can be greater when a separate material (not shown) is used to bond the lid to the substrate 38 or to bond two substrates 38 together. As further examples, the depth of each flow channel 40 can be approximately 1 µm, approximately 10 µm, approximately 50 µm, approximately 100 µm, or greater. In one example, the depth can range from approximately 10 µm to approximately 100 µm. In another example, the depth can range from approximately 10 µm to approximately 30 µm. In yet another example, the depth is approximately 5 µm or less. It should be understood that the depth of each flow channel 40 is greater than, less than, or between the values ​​specified above. The different examples of the architecture within the flow channels 40 of flow cell 36 are illustrated in Figure 5B and Figure 5C. In the example shown in Figure 5B, the flow cell 36 includes a single-layer substrate 38A and the flow channel 40 defined at least partially in the single-layer substrate 38A. A polymeric hydrogel 42 is present in the flow channel 40. An example of the polymeric hydrogel 42 includes an acrylamide copolymer, such as poly(N-(5-azidoacetamidipentyl)acrylamide-coacrylamide, PAZAM. PAZAM and some other forms of the acrylamide copolymer are represented by the following structure (I): ινΐΛ / a / zuzi / ui ¿uio where: RAse is selected from the group consisting of azide, optionally substituted amino, optionally substituted alkenyl, optionally substituted alkyne, halogen, optionally substituted hydrazone, optionally substituted hydrazine, carboxyl, hydroxy, optionally substituted tetrazol, optionally substituted tetrazine, nitrile oxide, nitrone, sulfate, and thiol; RBes H or optionally substituted alkyl; Rc, RDy REse are selected independently of the group consisting of H and optionally substituted alkyl; each of the -(CH2)P- groups can be optionally substituted; p is an integer in the range of 1 to 50; n is an integer in the range of 1 to 50,000; ym is an integer in the range of 1 to 100,000. An expert in the technique will recognize that the arrangement of recurring “n” and “m” features in structure (I) is representative, and the monomeric subunits may be present in any order in the polymer structure (e.g., random, block, patterned, or a combination of these). The molecular weight of PAZAM and other forms of acrylamide copolymer can range from approximately 5kDa to approximately 1500 kDa or from approximately 10kDa to approximately 1000 kDa, or can be, in one specific example, approximately 312 kDa. In some examples, PAZAM and other forms of acrylamide copolymer are linear polymers. In some other examples, PAZAM and other forms of acrylamide copolymer are slightly crosslinked polymers. In other examples, polymeric hydrogel 42 can be a variation of structure (I). In one example, I o., N.^ the acrylamide unit can be replaced by N,N-dimethylacrylamide ( and In this example the acrylamide unit in structure (I) can be replaced with MA / a / ZUZI / u ι zu i orDr8 , where RD, RE and RF are each a C1-C6 alkyl, and RG and RH are each a C1-C6 alkyl (instead of H, as is the case with acrylamide). In this example, q can be an integer in the range of 1 to 100,000. In another example, N,N-dimethylacrylamide can be used in addition to the acrylamide unit. In this example, the structure (I) can include 0Λ- G r r0 R-Vn plus the recurring features of “n” and “m”, where RD, RE and RF are RDRP7 and each is H or a C1-C6 alkyl, and RG and RH are each a C1-C6 alkyl. In this example, q can be an integer in the range of 1 to 100,000. As another example of the initial polymeric hydrogel, the recurring feature “n” in structure (I) can be replaced by a monomer that includes a heterocyclic azide group having structure (II): O,,, ,N. , ''r ^Z' 'R. wherein R1 is H or a C1-C6 alkyl; R2 is H or a C1-C6 alkyl; L is a linker including a linear chain with 2 to 20 atoms selected from the group consisting of carbon, oxygen, and nitrogen, and 10 optional substituents on the carbon and any nitrogen atom in the chain; E is a linear chain including 1 to 4 atoms selected from the group consisting of carbon, oxygen, and nitrogen, and optional substituents on the carbon and any nitrogen atom in the chain; A is an N-substituted amide with an H or a C1-C4 alkyl coupled to N; and Z is a nitrogen-containing heterocycle. Examples of Z include 5 to 10 ring members present as a single cyclic structure or a condensed structure. ML / a / ZUZ 1 / u 1 zu 1 o As yet another example, polymeric hydrogel 42 can include a recurring unit of each of structures (III) and (IV): MA / a / ZUZI / u ι zu io wherein each of R1a, R2a, R1by R2b is independently selected from hydrogen, an optionally substituted alkyl or optionally substituted phenyl; each of R3ay R3b is independently selected from hydrogen, an optionally substituted alkyl, an optionally substituted phenyl or an optionally substituted C7-C14 aralkyl; and each of L1y L2 is independently selected from an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker. It should be understood that other molecules can be used to form the polymeric hydrogel 42, provided they are functionalized to graft oligonucleotide primers 44,46 onto it. Other examples of suitable polymeric layers include those having a colloidal structure, such as agarose; or a polymeric mesh structure, such as gelatin; or a cross-linked polymeric structure, such as polyacrylamide polymers and copolymers, silane-free acrylamide (SFA), or an azolized version of SFA. Examples of suitable polyacrylamide polymers can be synthesized from acrylamide and an acrylic acid or an acrylic acid containing a vinyl group, or from monomers that undergo [2+2] photocycloaddition reactions. Still other examples of suitable polymeric hydrogels 42 include mixed copolymers of acrylamides and acrylates.In the examples described herein, a variety of polymer architectures containing acrylic monomers (e.g., acrylamides, acrylates, etc.) can be used, such as branched polymers, including star polymers, star-shaped or star-block polymers, dendrimers, and the like. For example, the monomers (e.g., acrylamide, catalyst-containing acrylamide, etc.) can be incorporated, either randomly or in blocks, into the branches (arms) of a star-shaped polymer. To introduce the polymeric hydrogel 42 into the flow channel 40, a mixture of the polymeric hydrogel 42 can be generated and then applied to the substrate 38A (which has the flow channel 40 defined at least partially therein). In one example, the polymeric hydrogel 42 can be present in a mixture (e.g., with water or with ethanol and water). The mixture can then be applied to the substrate surfaces (including the flow channel(s) 40) using spin coating, dip coating, spray coating, or flow of the material under positive or negative pressure, or another suitable technique. These types of techniques deposit the polymeric hydrogel 42 onto the substrate 38A (e.g., in the flow channel 40 and in the interstitial regions 48 surrounding the flow channel 40). Other selective deposition techniques (e.g., involving a mask, controlled printing techniques, etc.) may also be used.) can be used to specifically deposit the polymeric hydrogel 42 in the flow channel 40 and not in the interstitial regions 48. In some examples, the substrate surface (including the portion exposed in the flow channel 40) can be activated, and then the mixture (including the polymeric hydrogel 42) can be applied to it. In one example, a silane or silane derivative (e.g., norbornene silane) can be deposited onto the substrate surface using vapor deposition, spin coating, or other deposition methods. In another example, the substrate surface can be exposed to plasma incineration to generate the surface activating agent(s) (e.g., -OH groups) that can adhere to the polymeric hydrogel 42. Depending on the polymer hydrogel 42, the applied mixture may be exposed to a curing process. For example, curing may take place at a temperature ranging from room temperature (e.g., approximately 25 °C) to approximately 95 °C for a time ranging from approximately 1 millisecond to approximately several days. In some examples, polishing can be performed to remove the polymeric hydrogel 42 from the interstitial regions 48 on the perimeter of the channel or flow channels 40, while leaving the polymeric hydrogel 42 on the surface of the channel or flow channels 40 at least substantially intact. The 36p flow cell also includes amplification primers 44, 46. A grafting process can be performed to graft the amplification primers 44, 46 onto the polymeric hydrogel 42 in the flow channel 40. In one example, the amplification primers 44, 46 can be immobilized on the polymeric hydrogel 42 by single-point covalent coupling at or near the 5' end of the primers 44, 46. This coupling leaves (i) a specific portion of the adapter of the primers 44, 46 free to hybridize with its corresponding nucleic acid fragment (e.g., the P5' portion bound to the 14, 14' fragment) and (ii) the 3' hydroxyl group free for primer extension. Any suitable covalent coupling can be used for this purpose. Examples of finished primers that IVIA / a / ZUZΊ / UI ¿UI or can be used include alkyne-terminated primers, which can bind to an azide entity of the polymeric hydrogel 42. Specific examples of suitable primers 44, 46 include the P5 and P7 primers used on the surface of commercial flow cells sold by Illumina Inc. for sequencing on HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NEXTSEQ™, NEXTSEQDX™, NOVASEQ™, GENOME AnalyzeR™, ISEQ™, and other instrument platforms. In one example, grafting may involve flow-through deposition (e.g., using a temporarily attached or permanently attached cap), dip coating, spray coating, puddle dispensing, or another suitable method that will couple the primer(s) 44, 46 to the polymer hydrogel 42 in the flow channel 42. Each of these illustrative techniques may use a primer solution or mixture, which may include the primer(s) 44, 46, water, a regulator, and a catalyst. With any of the grafting methods, the primers 44, 46 react with reactive groups of the polymer hydrogel 42 in the flow channel 40 and have no affinity for the surrounding substrate 38A. As such, the primers 44, 46 are selectively grafted onto the polymer hydrogel 42 in the flow channel 40. In the example shown in Figure 5C, the flow cell 38 includes a multilayer substrate 38B, which includes a support 52 and a patterned material 50 positioned on the support 52. The patterned material 50 defines depressions 54 separated by interstitial regions 48. The depressions 54 are located within each of the flow channels 40. In the example shown in Figure 5C, the pattern material 50 is placed on support 52. It should be understood that any material that can be selectively deposited, or deposited and shaped to form the depressions 54 and interstitial regions 48, can be used for the pattern material 50. As an example, an inorganic oxide can be selectively applied to substrate 52 by vapor deposition, spray printing, or inkjet printing. Examples of suitable inorganic oxides include tantalum oxide (e.g., Ta₂O₃), aluminum oxide (e.g., Al₂O₃), silicon oxide (e.g., SiO₂), hafnium oxide (e.g., HfO₂), etc. As another example, a resin can be applied to substrate 52 and then shaped. Suitable deposition techniques include chemical vapor deposition, dip coating, spray coating, pallet delivery, ultrasonic spray coating, scraper coating, spray printing, screen printing, micro-contact printing, etc. Suitable modeling techniques include photolithography, nanoimprint lithography (NIL), stamping techniques, embossing techniques, molding techniques, micro-embossing techniques, printing techniques, etc. Examples of suitable resins include a polyhedral oligomeric silsesquioxane (POSS) resin-based resin, a non-POSS epoxy resin, a poly(ethylene glycol) resin, a polyether resin (e.g., open-ring epoxies), an acrylic resin, an acrylate resin, a methacrylate resin, an amorphous fluoropolymer resin (e.g., Bellex's CYTOP®), and combinations thereof. As used herein, the term “polyhedral oligomeric silsesquioxane” (POSS) refers to a chemical composition that is a hybrid intermediate (e.g., RSiO1.s) between silica (SiO2) and silicon (R2SiO2). An example of POSS is described in Kehagias et al., Microelectronics Engineering 86 (2009), pp. 776–778, which is incorporated herein by reference. In one example, the composition is an organosilicon compound with the chemical formula [RSiO3 / 2]n, where the R groups may be the same or different. Illustrative R groups for POSS include epoxy, azide / azide, a thiol, a poly(ethylene glycol), a norbornene, a tetrazine, acrylates and / or methacrylates, or, for example, alkyl, aryl, alkoxy, and / or haloalkyl groups. The resin composition described herein may comprise one or more different cage or core structures as monomeric units. The polyhedral structure may be a T8 structure, such as: r7 ΜΛ / a / ZUZ 1 / u ι zu ioy represented by: T8 This monomeric unit typically has eight arms of functional groups Ri to Rs. The monomeric unit can have a cage-like structure with 10 silicon atoms and 10 R groups, called Thio, such as: Uncle MA / a / ZUZ 1 / UI ¿UI or , or it can have a cage-like structure with 12 silicon atoms and 12 R groups, called T12, such as: T12 The POSS-based material may alternatively include T6, Tu, or Ti6 cage structures. The average cage content may be adjusted during synthesis and / or controlled by purification methods, and a cage size distribution of the monomeric unit(s) may be used in the examples described herein. As shown in Figure 5C, the patterned material 50 includes the depressions 54 defined therein, and the interstitial regions 48 that separate adjacent depressions 54. Many different arrangements of the depressions 54 are possible, including regular, repeating, and irregular patterns. In one example, the depressions 54 are arranged in a hexagonal grid for close packing and improved density. Other arrangements may include, for example, rectilinear (rectangular) arrangements, triangular arrangements, and so on. In some embodiments, the arrangement or pattern may be an xy format of the depressions 54 arranged in rows and columns. In some other examples, the arrangement or pattern may be a repeating arrangement of the depressions 54 and / or interstitial regions 48. In still other examples, the arrangement or pattern may be a random arrangement of the depressions 54 and / or interstitial regions 48.The pattern may include dots, pads, stripes, swirls, lines, triangles, rectangles, circles, arcs, vests, planes, diagonals, arrows, squares and / or cross patterns. The arrangement or pattern of depressions 54 can be characterized with respect to the density of depressions 54 (number of depressions 54) in a defined area. For example, depressions 54 may be present at a density of approximately 2 million per mm². The density can be adjusted to different densities, including, for example, approximately 100 per mm², approximately 1000 per mm², approximately 0.1 million per mm², approximately 1 million per mm², approximately 2 million per mm², approximately 5 million per mm², approximately 10 million per mm², approximately 50 million per mm², or more or less. It should be further understood that the density of depressions 54 in the patterned material 50 may fall between one of the lower and one of the upper values ​​selected from the ranges above.As examples, a high-density array might be characterized by having 54 depressions separated by less than approximately 100 nm, a medium-density array might be characterized by having 54 depressions separated by approximately 400 nm to approximately 1 pm, and a low-density array might be characterized by having 54 depressions separated by more than approximately 1 pm. While illustrative densities have been provided, it should be understood that any suitable density may be used. The density of the 54 depressions may depend, in part, on the depth of the 54 depressions. In some cases, it may be desirable for the separation between the 54 depressions to be even greater than the examples listed herein. The arrangement or pattern of the depressions 54 can also or alternatively be characterized in terms of the average pitch, or the spacing from the center of one depression 54 to the center of an adjacent depression 54 (center-to-center spacing) or from the edge of one depression 54 to the edge of an adjacent depression 54 (edge-to-edge spacing). The pattern may be regular, such that the coefficient of variation around the average pitch is small, or the pattern may be irregular, in which case the coefficient of variation may be relatively large. In either case, the average pitch may be, for example, approximately 50 nm, approximately 0.1 pm, approximately 0.5 pm, approximately 1 pm, approximately 5 pm, approximately 10 pm, approximately 100 pm, or more or less.The average step for a particular pattern of depressions 54 may fall between one of the lower and one of the upper values ​​selected from the ranges above. In one example, depressions 54 have a step (center-to-center spacing) of approximately 1.5 pm. While illustrative average step values ​​have been provided, it should be understood that other average step values ​​may be used. The size of each depression 54 can be characterized by its volume, opening area, depth and / or diameter. Each depression 54 can have any volume capable of confining a fluid. The minimum or maximum volume can be selected, for example, to accommodate the expected productivity (e.g., multiplexing), resolution, nucleotides, or analyte reactivity for downstream applications of flow cell 38. For example, the volume can be at least approximately 1 × 10⁻³ pm³, at least approximately 1 × 10⁻² pm³, at least approximately 0.1 pm³, at least approximately 1 pm³, at least approximately 10 pm³, at least approximately 100 pm³, or more. Alternatively or additionally, the volume can be at most approximately 1 × 10⁴ pm³, at most approximately 1 × 10³ pm³, at most approximately 100 pm³, at most MA / S / ZUZI / UI ZU IO approximately 10pm3, at most approximately 1 pm3, at most approximately 0.1 pm3, or less. The area occupied by each depression opening can be selected based on criteria similar to those outlined above for volume. For example, the area for each depression opening can be at least approximately 1 x 10³ pm², at least approximately 1 x 10² pm², at least approximately 0.1 pm², at least approximately 1 pm², at least approximately 10 pm², at least approximately 100 pm², or more. Alternatively, or additionally, the area can be at most approximately 1 x 10³ pm², at most approximately 100 pm², at most approximately 10 pm², at most approximately 1 pm², at most approximately 0.1 pm², at most approximately 1 x 10² pm², or less. The area occupied by each depression opening can be greater than, less than, or fall within the values ​​specified above. The depth of each depression 54 can be large enough to accommodate part of the polymeric hydrogel 42. In one example, the depth can be at least approximately 0.1 pm, at least approximately 0.5 pm, at least approximately 1 pm, at least approximately 10 pm, at least approximately 100 pm, or greater. Alternatively or additionally, the depth can be at most approximately 1 x 10³ pm, at most approximately 100 pm, at most approximately 10 pm, or less. In other examples, the depth is approximately 0.4 pm. The depth of each depression 54 can be greater, less, or fall between the values ​​specified above. In some cases, the diameter or length and width of each depression 54 may be at least approximately 50 pm, at least approximately 0.1 pm, at least approximately 0.5 pm, at least approximately 1 pm, at least approximately 10 pm, at least approximately 100 pm, or more. Alternatively or additionally, the diameter or length and width may be at most approximately 1 x 10³ pm, at most approximately 100 pm, at most approximately 10 pm, at most approximately 1 pm, at most approximately 0.5 pm, at most approximately 0.1 pm, or less (e.g., approximately 50 nm). In some examples, the diameter or length and width is approximately 0.4 pm. The diameter or length and width of each depression 54 may be larger, smaller, or fall between the values ​​specified above. In the example shown in Figure 5C, the polymeric hydrogel 42 is placed within each of the depressions 54. The polymeric hydrogel 42 can be applied as described with reference to the MA / a / ZUZI / u ι zu io Figure 5B, so that the polymeric hydrogel 42 is present in the depressions 54 and is not present in the surrounding interstitial regions 48. Although not shown in Figure 5A, Figure 5B, or Figure 5C, it should be understood that the flow cell 36 may also include a lid attached to the substrate 38. In one example, the lid may be attached to at least a portion of the substrate 38, e.g., in some of the interstitial regions 48. The bond formed between the lid and the substrate 38 may be a chemical bond, or a mechanical bond (e.g., using a fastener, etc.). The lid can be made of any material transparent to excitation light directed toward the substrate.38 Examples include glass (e.g., borosilicate, fused silicon, etc.), plastic, or similar materials. A commercially available example of suitable glass is D263® borosilicate glass, available from Schott North America, Inc. Commercially available examples of suitable plastic materials, namely cycloolefin polymers, are the ZEONOR® products available from Zeon Chemicals LP. The lid can be bonded to substrate 38 using any suitable technique, such as laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma-activated bonding, glass frit bonding, or other methods known in the art. In one example, a separator layer can be used to bond the lid to substrate 38. The separator layer can be made of any material that will seal at least part of the substrate 38 and the lid from each other. In some examples, the separator layer can be a radiation-absorbing material that helps bond the substrate 38 and the lid. In other examples, the flow cell 36 may also include an additional substrate with or without pattern 38 attached to the substrate 38. The substrates 38 may be attached as described in this description. Methods The method generally involves generating a series of time-based clustering images for a plurality of contiguous 14,14' conserved library fragments from a genome sample. Each time-based clustering image in the series is generated sequentially by introducing, into a flow cell 36, a respective sample that includes some of the contiguous 14,14' conserved library fragments, wherein some of the contiguous 14,14' conserved library fragments are attached to a solid support 16 (Figure 1) or are joined together (e.g., joined fragments 34 shown in Figure 3); initiating the release of the contiguous 14,14' conserved library fragments MA / a / ZUZI / u ι zu io 14' of the solid support 16 or between each other 34; amplify the contiguous conserved gene library fragments 14, 14' to generate a plurality of respective template strings; stain the respective template strings; and take images of the respective template strings. Using the method(s) described herein, contiguously conserved gene library fragments 14, 14' are used in combination with amplification and sequential imaging to generate a series of time-based clustering images. Each time-based clustering image records the location and spatial orientation of the template strings generated using the particular sample. These images can then be used to identify clustering locations (template strings), where the clustering originates from a particular sample introduced into the flow cell at a specific time. The methods may vary slightly depending on whether complexes 10 are introduced into flow cell 36 or whether joined fragments 34 are introduced into flow cell 34. The various examples will be described below. Methods that use the 10 or 10' complex In this example, a genome sample (e.g., sample 30) is fragmented to form a plurality of contiguously conserved fragments 14, 14', each of which is attached to a solid support 16. It should be understood that all contiguously conserved fragments 14, 14' of genome sample 30 may not be attached to the same solid support 16; rather, the contiguously conserved fragments 14, 14' associated with particular portions of genome sample 30 may be attached to the respective solid supports 16. As such, genome sample 30 is fragmented to form a plurality of the complexes 10 or 10'. This can be achieved as described with reference to Figure 1, Figure 2A to Figure 2C, Figure 3, or by using any other contiguity preservation method that incorporates adapters 18, 22 or 18', 22 to each of the fragments 14,14' so that they are sequencing-ready fragments 12, 12'. The 10 or 10' complexes formed using genome sample 30 could be incorporated into a mixture. As such, each of the plurality of contiguous 14,14' conserved fragments is also incorporated into the mixture. The liquid carrier for the mixture can be a buffer, such as a Tris-HCl buffer or a 0.5x sodium citrate saline (SSC) buffer. IVIA / a / ZUZΊ / UI ¿UI o The liquid carrier can be added to the plurality of contiguous preserved fragments 14, 14' (e.g., present in complexes 10 or 10') to initially form the mixture, and then the mixture can be diluted with additional liquid carrier to generate a predetermined number of dilution samples to be individually introduced into flow cell 36 (or an individual lane 40 thereof). The final volume of the resulting mixture, and therefore its dilution, can be controlled in any desired manner. In some cases, the dilution may depend on the volume of flow cell 36 (or, for example, each channel 40 of a multichannel flow cell) and the desired number of samples to be introduced into flow cell 36. In one example, the volume of flow cell 36 (or channel 40 thereof) can be used as the limiting dilution factor. As such, in some examples, carrier fluid can be added to dilute the mixture to a predetermined volume, where the predetermined volume is based on i) the volume of flow cell 36 as the limiting dilution and ii) the predetermined number of dilution samples to be introduced into flow cell 36. As an example, flow cell 36 or a lane 40 of flow cell 36 may have a volume of approximately 50 pL, and the desired number of samples may be 200.In this example, the mixture can be diluted to approximately 10,000 pL. As another example, flow cell 36 or lane 40 of flow cell 36 may have a volume of approximately 100 pL, and the desired number of samples may be 384. In this example, the mixture can be diluted to approximately 38,400 pL. The desired number of dilution samples may depend, for example, on the volume of flow cell 36 and the desired resolution of the individual template strands in the respective time-based clustering images. With smaller-volume flow cells 38, it may be desirable to have a more dilute mix so that each individual dilution sample introduced into flow cell 36 contains fewer contiguous 14,14' conserved library fragments (than if a less dilute mix were used). In this type of flow cell 36, fewer contiguous 14,14' conserved library fragments will lead to fewer template strands, which can improve the resolution of the template strands in the respective time-based clustering images. In one example, the desired number of samples may range from approximately 100 to approximately 1000.In other examples, the desired number of dilution samples to be prepared from the mixture may be greater than 1000. The upper limit of the number of dilution samples may depend, in part, on the desired time frame in which the overall method will occur. The mixture can then be divided into the predetermined number of dilution samples. For example, the diluted mixture can be divided so that all the dilution samples are generated. MA / S / ZUZI / UI ZU IO at the same time. In another example, the predetermined volume of any dilution sample can be separated from the bulk mixture when it is time for that sample to be introduced into flow cell 36. Figures 6A to 6D illustrate an example of a patternless flow cell 36 (e.g., as shown in Figure 5B) from a top view during different stages of time-based clustering image generation. As shown schematically in Figure 6A to Figure 6D, the flow cell 36 can be introduced into a system that includes a flow cell receptacle 35; a fluid control system 37 that includes fluid supply 39 to supply respectively a dilution sample 56A and a dye (not shown) to a flow cell 36 positioned in the flow cell receptacle 35; an illumination system 62 positioned to illuminate the flow cell 36 positioned in the flow cell receptacle 35; and a detection system 64 positioned to capture an image of the flow cell 36 positioned in the flow cell receptacle 35. and a controller 41 in operational communication with the fluidic control system 37, the lighting system 62 and the detection system 64, the controller 41 causes the fluidic supply 39 to introduce the dilution sample 56A into the flow cell 36 positioned in the flow cell receptacle 35;causing delivery fluids 39 to introduce the dye into flow cell 36 positioned in flow cell receptacle 35 after template strands 58A are generated in flow cell 36 positioned in flow cell receptacle 35 from contiguously conserved library fragments present in dilution sample 56A; causing illumination system 62 to illuminate the dyed template strands in flow cell 36 positioned in flow cell receptacle 35; and causing detection system 64 to image the illuminated dyed template strands in flow cell 36 positioned in flow cell receptacle 35. When in position, the flow cell 36 is in continuous communication with the fluidic control system 39 (e.g., pumps, valves, and the like) and is in optical communication with a lighting system 62 and a detection system 64. In Figure 6A, a first of the dilution samples 56A (which include some of the 10 or 10' complexes, shown as 10A in Figure 6A) is introduced into the flow cell 36. The introduction of any of the respective dilution samples 56A (or e.g., 56B in Figure 6C) involves fluidly directing one of the dilution samples 56A or 56B into the flow cell 36. The dilution sample 56A can be introduced, e.g., into a cartridge 45, and the system IVIA / a / ¿U¿Ί / UI ¿UI or fluid control 37 can smoothly transport the dilution sample 56A from cartridge 45 to flow channel 40 of flow cell 36 using supply fluid 39 (e.g., pumps, valves, and the like). Given the concentration of the 10A complexes in the dilution sample 56A, most, if not all, of the 10A complexes will settle on the polymer hydrogel 42 and any primers 44, 46 in it (which are not shown in Figures 6A to 6D). In some instances, the 10A complexes may settle and remain in the flow channel 40 or depressions 54 due to the depth of the flow channel 40 or depressions 54. In other instances, the flow channel 40 or depressions 54 may include a capture site to which the 10A complexes adhere. It should be understood that some 10A, 40B, or 40C complexes may not settle, and these 10A, 40B, or 40C complexes will be removed from flow cell 36 before further processing. Examples of this method include removing the untrapped 10A complexes from flow cell 36. Washing may involve introducing a fluid into flow cell 36. The flow may then push any 10A complexes that have not settled and / or adhered through an outlet port of flow cell 36. This example of the method then includes initiating the release of contiguous 14,14' conserved library fragments from the respective solid supports 16 to which they are attached. In this example, the sequencing-ready nucleic acid fragments 12,12' (including the contiguous 14,14' conserved library fragments and the adapters 18,22 or 18',22 attached to them) are released from the respective solid supports 16. In Figure 6A, the release of the sequencing-ready nucleic acid fragments 12,12' from the solid supports 16 is represented by the arrows pointing outward from each solid support 16. The release of nucleic acid fragments ready for sequencing 12,12' can be initiated in several different ways. In one example, initiating release involves heating the flow cell 36. In this example, the system may include a heater 43. The controller 41 can cause the heater 43 to initiate the release of some of the contiguous library fragments 12,12' from the solid support 16 or from each other. As an example, temperatures greater than 70 °C can be used to at least partially break the bonds and thus initiate the release of the nucleic acid fragments ready for sequencing 12,12'. In another example, initiating release involves introducing a cleavage agent into the flow cell 36. The fluidic control system 37 can be used to deliver the cleavage agent.The cleavage agent can initiate the chemical, enzymatic, or photochemical release of nucleic acid fragments ready for sequencing 12, 12' from the solid support. IVIA / a / ¿U¿Ί / UI ¿UI o 16. In these examples, another stimulus, such as heat or light, can activate the cleavage agent to release the nucleic acid fragments ready for sequencing 12, 12' from the solid support 16. As an example, free biotin can be introduced as the cleavage agent, and heating to approximately 92 °C can be used to induce the release of biotin-oligo from the solid support 16. The sequencing-ready nucleic acid fragments 12,12' released are transported from the solid support 16 and seeded onto the polymeric hydrogel 42. More specifically, amplification primers 46, 48 seed the sequencing-ready nucleic acid fragments 12,12' released in a relatively confined manner. In one example, seeding is achieved through hybridization between the first or second sequence of the 12,12' fragment and a complementary sequence of primers 46, 48 on the polymeric hydrogel 42 in the flow cell 36. Seeding can be performed at a hybridization temperature suitable for the sequencing-ready nucleic acid fragments 12,12' and primer(s) 46, 48. The heater 43 can be controlled to bring the flow cell 36 to the seeding temperature. A washing process can be performed to remove the globules. The seeded 12,12' sequencing-ready nucleic acid fragments can then be amplified using any suitable method, such as cluster generation. In one example of cluster generation, the released 12,12' sequencing-ready nucleic acid fragments are copied from the hybridized primers 46, 48 by extending the 3' end using a high-fidelity DNA polymerase. The original 12,12' sequencing-ready nucleic acid fragments are denatured, leaving the immobilized copies within the flow channel 40 or some of the depressions 54. Any clonal amplification process can be used. For example, isothermal bridging amplification can be used to amplify the immobilized copies.For example, the copied templates coil up to hybridize with an adjacent complementary primer 46, 48, and a polymerase copies the copied templates to form double-strand bridges, which denature to form two single-stranded chains. These two chains coil up and hybridize with adjacent complementary primers 46, 48 and extend again to form two new double-stranded loops. The process is repeated for each template copy in cycles of isothermal denaturation and amplification to create dense clonal clusters. Each cluster of double-strand bridges is denatured. In one example, the reverse strand is removed by specific base cleavage, leaving forward strands of template polynucleotides. It should be understood that clustering results in the formation of multiple template strands 58A in the flow channel 40 or some of the depressions 54.In some examples, controller 41 causes heater 43 iviA / a / zuzi / ui ¿uio to run a thermal cycle to amplify the seeded nucleic acid fragments ready for sequencing 12, 12'. Figure 6B illustrates 60A clusters of 58A template strands generated from the 10A complexes of the first dilution sample 56A (compartment). The 60A clusters in Figure 6B are outlined for clarity. Although Figure 6B illustrates four 60A clusters, it should be understood that the number of 60A clusters will depend on the number of 10A complexes introduced into sample 56A, as well as the number of sequencing-ready nucleic acid fragments 12,12' released from each solid support 16. One 60A cluster is generated from each of the released sequencing-ready nucleic acid fragments 12,12'. In addition, the released sequencing-ready nucleic acid fragments 12,12' can diffuse across the flow cell surface, and therefore 60A clusters can be generated across the flow cell surface. After generating the 60A groups for the first dilution sample 56A, a dye is introduced into flow cell 36. Any fluorescent dye capable of staining the template strands 58A may be used. Examples of suitable fluorescent dyes include the SYBR® family of dyes from Molecular Probes, Inc. (e.g., SYBR® Green, SYBR® Gold, SYBR® Safe, etc.), ethidium bromide, propidium iodide, crystal violet, EVAGREEN® dye (from Biotium), DAPI (4',6-diamidine-2-phenylindole), or similar dyes. The dye is introduced into flow cell 36, e.g., from a second cartridge (not shown), incubated for a time sufficient to stain the template strands 58A, and then discharged from flow cell 36. The illumination system 62 can then be used to illuminate the dyed template strings 58A in the flux cell 36. The illumination system can include a light source and a plurality of optical components. Examples of light sources can include lasers, arc lamps, LEDs, or laser diodes. Optical components can be, for example, reflectors, dichroic reflectors, beam splitters, collimators, lenses, filters, wedges, prisms, mirrors, detectors, and the like. The illumination system can be operatively positioned to direct excitation light onto the flux cell surface corresponding to the dye used. The detection system 64 can be used to capture an h-image of the fluorescent template strings 58A. This h-image is the time-based clustering image for dilution sample 56A because it represents the spatial location and orientation of the template strings 58A associated with dilution sample 56A. Any suitable camera can be used to capture an h-image of the 60A clusters in flow cell 36. MA / a / ZUZl / u ι zu io Image h can be stored electronically for retrieval and later use. Examples of systems include an electronic storage component 47 for storing image h. In the electronic record, image h can be linked to dilution sample 56A. Image h can also be assigned a time record. Examples of this method include assigning each time-based cluster image li in the series a time record of the respective sample introduction, which includes some of the contiguous preserved gene library fragments. The time record can include a timestamp indicating when dilution sample 56A was introduced and / or captured, a step number in an introduction and / or imaging sequence (e.g., sample 1 of 200, sample 2 of 200, ... sample X of 200), or combinations thereof. A wash may take place after imaging of groups 60A in flow cell 36. Water, a buffer, or another mild wash solution may be used. The processes illustrated and described with reference to Figure 6A and Figure 6B are then repeated with a second dilution sample 56B (illustrated in Figure 6C). In Figure 6C, the second dilution sample 56B is introduced into the flow cell 36. The 10B complexes (which may be 10 or 10j complexes) are fixed and / or adhered to the surface of the flow cell. As shown in Figure 6C, the method includes initiating the release of contiguous 14,14' conserved library fragments from the respective solid supports 16 to which they are attached. In this example, the nucleic acid fragments ready for sequencing 12,12' (including the contiguous 14,14' conserved library fragments and the adapters 18,22 or 18',22 attached to them) are released from the respective solid supports 16. The sequencing-ready nucleic acid fragments 12,12' released are transported from the solid support 16 and seeded onto the polymeric hydrogel 42. The seeded sequencing-ready nucleic acid fragments 12,12' can then be amplified using any suitable method, such as cluster generation. It should be understood that this clustering round results in the formation of several additional template strands 58B in the flow channel 40 or some of the depressions 54. Figure 6D illustrates 60B clusters of 58B template chains generated from the respective 10B complexes of the second dilution sample 56B (compartment). The 60A and 60B clusters in Figure 6D are... Figure 6D is outlined for clarity. Although Figure 6D illustrates three 60B groups, it should be understood that the number of 60B groups will depend on the number of 10B complexes introduced into the 56B sample, as well as the number of nucleic acid fragments ready for sequencing 12,12' released from each solid support 16. After generating the 60B groups for the second dilution sample 56B, the dye is reintroduced into flow cell 36. The same dye used to dye template strands 58A can be used to dye template strands 58B and any template strands generated subsequently. The illumination system 62 can then be used to illuminate the dyed template strings 58A and 58B in the flux cell 36. The detection system 64 can be used to capture an image of the fluorescent template strings 58A and 58B in the respective groups 60A and 60B. Image l2 can also be stored electronically for retrieval and later use. In the electronic record, image l2 can be linked to dilution sample 56B. Image l2 can also be assigned a timestamp. The timestamp can include a timestamp indicating when the image of dilution sample 56B was captured, a step number in a sequence (e.g., sample 2 of 200), or combinations of these. A wash may take place after imaging of groups 60A, 60B is obtained in flow cell 36. Water, a buffer, or another mild wash solution may be used. The processes illustrated and described with reference to Figure 6A and Figure 6B can then be repeated, e.g., using the described system, for the number of dilution samples derived from the original mixture. Each additional image l3, l4, ... Ix will illustrate new groups 60C, 60D, ... 60X from the template supports 58C, 58D, ... 58X generated by introducing a respective dilution sample 56C, 56D, ...56X. All images h, l2, ... Ix obtained for the respective dilution samples 56A, 56B, ...56X are associated with a particular mixture and therefore a particular longer nucleic acid molecule 30. Because each sequential image l2, h, l4, ... L represents a newly formed group 60B, 60C, 60D, ... 60X with respect to the immediately preceding image h, l2, l3, ... lx, image subtraction can be used to generate a resolved cluster image for each sample introduced after the first sample 56A. Some examples of resolved cluster images Rlx are shown in Figure 6. iviA / a / zuz 1 / u 1 zu 1 o Figure 7 represents the h, l2, k, L, k, k images taken for six different dilution samples 56A, 56B, 56C, 56D, 56E, 56F that are sequentially introduced, amplified, stained, and captured as described with reference to Figure 6A and Figure 6B. As illustrated, new groups 60A, 60B, 60C, 60D, 60E, and 60F are generated respectively for each of the newly introduced and processed dilution samples 56A, 56B, 56C, 56D, 56E, 56F. Since image h for the first sample 56A includes 60A groups from one sample, it is not necessary to generate a resolved cluster image, as the original image l· can be used for the spatial identification of the 60A groups. For each sample introduced after the first sample 56A, a resolved cluster image Rl2, Rl3, RL, Rls, Rk is generated for each of the other images l2, l3, L, k, k. As an example, image h (representing template strings 58A in 60A groups) can be subtracted from image l2 (representing template strings 58A in 60A groups and template strings 58B in 60B groups) to generate a resolved cluster image Rl2 for the second sample 56B. This resolved clustering image Rl2 represents a spatial location and orientation of template strings 58B in clusters 60B associated with the second sample 56B.For another example, image h (representing template strings 58A in groups 60A) and image l2 (representing template strings 58A in groups 60A and template strings 58B in groups 60B) can be subtracted from image l3 (representing template strings 58A in groups 60A, template strings 58B in groups 60B, and template strings 58C in groups 60C) to generate a resolved cluster image Rl3 for a third sample, e.g., 56C. This resolved cluster image Rl3 represents a spatial location and orientation of template strings 58C in groups 60C associated with a third sample 56C. The resolved cluster image RL,Rk, and Rk can be generated similarly by subtracting any of the preceding images. It should be understood that image subtraction can be performed for any of the h, l2, l3, · lx in a series. The resulting resolved cluster image Rlx for any given 56X dilution sample represents the location and spatial orientation of the 58X template strings associated with that 56X dilution sample. The resolved clustering images can be stored for later analysis. Methods that use joined fragments 34 In this example, a genome sample is fragmented to form a plurality of contiguous conserved fragments 14, 14' that are joined together, e.g., as joined fragments ινΐΛ / a / zuz i / u 1 ¿u 1 o 34. It should be understood that all contiguous conserved fragments 14, 14' of the genome sample may not be joined together; rather, the process shown in Figure 4 may result in the formation of several joined fragments 34. The joined fragments 34 formed using the genome sample can be incorporated into a mixture. As such, each of the plurality of contiguous conserved fragments 14, 14' is also incorporated into the mixture. The liquid carrier for the mixture can be a buffer, such as a Tris-HCl buffer or a 0.5x sodium citrate saline (SSC) buffer. The liquid carrier can be added to a plurality of bonded fragments 34 to initially form the mixture, and then the mixture can be diluted with additional liquid carrier to generate a predetermined number of dilution samples to be introduced individually into the flow cell 36 (or an individual lane 40 thereof). The final volume of the mixture that is generated, and therefore the dilution of the mixture, can be controlled in any desired manner and as described in this description. The mixture can then be divided into a predetermined number of dilution samples. In one example, the dilute mixture can be divided so that all dilution samples are generated simultaneously. In another example, a predetermined volume of any given dilution sample can be separated from the bulk mixture when it is time for that sample to be introduced into flow cell 36. In this illustrative method, the bonded fragments 34 shown in Figure 4 are introduced into the flow channel as part of one of the dilution samples. An example of this dilution sample 66A is shown in Figure 8. As shown in Figure 8, within the flow cell 36, the transposases 32 are removed from the linked fragments 34. This can be achieved, for example, using SDS or proteinase. The removal of the transposases 32 releases contiguously conserved fragments 14, 14' (and any 26', 28' chains directly or indirectly linked to these) from adjacent contiguously conserved fragments 14, 14'. In other words, the subfragments 72 of the linked fragments 34 are released and are able to seed into the polymeric hydrogel 42 via the 26' transferred chains. As shown schematically in Figure 8, the 26' transferred chains hybridize to the complementary and respective primers 46 on the surface of the flow cell 36. In some cases, heat may be applied during hybridization. MA / a / ZUZI / u ι zu io application of heat may depend on the melting temperature of the transferred chains 26'. As an example, the P5' portion of the transferred chain 26' is hybridized to the complementary P5 amplification primer 46 attached to the polymeric hydrogel 42. A wash solution can be flowed through the flow channel of flow cell 36 to remove transposases 32 from flow cell 36. An example of a suitable wash solution includes SDS, which can remove the transposase. A second wash solution, such as TRIS or a hybridization wash regulator, can be used to rinse flow cell 36. Prior to amplification, this example of the method also includes introducing a second portion of the sequence (e.g., 28' non-transferred strands) into each of the contiguous conserved library fragments hybridized 14, 14' at an end opposite the hybridized end.As such, in some examples of the method, each of the contiguous conserved library fragments 14,14' includes a first sequence portion at a first end that hybridizes to a first primer sequence 46 on a flow cell surface 26; and prior to amplification, the method further comprises attaching a second sequence portion to each of the hybridized conserved library fragments 14,14' at a second end opposite the first end, the second sequence portion being identical to a second primer sequence 48 on the flow cell surface 36, so that the copy of the second sequence portion can hybridize to the second primer sequence 48. The introduction of the second sequence portion can be performed using extension ligation. In one example, extension ligation (as represented by arrows 74 in Figure 8) can be initiated to join the non-transferred 28' chains to the corresponding 14, 14' fragments. In one example, extension ligation can be initiated by introducing an extension ligation mixture into the flow cell 36 and heating it to a temperature suitable for enzymatic activity (e.g., in the range of approximately 37 °C to approximately 50 °C). The extension ligation mixture may include a ligation enzyme (e.g., DNA ligase) that catalyzes the formation of a link between a 28' non-transferred strand and its corresponding 14' or 14' fragment. As described with reference to Figure 4, the 28' non-transferred strands include a second sequencing primer sequence (e.g., a sequencing primer sequence for reading 2) and a second sequence (P7) that is identical to at least a portion of another amplification primer (P7) on the flow cell surface. This second sequence allows the generation of a complementary copy, e.g., P7', during amplification, which can hybridize to amplification primer (P7) on the flow cell surface during the ML / a / ZUZ 1 / UI ¿UI or clustering. As such, ligation results in the formation of the sequencing of new 12, 12' library fragments attached to the surface of the flow cell. The extension ligation mixture may also include a blocking group that is attached to the exposed ends of the primers 46 to prevent unwanted extension of these primers 46. Alternatively, the primers 46 may be surface-grafted with blocking groups (e.g., a phosphate at 3j attached to it). In yet another example, blocking groups may not be used. Since the resulting fragments 12,12' bind together, heating can be used to dissociate the 12' fragments from the 12 fragments. The 12' fragments that do not hybridize with the primers 46 can be removed from the flow cell 36 by washing. When used, any locked primer 46 can be unlocked (e.g., using kinase or another suitable unlocking agent) so that amplification can be performed. In this example, amplification can be carried out using any suitable method, such as cluster generation. Cluster generation can be performed as described herein with reference to Figure 6A. The system described with reference to Figure 6A can be used. Figure 9A illustrates the 70A groups of template strands 68A generated from the attached fragments 34 of the dilution sample 66A (shown in Figure 8). The 70A groups in Figure 9A are outlined for clarity. Although Figure 9A illustrates four 70A groups, it should be understood that the number of 70A groups will depend on the number of attached fragments 34 introduced into sample 66A, as well as the number of nucleic acid fragments ready for sequencing 12,12' released from the attached fragments 34. After generating the 70A groups for the first dilution sample 66A, a dye is introduced into flow cell 36 as described with reference to Figure 6B. The dye is introduced into flow cell 36, allowed to incubate for a suitable period of time to stain the template strands 68A, and then discharged from flow cell 36. The illumination system 62 can then be used to illuminate the dyed template strings 68A in the flow cell 36, and the detection system 64 can be used to capture an h-image of the fluorescent template strings 68A. This h-image is the time-based cluster image for the dilution sample 66A because it represents a spatial location and orientation of the template strings 68A associated with the dilution sample 66A. ML / a / ZUZ 1 / u ι zu io Image h can be stored electronically for retrieval and later use. In the electronic record, image h can be linked to dilution sample 66A. Image h can also be assigned a timestamp. The timestamp can include a timestamp indicating when dilution sample 66A was introduced and / or acquired, a step number in an introduction and / or imaging sequence (e.g., sample 1 of 200, sample 2 of 200, ... sample X of 200), or combinations thereof. A wash may take place after imaging of groups 70A in flow cell 36. Water, a buffer, or another mild wash solution may be used. The processes illustrated and described with reference to Figure 8 and Figure 9A are then repeated with a second dilution sample 66B (illustrated in Figure 9B). In Figure 9B, the second dilution sample 66B is introduced into flow cell 36. The joined fragments 34 can be split into subfragments 72 as described with reference to Figure 8. A transferred strand 26 from at least some of the subfragments 72 will be hybridized to the complementary amplification primers 46 in flow cell 36. Extension ligation and the other processes described with reference to Figure 8 can then be performed, resulting in nucleic acid fragments ready for sequencing 12 attached to the surface of the flow cell. Group generation can be performed as described in the present description with reference to Figure 6A. Figure 9C illustrates the 70B groups of template strands 68B generated from the attached fragments 34 of the second dilution sample 66B. Groups 70A and 70B in Figure 9C are outlined for clarity. Although Figure 9C illustrates two 70B groups, it should be understood that the number of 70B groups will depend on the number of attached fragments 34 introduced into sample 66B, as well as the number of nucleic acid fragments ready for sequencing 12,12' released from the attached fragments 34. After generating the 70B groups for the second dilution sample 66B, the dye is reintroduced into flow cell 36. The same dye used to dye template strands 68A can be used to dye template strands 68B and any template strands generated subsequently. MA / a / ZUZI / u ι zu io The illumination system 62 can then be used to illuminate the dyed template strings 68A and 68B in the flux cell 36. The detection system 64 can be used to capture an image of the fluorescent template strings 68A and 68B in the respective groups 70A and 70B. Image l2 can also be stored electronically for retrieval and later use. In the electronic record, image l2 can be linked to dilution sample 66B. Image l2 can also be assigned a timestamp. The timestamp can include a timestamp indicating when the image of dilution sample 56B was captured and / or imported, a step number in a sequence (e.g., sample 2 of 1000), or combinations thereof. A wash may take place after imaging of groups 70A, 70B is obtained in flow cell 36. Water, a buffer, or another mild wash solution may be used. The processes illustrated and described with reference to Figure 9B and Figure 9C can then be repeated for the number of dilution samples derived from the original mixture. Each additional image h, l4, ... Ix will illustrate new groups 70C, 70D, ... 70X from the template supports 68C, 68D, ... 68X generated with the introduction of a respective dilution sample 66C, 66D, ...66X. All images h, l2, ... Ix obtained for the respective dilution samples 66A, 66B, ...66X are associated with a particular mixture and therefore a particular longer nucleic acid molecule 30. Because each sequential image l2, l3, l4, ... L represents a newly formed group 70B, 70C, 70D, ... 70X with respect to the immediately preceding image h, l2, l3, ... lx, image subtraction can be used to generate a resolved cluster image for each sample introduced after the first sample 66A. The resolved cluster images Rlx can be generated as described with reference to Figure 7. It should be understood that image subtraction can be performed for any of the l1, l2, l3, ... Ix in a series. The resulting resolved cluster image Rlx for any given 66X dilution sample represents the location and spatial orientation of the 68X template strings associated with that 66X dilution sample. The resolved clustering images can be stored for later analysis. IVIA / a / ZUZΊ / UI ¿UI o Other methods Instead of introducing individual dilution samples 56A, 66A, the dilute mixture can be diffused into the flow cell in predetermined volumes, and processing in flow cell 38 can be carried out as described herein to amplify, stain, and record the generated template string images 58X, 68X. Diffusion can be controlled so that a predetermined volume is introduced at a time. As such, some examples of the method include generating a time-based clustering image for each of the limiting dilution samples introduced into flow cell 36 by: controlling the diffusion of the mixture into the flow cell so that one of the limiting dilution samples is introduced into flow cell 36 at a time; initiating the release of contiguously conserved library fragments 14, 14' from a solid support 16 or from each other (e.g., from the linked fragments 34) into one of the limiting dilution samples in flow cell 36; amplifying the contiguously conserved library fragments 14, 14' to generate a plurality of respective template strands 58A, 68A; staining the respective template strands 58A, 68A; and imaging the respective template strands 58A, 68A. Sequencing and analysis When all dilution samples of a mixture are amplified and imaged as described herein, the flow cell 36 is ready for sequencing. A variety of sequencing approaches or technologies can be used, including techniques often referred to as sequencing by synthesis (SBS), cyclic array sequencing, ligation sequencing, pyrosequencing, etc. As an example, a sequencing-by-synthesis (SBS) reaction can be performed on a system such as HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NOVASEQ™, NEXTSEQDX™, ISEQ™, NEXTSEQ™, or other Illumina sequencing systems (San Diego, CA). In SBS, the extension of sequencing primers along template strands 58A, 58B, ... 58X is monitored to determine the nucleotide sequence in the templates. The 3' ends of template chains 58A, 58B, ... 58X and any primers attached to flow cell 46, 48 (not coupled to template chains 58A, 58B, ... 58X) can be blocked to prevent interference with the sequencing reaction and in particular to prevent unwanted priming. MA / a / ZUZI / u ι zu io A sequencing primer can be introduced that hybridizes to a complementary sequence in the 58A, 58B, ... 58X template strands. This sequencing primer produces the 58A, 58B, ... 58X template strands ready for sequencing. The underlying chemical process can be polymerization (e.g., catalyzed by a polymerase enzyme) or ligation (e.g., catalyzed by a ligase enzyme). In a polymerase-based SBS process, fluorescently labeled nucleotides are added to the sequencing primer in a template-dependent manner, such that the order and type of nucleotides added to the sequencing primer can be used to determine the template sequence. For example, to initiate a first SBS cycle, one or more labeled nucleotides, DNA polymerase, etc., can be supplied into / through flow cell 36, etc., where extending the sequencing primer causes a labeled nucleotide to be incorporated. This incorporation can be detected through an imaging event. During an imaging event, the illumination system 62 can provide excitation light to flow cell 36. In some examples, fluorescently labeled nucleotides may also include a reversible termination property that stops further primer extension once a nucleotide has been added to the template. For example, a nucleotide analog with a reversible terminator can be added to a template so that no further extension can occur until a release agent is administered to remove the terminator. Therefore, for examples using reversible termination, a release reagent can be supplied to flow cell 36, etc. (after detection has occurred). The washing may take place between the various fluid delivery stages. Afterward, the SBS cycle may be repeated n times to extend the template by n nucleotides, thereby detecting a sequence of length n. Although the SBS has been described in detail, it should be understood that the flow cell 36 described herein may be used with another sequencing protocol, for genotyping, or in other chemical and / or biological applications. The sequencing reads obtained during the sequencing operation can be grouped together based on the resolved clustering images Ría, Rl3,... RL. The grouped sequencing reads can then be linked to a respective sample from the dilution samples based on the resolved clustering images Rl2, Rh, ... Rlx. It can be inferred that the grouped and linked sequencing reads originated from the same acid sample. IVIA / a / ¿U¿Ί / UI ¿UI or longer nucleic 30. As such, some examples of the method include performing a sequencing operation in flow cell 26 that includes the respective template strings for each of the plurality of library fragments; and grouping sequencing reads together into different groups based on the resolved clustering images RI, Rl2, Rh, Rlx. To further illustrate the present description, examples are provided herein. It is understood that these examples are provided for illustrative purposes and should not be interpreted as limiting the scope of the present description. Examples Example 1 Barcode oligonucleotides (e.g., adapter 18) were linked via a biotin connector to streptavidin M280 globules (Thermofisher) to form a globule mixture. A universal transposome was hybridized to the complementary sequence at the end of the oligos to form a globule-linked transposome (BLT). The BLTs were then washed in a wash buffer, resuspended in a working buffer, and accessory proteins (single-stranded binding protein (Thermofisher) and double-stranded binding protein (Illumina)) were added. High molecular weight NA12878 DNA (extracted from cultured cells using the Qiagen MAGATTRACT® HMW DNA extraction protocol) was added to the BLT mixture, and the tubes were gently inverted to mix. The tubes were then incubated at room temperature for approximately 15 minutes to allow the DNA to wrap around the globules. A labeling buffer (containing magnesium chloride and tris acetate) was then added to each tube, and the samples were incubated for approximately 10 minutes at approximately 55°C. During this step, the transposome labeled the DNA, and the labeled DNA bound to the BLTs. After the labeling reaction, sodium dodecyl sulfate (SDS) was added, and the samples were incubated at room temperature for approximately 5 minutes to denature the transposase. The tubes were then placed on a magnet, and the supernatant was removed. The globules were washed with the wash buffer.After the final wash, the globules were resuspended in a ligase mixture (containing T7 ligase and its associated regulator, NEB T7 ligase regulator). The samples were then mixed and incubated for approximately 45 minutes at room temperature. During this time, the separation on the transfer support (where the transposome initially hybridized) was ligated. IVIA / a / ZUZΊ / UI ¿UI or physically attached the labeled DNA to the sphere. Then the samples were placed on a magnet, the supernatant was removed, and the globules were washed again in the wash regulator. The labeled cells were then divided into two groups to remove non-transferred strands and introduce a sample index (e.g., adapter 22). One group (comparative group) was exposed to a comparative workflow in which heat was used to remove non-transferred strands. The other group (example group) was exposed to an example workflow in which an exonuclease was used to remove non-transferred strands. The comp. group was resuspended in the wash buffer and heated to approximately 80 °C for about 5 minutes to denature the untransferred chains. The tubes containing the comp. group were then placed on a magnet, the supernatant was discarded, and the globules were washed. The sample index, diluted in the wash buffer, was then added to the comp. group globules, and this mixture was incubated at approximately 80 °C for about 1 minute, followed by a slow temperature reduction. The sample group was suspended in a T7 exonuclease mixture (containing T7 exonuclease and NEB 4 regulator) and incubated at room temperature for approximately 10 minutes. The 5' to 3' exonuclease activity of the T7 exonuclease digested the untransferred strands. The tubes containing the sample group were then placed on a magnet, the supernatant was discarded, and the cells were washed. The sample index, diluted in the wash regulator, was then added to the sample group cells, and this mixture was incubated at approximately 55 °C for approximately 5 minutes to allow annealing of the sample index. The tubes containing the comp. and ex. groups, respectively, were placed on a magnet, and the supernatant was removed. A spreading binder was added, and the samples were incubated for approximately 5 minutes at approximately 37 °C. The tubes were then placed on a magnet again, the supernatant was removed, and the beads from the comp. and ex. groups were washed in the wash buffer. After the final wash, the globules were resuspended in the wash buffer. Some cells from the comp. group and some cells from the ex. group were subsampled and used in a PCR reaction. In addition to the respective cells, each PCR mix consisted of PCR mix, EPM illumina, and P5 and P7 oligos. Each sample was amplified using PCR. MA / a / ZUZI / u ι zu io After PCR, a subsample of the PCR supernatant from each comp. group and the ex. group were transferred to a new tube, and size selection (0.50x–0.62x solid-phase reversible immobilization, SPRI) was performed using sample purification globules. The resulting libraries were eluted in a resuspension regulator. These libraries were sequenced in single-cell 2500 HISEQ™ fast flow cells (using a standard read length of 2 x 10¹ cycles). After Fastq generation, the samples were aligned to the human genome (hg38), and the data were imported into IGV. Figure 10 shows the coverage obtained, using the library of each comp. group (labeled thermal denaturation) and the ex. group (labeled T7 exonuclease), for an AT-rich region of the human genome known to be negatively affected by the high-temperature steps in the library preparation protocol. The results for library fragments from the comp. group whose non-transferred strands were removed by heat denaturation are shown at the top of Figure 10, and the results for library fragments from the ex. group whose non-transferred strands were removed by exonuclease digestion are shown at the bottom of Figure 10. As illustrated, there was complete coverage of the AT-rich region for the library fragments from the ex. group, while there was only partial coverage of the same region for the library fragments from the comp. group.These results indicate that the use of the enzymatic digestion method described in this description increases the coverage of the gene library and improves sequencing in rich regions of the genome compared to heat denaturation. Example 2 The complexes were prepared as described in the method of Figure 2A to Figure 2C (multi-stage ligation and digestion) and in the method of Figure 3 (single-vessel ligation and digestion). BLT generation, DNA binding, labeling, and SDS exposure were performed as described in Example 1. After transposase removal and associated washing, the labeled globules were divided into two groups to remove non-transferred strands by means of multi-stage ligation and digestion (referred to as the e.g. 2 group) or by means of single-pot ligation and digestion (referred to as the e.g. 3 group). The Example 2 group was resuspended in a mixture of e. Cali DNA ligase, which includes e. Cali DNA ligase and its associated regulator, both from NEB. The Example 2 group samples were then pooled and incubated for approximately 15 minutes at approximately 16 °C. The tubes containing the Example 2 samples were then placed on a magnet, the supernatant was discarded, and the MA / a / ZUZl / u ι zu io globules from Example 2 were washed in the wash regulator. After washing, the globules from the ex. 2 group were resuspended in a mixture containing exonuclease T7 and NEB 4 regulator and incubated at approximately 25 °C for approximately 10 minutes. Group 3 was resuspended in a combined ligase and exonuclease mixture including e. coli DNA ligase, T7 exonuclease, NAD+ and a CUTSMART™ regulator (from NEB) at approximately 25°C for approximately 15 minutes. The tubes containing sample group 2 and sample group 3 were then placed on a magnet, the supernatant was removed, and the respective cells were washed. The sample index, diluted in the wash regulator, was then added to the cells of each sample group, and these mixtures were incubated at approximately 55 °C for approximately 5 minutes to allow annealing of the sample index. The tubes containing sample group 2 and sample group 3, respectively, were placed on a magnetic stir bar and the supernatant was removed. A spreading binder was added, and the samples were incubated for approximately 5 minutes at approximately 37°C. The tubes were then placed on a magnetic stir bar again, the supernatant was removed, and the beads from the compound group and sample group were washed in the wash buffer. After the final wash, the globules were resuspended in the wash buffer. Some cells from group 2 and some cells from group 3 were subsampled and used in a PCR reaction. In addition to the respective cells, each PCR mix consisted of PCR mix, EPM illumina, and P5 and P7 oligos. Each sample was amplified using PCR. After PCR, a subsample of the PCR supernatant from group 2 and group 3 was transferred to a new tube. Sample purification pellets were added, and a 2.5x SPRI was performed. The resulting library was eluted in a resuspension buffer. The cleaned libraries were then run on a high-sensitivity BioAnalyzer 2100 chip, and the trace was obtained as shown in Figure 11. The results show that the size profiles and yields of the libraries were comparable for multi-step ligation and digestion and for single-step ligation and digestion. These results indicate that the combined reagent formulation did not adversely affect ligation and did not result in digestion of the transferred fragment or strand. The gene library fragments bound to illustrative globules (complexes) obtained using the gene library preparation methods described in Examples 1 and 2 can be divided into subsamples and diluted to form a plurality of dilution samples as described herein. The method described with reference to Figures 6A to 6D (which includes clustering, staining, and imaging) can then be performed on each of the dilution samples to generate a series of time-based clustering images. When all the dilution samples are amplified and imaged, the flow cell is ready for sequencing. The library preparation techniques and time-based imaging techniques described herein can be used together to efficiently and reliably reconstitute a long DNA fragment. Additional notes Furthermore, it should be understood that the ranges provided in this description include the stated range and any values ​​or subranges within that range, as if explicitly stated. For example, a range represented by approximately 2 mm to approximately 300 mm should be understood to include not only the explicitly stated limits of approximately 2 mm to approximately 300 mm, but also individual values ​​such as approximately 15 mm, 22.5 mm, 245 mm, etc., and subranges such as approximately 20 mm to approximately 225 mm, etc. It should be noted that all combinations of the foregoing concepts and additional concepts described in greater detail below (provided such concepts are not mutually inconsistent) are considered part of the subject matter of the invention described herein. In particular, all combinations of the claimed subject matter appearing at the end of this description are considered part of the subject matter of the invention described herein. It should also be noted that terminology used explicitly in this description, which may also appear in any description incorporated by reference, should have a meaning consistent with the particular concepts described herein. While several examples have been described in detail, it is understood that the examples described may be modified. Therefore, the above description is not intended to be limiting.

Claims

1. A method comprising: generating a series of time-based clustering images for a plurality of contiguous conserved library fragments from a genome sample, characterized in that each time-based clustering image in the series is generated sequentially by: introducing, into a flow cell, a respective sample including some of the contiguous conserved library fragments, wherein some of the contiguous conserved library fragments are attached to a solid support or are attached to each other; initiating the release of some of the contiguous conserved library fragments from the solid support or from each other; amplifying some of the contiguous conserved library fragments to generate a plurality of respective template strands; staining the respective template strands; and imaging the respective template strands.

2. The method as defined in claim 1, further comprising assigning to each time-based clustering image in the series a time record of the introduction of the respective sample that includes some of the contiguous preserved gene library fragments.

3. The method as defined in claim 2, characterized in that the time record is a timestamp or a number of steps in a sequence.

4. The method as defined in any one of claims 1 to 3, further comprising using image subtraction to generate a resolved cluster image for each of the respective samples, characterized in that each resolved cluster image records a location and spatial orientation of the respective template strings associated with one of the different samples.

5. The method as defined in claim 4, further comprising storing the resolved clustering images. iviA / a / zuzi / ui ¿uio 6. The method as defined in claim 4 or 5, further comprising: performing a sequencing operation in the flow cell that includes the respective template strings for each of the plurality of library fragments; and grouping sequencing reads together into different groups based on the resolved clustering images.

7. The method as defined in claim 6, further comprising linking the different groups to each of the different samples based on the resolved clustering images.

8. The method as defined in any one of claims 1 to 6, characterized in that: prior to generating the series of time-based clustering images, the method further comprises: adding a liquid carrier to the plurality of contiguously conserved gene library fragments to form a mixture; and diluting the mixture with the liquid carrier to generate a predetermined number of dilution samples to be introduced into the flow cell; and the introduction of the respective sample involves fluidly directing one of the dilution samples into the flow cell.

9. The method as defined in claim 8, characterized in that the mixture including the plurality of contiguously conserved gene library fragments is diluted to a predetermined volume based on i) a flow cell volume as a limiting dilution and ii) the predetermined number of dilution samples.

10. The method as defined in any one of claims 1 to 9, characterized in that: each contiguously conserved gene library fragment is attached to the solid support; and release involves heating.

11. The method as defined in any one of claims 1 to 9, characterized in that: each of the contiguous conserved library fragments includes a first sequence portion at a first end that hybridizes to a first primer sequence on a flow cell surface; and prior to amplification, the method further comprises attaching a second sequence portion to each of the hybridized conserved library fragments at a second end opposite the first end, the second sequence portion being identical to a second primer sequence on the flow cell surface.

12. The method as defined in any one of claims 1 to 9, characterized in that: each contiguously conserved library fragment is attached to the solid support; the solid support has a plurality of adapters attached thereto; and the method further comprises preparing the contiguously conserved library fragment attached to the solid support by: labeling the genome sample in the presence of the solid support and a plurality of L-adapters, each L-adapter comprising a transferred strand and a non-transferred strand, thereby generating a plurality of sample fragments, wherein a respective transferred strand is incorporated into a 5' end of each sample fragment and a respective non-transferred strand is hybridized to a portion of each adapter; ligating the respective transferred strands to a respective adapter of the plurality of adapters; digesting the non-transferred strand using a 5'-3' exonuclease;and attach a partial Y-adapter to each of the transferred strings.

13. The method as defined in claim 12, characterized in that the ligation and digestion occur in a single-vessel protocol.

14. A method comprising: preparing a mixture that includes a plurality of contiguously conserved library fragments from a genome sample, the plurality of contiguously conserved library fragments being attached to solid supports or attached to each other; diluting the mixture to generate a predetermined number of dilution samples to be introduced into a flow cell; and generating a time-based clustering image for at least one of the contiguously conserved library fragments by: introducing a first of the dilution samples including some of the contiguously conserved library fragments into the flow cell; initiating the release of some of the contiguously conserved library fragments from the solid support or from each other; amplifying some of the contiguously conserved library fragments to generate a plurality of template strands; and staining the plurality of template strands.and take images of the plurality of template strings. MA / a / ZUZI / u ι zu io; 15. The method as defined in claim 14, characterized in that the mixture is diluted to a predetermined volume based on i) a volume of the flow cell as a limiting dilution and ii) the predetermined number of dilution samples to be introduced into the flow cell.

16. The method as defined in claim 14 or 15, further comprising: generating a second time-based clustering image for some other contiguous conserved library fragments by: introducing a second of the dilution samples including the other contiguous conserved library fragments into the flow cell; initiating the release of some other contiguous conserved library fragments from the solid support or from each other; amplifying some other contiguous conserved library fragments to generate a second plurality of template strings; staining the second plurality of template strings; and imaging the second plurality of template strings.

17. The method as defined in claim 16, further comprising generating a predetermined number of time-based clustering images for a predetermined number of contiguous conserved library fragments by repeatedly introducing, initiating, amplifying, staining, and imaging each other from a predetermined number of dilution samples.

18. The method as defined in claim 17, further comprising assigning to each predetermined number of time-based clustering images a time record of the introduction of the respective dilution sample.

19. The method as defined in claim 18, characterized in that the time record is a timestamp or a number of steps in a sequence.

20. The method as defined in any one of claims 17 to 19, further comprising using image subtraction to generate a resolved cluster image for each of the dilution samples introduced into the flow cell, characterized in that each resolved cluster image records a spatial location and orientation of the plurality of template strings associated with a different dilution sample. MA / a / ZUZI / u ι zu io 21. The method as defined in any one of claims 14 to 20, characterized in that: each contiguous conserved library fragment is attached to the solid support; the solid support has a plurality of adapters attached thereto; and the method further comprises preparing the contiguous conserved library fragment attached to the solid support by: labeling the genome sample in the presence of the solid support and a plurality of L-adapters, each L-adapter comprising a transferred strand and a non-transferred strand, thereby generating a plurality of sample fragments, wherein a respective transferred strand is incorporated into a 5' end of each sample fragment and a respective non-transferred strand is hybridized to a portion of each adapter; ligating the respective transferred strands to a respective adapter of the plurality of adapters; digesting the non-transferred strand using a 5'-3' exonuclease;_and attach a partial Y-adapter to each of the transferred strings.; 22. The method as defined in claim 21, characterized in that the ligation and digestion occur in a single-vessel protocol.

23. A system comprising: a flow cell receptacle; a fluidic control system including fluid supply for respectively supplying a dilution sample and a dye to a flow cell positioned in the flow cell receptacle; an illumination system positioned to illuminate the flow cell positioned in the flow cell receptacle; a detection system positioned to capture an image of the flow cell positioned in the flow cell receptacle; and a controller in operational communication with the fluidic control system, illumination system, and detection system, the controller for: causing the supply fluids to introduce the dilution sample to the flow cell positioned in the flow cell receptacle;causing the delivery fluids to introduce the dye into the flow cell positioned in the flow cell receptacle after template strings are generated in the flow cell positioned in the flow cell receptacle from contiguously conserved library fragments present in the dilution sample; MA / a / ZUZl / u ι zu io causing the illumination system to illuminate the dyed template strings in the flow cell positioned in the flow cell receptacle; and causing the detection system to capture the image of the dyed template strings, illuminated in the flow cell positioned in the flow cell receptacle.

24. The system as defined in claim 23, characterized in that the flow cell receptacle is part of a sequencer that includes a heater, wherein the controller is for: causing the heater to initiate the release of some of the contiguous conserved library fragments from a solid support or from each other; causing the heater to execute a thermal cycle to amplify some of the contiguous conserved library fragments to generate template strands.

25. The system as defined in one of claims 23 or 24, further comprising: a first cartridge containing the dilution sample; and a second cartridge containing the dye.

26. The system as defined in any one of claims 23 to 25, further comprising an electronic storage component for storing the image.

27. The system as defined in claim 23, characterized in that the image is a time-based clustering image in a series of time-based clustering images for a plurality of contiguously conserved gene library fragments from a genome sample, wherein each time-based clustering image in the series is generated sequentially.