Time-based cluster imaging of amplification proximity retention library fragments of genomic DNA

Abstract

In one exemplary method, a series of time-based clustered images are generated for a plurality of library fragments from a genomic sample. Each time-based clustered image in the series is generated in order. To generate each time-based clustered image in the series: i) introducing a respective sample into the flow cell, the respective sample comprising a proximity-preserved library fragment of the plurality of library fragments, where the proximity-preserved library fragments are attached to a solid support or to each other; ii) releasing the proximity retention library fragments from the solid support or from each other; iii) amplifying the proximity preserved library fragment to generate a plurality of corresponding template strands; iv) staining the corresponding template chain; and v) imaging the corresponding template chain.

Description

[0001] This application is a divisional application of the PCT international application PCT / EP2020 / 084157 filed on December 1, 2020, which entered the Chinese national phase on September 28, 2021, with application number 202080025531.X and invention name "Time-based cluster imaging of amplified proximity-retained library fragments of genomic DNA".

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 942,563, filed December 2, 2019, the contents of which are incorporated herein by reference in their entirety. Background Art

[0004] There are multiple methods and applications of libraries of fragmented and labeled deoxyribonucleic acid (DNA) molecules that wish to be produced by double-stranded DNA (dsDNA) molecules. Generally, the purpose is to generate smaller DNA molecules (e.g., DNA fragments) by larger dsDNA molecules to be used as templates in DNA sequencing reactions. These templates can make it possible to obtain short read lengths. Short sequence reads are usually overlapped with multiple other short sequence reads to provide redundant coverage on different parts of an overall longer sequence. During data analysis, overlapping sequences from multiple reads can be used to piece together longer sequence information. Therefore, overlapping short sequence reads can be compared to rebuild longer nucleic acid sequences. In some cases, a pre-sequencing step (such as barcoding a specific nucleic acid molecule) can be used to simplify data analysis. Summary of the invention

[0005] The first aspect disclosed herein is a method comprising generating a series of time-based cluster generation images for a plurality of proximity-preserving library fragments from a genomic sample, wherein each time-based cluster generation image in the series is sequentially generated by: introducing a corresponding sample including some of the proximity-preserving library fragments into a flow cell, wherein the some of the proximity-preserving library fragments are attached to a solid support or to each other; triggering the release of the some of the proximity-preserving library fragments from the solid support or from each other; amplifying the some of the proximity-preserving library fragments to generate a plurality of corresponding template chains; staining the corresponding template chains; and imaging the corresponding template chains.

[0006] The second aspect disclosed herein is a method comprising preparing a mixture of multiple proximity-preserving library fragments comprising a genomic sample, the multiple proximity-preserving library fragments being attached to a solid support or to each other; diluting the mixture to generate a predetermined number of diluted samples to be introduced into a flow cell; and generating a time-based cluster generation image for at least one of the proximity-preserving library fragments by: introducing a first sample of the diluted samples comprising some of the proximity-preserving library fragments into the flow cell; triggering release of the some of the proximity-preserving library fragments from the solid support or from each other; amplifying the some of the proximity-preserving library fragments to generate multiple template chains; staining the multiple template chains; and imaging the multiple template chains.

[0007] The third aspect disclosed herein is a system comprising: a circulation cell receptacle; a fluid control system comprising a fluid delivery device for delivering a diluted sample and a stain, respectively, to a circulation cell positioned in the circulation cell receptacle; an illumination system positioned to illuminate the circulation cell positioned in the circulation cell receptacle; a detection system positioned to capture an image of the circulation cell positioned in the circulation cell receptacle; and a controller operably communicating with the fluid control system, the illumination system, and the detection system, the controller: causing the fluid delivery device to introduce the diluted sample into the circulation cell positioned in the circulation cell receptacle; causing the fluid delivery device to introduce the stain into the circulation cell positioned in the circulation cell receptacle after generating a template chain from proximity-retained library fragments present in the diluted sample in the circulation cell positioned in the circulation cell receptacle; causing the illumination system to illuminate the stained template chain in the circulation cell positioned in the circulation cell receptacle; and causing the detection system to image the illuminated stained template chain in the circulation cell positioned in the circulation cell receptacle.

[0008] It should be understood that any features of the first method and / or second method and / or system disclosed herein may be combined in any desired manner and / or configuration and / or with any of the embodiments disclosed herein to achieve the benefits as described in the present disclosure, including, for example, using parsed cluster images to identify specific groups of template chains. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] By referring to the following detailed description and accompanying drawings, the features of the examples of the present disclosure will become apparent, wherein like reference numerals correspond to similar but possibly not identical parts. For the sake of brevity, reference numerals or features having previously described functions may or may not be described in conjunction with other drawings in which they appear.

[0010] Figure 1is a schematic diagram of an example of an example method for preparing a complex comprising proximity-retaining library fragments attached to a solid support;

[0011] FIG. 2A to FIG. 2C are schematic diagrams that together illustrate another example of a method for preparing another example of a complex comprising proximity-retaining library fragments attached to a solid support;

[0012] Figure 3 It occurs in a single, single-pot reaction. FIG. 2A to FIG. 2C Schematic diagram of the ligation and digestion of the method;

[0013] Figure 4 is a schematic diagram of an example of a portion of a library preparation process for generating attached proximity-preserving library fragments;

[0014] Figure 5A is a top view of an example of a flow cell;

[0015] Figure 5B is an enlarged partial cross-sectional view of an example of a flow channel of a flow cell;

[0016] Figure 5C is an enlarged partial cross-sectional view of another example of a flow channel of a flow cell;

[0017] FIG. 6A to FIG. 6D is a schematic diagram of several steps in a method for generating a series of time-based cluster generation images;

[0018] Figure 7 are images taken after clustering was performed on different samples (in Figure 7 ) and is a schematic diagram of the resolved cluster images generated for each sample in the sample (on the left Figure 7 Schematic diagram of the right side of the figure;

[0019] Figure 8 It is used on the circulation pool Figure 4 Schematic diagram of an example of a portion of a library preparation process for attaching proximity-preserving library fragments;

[0020] 9A to 9C is a schematic diagram of several steps in a method for generating a series of time-based cluster generation images;

[0021] Fig.10 is an original color screenshot from the Integrative Genomics Viewer (IGV) browser reproduced in black and white, depicting the results for the AT-rich region of the INTS4P1 gene when heat denaturation is used to remove the non-transferred strand and when T7 exonuclease conditions are used to remove the non-transferred strand; and

[0022] Fig.11 is depicted through FIG. 2A to FIG. 2C and Figure 3 Graph of fluorescence (Y-axis, fluorescence units) versus size (X-axis, base pairs) of PCR-amplified library fragments generated by the method shown in . DETAILED DESCRIPTION

[0023] Library fragments are deoxyribonucleic acid (DNA) pieces of similar size (e.g., <1000 bp) to larger or longer DNA fragments. If it can be identified that the long DNA fragments originate from a common source compartment, the library fragments can be combined together in the sequencing data. Compartmentalization of DNA fragments of different lengths may be desired in order to achieve subhaploid genomic content within each compartment of synthetic long reads. When multiple short fragments can be combined together based on the identification that the long DNA fragments originate from a compartment, synthetic long reads (or linked short reads) are enabled. Compartmentalization has been physically implemented, for example, using wells, beads, droplets, or other physical compartments.

[0024] The principle common to all these compartmentalization methods is the use of a barcode sequence or index sequence to identify the compartment from which the long DNA fragment originates. The barcode sequence attached to each of the shorter fragments can be unique to a specific long DNA fragment and can therefore help to label different compartments during library preparation. It is the barcode sequence that is used to group short reads together to form a synthetic long read based on the assumption that the short reads all originate from the same compartment.

[0025] The exemplary methods disclosed herein achieve compartmentalization of different library fragments without the need to incorporate unique barcode sequences. The method utilizes proximity retention of library fragments and imaging to form a series of time-based clustering images. Each time-based clustering image can be used to identify the sample (compartment) that generated a specific set of template strands. In addition, each sequence read can be combined using the time-based clustering image. This combination allows reads to be linked, thereby achieving reconstruction of long DNA fragments.

[0026] definition

[0027] Unless otherwise indicated, the terms used herein should be understood to have their ordinary meanings in the relevant art. Several terms used herein and their meanings are listed below.

[0028] As used herein, the singular forms "a", "an", and "the" include both the singular and the plural, unless the context clearly dictates otherwise. As used herein, the term "comprising" is synonymous with "including", "containing", or "characterized by", and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0029] The references to "one example", "another example", "example", etc. throughout this specification mean that the specific elements (e.g., features, structures, compositions, configurations, and / or characteristics) described in conjunction with the example are included in at least one example described herein and may or may not be present in other examples. In addition, it should be understood that the elements used in any example may be combined in various examples in any suitable manner, unless the context clearly indicates otherwise.

[0030] The terms "substantially" and "approximately" used throughout this disclosure (including claims) are used to describe and illustrate small fluctuations, such as those caused by changes in processing. For example, these terms can refer to less than or equal to ±5% of a specified value, such as less than or equal to ±2% of a specified value, such as less than or equal to ±1% of a specified value, such as less than or equal to ±0.5% of a specified value, such as less than or equal to ±0.2% of a specified value, such as less than or equal to ±0.1% of a specified value, such as less than or equal to ±0.05% of a specified value.

[0031] Joint: A linear oligonucleotide sequence that can be fused to a nucleic acid molecule, for example, by connection or labeling. Suitable joint lengths can be in the range of about 10 nucleotides to about 100 nucleotides, or about 12 nucleotides to about 60 nucleotides, or about 15 nucleotides to about 50 nucleotides. Joints can include any combination of nucleotides and / or nucleic acids. In some examples, joints can include sequences complementary to at least a portion of a primer (e.g., a primer including a universal nucleotide sequence such as a P5 or P7 sequence). For example, a joint at one end of a fragment includes a sequence complementary to at least a portion of a first flow cell primer, and a joint at the other end of the fragment includes a sequence identical to at least a portion of a second flow cell primer. A complementary joint can hybridize with a first flow cell primer, and the same joint is a template for its complementary copy, which can hybridize with a second flow cell primer during cluster generation. In some examples, a joint can include a sequencing primer sequence or a sequencing binding site. A combination of different joints can be incorporated into a nucleic acid molecule such as a DNA fragment.

[0032] Capture site: A portion of the surface of a flow cell that has been physically modified and / or modified with chemical properties that allow for the localization of a complex. In one example, a capture site may include a chemical capture agent (i.e., a material, molecule, or moiety that is capable of attaching, retaining, or binding a target molecule (e.g., a complex)). An exemplary chemical capture agent includes a member of a receptor-ligand binding pair (e.g., avidin, streptavidin, biotin, lectin, carbohydrate, nucleic acid binding protein, epitope, antibody, etc.) that is capable of binding to a target molecule (or binding to a connecting portion attached to a target molecule). Another example of a chemical capture agent is a chemical reagent that is capable of forming an electrostatic interaction, hydrogen bond, or covalent bond (e.g., thiol-disulfide exchange, click chemistry, Diels-Alder, etc.) with a complex.

[0033] Complex: A support, such as a solid support, and a ready-to-sequence nucleic acid fragment attached to the support. The support may also include one member of a binding pair, the other member of which is part of a capture site.

[0034] Fragment: A portion or piece of genetic material (eg, DNA, RNA, etc.) Proximity-preserving library fragments are smaller pieces of a longer nucleic acid sample that have been fragmented, where the smaller fragments are held together in some way (eg, by beads, with transposomes, etc.).

[0035] Nucleic acid molecule or sample: A polymeric form of nucleotides of any length and may include ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term may refer to a single-stranded or double-stranded polynucleotide.

[0036] A "template" nucleic acid molecule (or strand) may refer to the sequence to be analyzed.

[0037] The nucleotides in the nucleic acid sample may include naturally occurring nucleic acids and functional analogs thereof. Examples of functional analogs can hybridize with nucleic acids in a sequence-specific manner or can be used as templates for replicating specific nucleotide sequences. Naturally occurring nucleotides typically have a backbone containing a phosphodiester bond. Similar structures may have alternative backbone bonds, including any of a variety of backbone bonds known in the art. Naturally occurring nucleotides typically have deoxyribose (e.g., present in DNA) or ribose (e.g., present in RNA). Similar structures may have alternative sugar moieties, including any of a variety of sugar moieties known in the art. Nucleotides may include natural or non-natural bases. Natural DNA may include one or more of adenine, thymine, cytosine and / or guanine, and natural RNA may include one or more of adenine, uracil, cytosine and / or guanine. Any non-natural bases may be used, such as locked nucleic acids (LNA) and bridge nucleic acids (BNA).

[0038] Primer: a nucleic acid molecule that can hybridize with a target sequence, such as a joint attached to a proximity retention library fragment. For example, an amplification primer can be used as a starting point for template amplification and cluster generation. For another example, a synthetic nucleic acid (template) chain may include a site to which a primer (e.g., a sequencing primer) can hybridize, so as to initiate the synthesis of a new chain complementary to the synthetic nucleic acid (template) chain. Any primer may include any combination of nucleotides or their analogs. In some examples, a primer is a single-stranded oligonucleotide or polynucleotide. The primer length can be any number of base lengths and can include a variety of natural and / or non-natural nucleotides. In one example, a sequencing primer is a short chain ranging from 10 to 60 bases, or 20 to 40 bases.

[0039] Ready-to-sequencing nucleic acid fragments: genetic material has a portion of adapters at the 3' and 5' ends (e.g., proximity-preserving library fragments). In the ready-to-sequencing nucleic acid fragments, each adapter includes a known universal sequence (e.g., which is complementary to at least a portion of a primer on a flow cell) and a sequencing primer sequence. Ready-to-sequencing nucleic acid fragments can be bound via insertion into a transposon bound to the surface of a solid support (e.g., a bead), or directly immobilized via a binding pair or other cleavable linker.

[0040] Solid support: A small body made of a rigid or semi-rigid material, whose shape can be characterized, for example, by spherical, ellipsoidal, microspherical, or other recognized particle shapes, whether of regular or irregular size. The solid support can have a sequencing library attached to it. Exemplary materials that can be used for solid supports include, but are not limited to, glass; plastics such as acrylic, polystyrene or a copolymer of styrene and another material, polypropylene, polyethylene, polybutylene, polyurethane, or polytetrafluoroethylene (available from The Chemours Co. ); polysaccharides or cross-linked polysaccharides, such as agarose or agarose gel; nylon; nitrocellulose; resins; silica or silica-based materials, including silicon and modified silicon; carbon fibers, metals; inorganic glass; fiber optic bundles, or a variety of other polymers. Exemplary solid supports include controlled pore glass beads, paramagnetic beads, thoria sols, Beads (agarose in the form of cross-linked beads, commercially available from Cytivia), nanocrystals and other solid supports known in the art, as described, for example, in the Microsphere Detection Guide from Bangs Laboratories, Fishers Ind.

[0041] Transposome: A complex formed between an integrase (eg, integrase or transposase) and a transferable strand, a non-transferable strand, or both a transferable strand and a non-transferable strand.

[0042] Proximity-preserving library fragments

[0043] In the examples disclosed herein, the library fragments introduced into the flow cell are proximity-preserving library fragments. Proximity-preserving library fragments are smaller pieces of a longer nucleic acid sample that have been fragmented, where the smaller pieces are physically held together in some manner. In some examples disclosed herein, solid supports can be used in library preparation to preserve proximity. In other examples disclosed herein, proximity can be preserved by forming fragments attached to each other by bound transposomes during initial library preparation, introducing the attached fragments into the flow cell, and then completing library preparation on the flow cell.

[0044] Figure 1 An example of a method for forming a complex 10 comprising a sequencing-ready nucleic acid fragment 12 comprising a fragment 14 from a larger nucleic acid sample whose proximity is preserved on a solid support 16 is depicted.

[0045] In order to form Figure 1 In an exemplary method of the complex 10 shown, the adapter sequence 18 is bound to the solid support 16 via one member of the binding pair 20. In one example, the adapter sequence 18 may include a first sequencing primer sequence (e.g., a read 1 sequencing primer sequence) and a first sequence (P5′) that is coupled to a flow cell (e.g., Figure 5A and Figure 5B The adapter sequence 18 is complementary to at least a portion of one of the amplification primers (e.g., P5) on the amplification primers shown in Figure 1. The adapter sequence 18 is bound to one member 20 of a binding pair (e.g., biotin) so that it can be bound to the surface of a solid support 16, which includes the other member of the binding pair (e.g., avidin, streptavidin, etc.).

[0046] like Figure 1 As shown, the transposome complex 24" may also be bound to the solid support 16. Before loading the transposome complex 24" on the solid support 16, a partial Y-linker 25' may be mixed with a transposase 32 (although not shown, it may include two Tn5 molecules) to form an example of a transposome complex 24". The partial Y-linker 25' may include two mosaic end sequences M1 and M2 that hybridize to each other. One of the mosaic end sequences M1 has a free end that can be attached to the fragmented DNA strand during labeling, and thus is similar to FIG. 2A to FIG. 2CThe transferred strand 26 in the mosaic end sequence. Another mosaic end sequence M2 in the mosaic end sequence can be attached to a second sequencing primer sequence (e.g., read 2 sequencing primer sequence) and a second sequence (P7) having a sequence identical to at least a portion of another amplification primer (P7) in the amplification primers on the flow cell, such that a copy thereof (e.g., P7′) is complementary to the amplification primer (P7). In this example, another mosaic end sequence M2 in the mosaic end sequence, the second sequencing primer sequence, and the second sequence constitute an adapter sequence 22. The adapter sequence 22 is not attached to the fragmented DNA strands during labeling, and thus is similar to FIG. 2A to FIG. 2C The non-transfer chain 28 in.

[0047] 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 suitable conditions for ligating the mosaic end M1 of the partial Y-linker 25' to the 3' end of the linker sequence 18. Figure 1 As shown, individual transposome complexes 24" can be attached to each of the linker sequences 18 on the solid support 16.

[0048] In this exemplary method for forming the complex 10, a labeling process may then be performed. A fluid (e.g., labeling buffer) including a longer nucleic acid sample 30 (e.g., DNA) may be added to a solid support 16 having an adapter sequence 18 and a transposome complex 24" bound thereto. When the sample contacts the transposome complex 24", the longer nucleic acid sample is labeled. During this labeling example, the sample 30 is fragmented into fragments 14, 14', and each of the fragments 14, 14' is tagged at its 5' end with a free end of the mosaic end M1 of a partial Y-adapter 25'.

[0049] like Figure 1 As shown, labeling of the longer nucleic acid sample 30 results in multiple bridging molecules between the transposome complexes 24". The bridging molecules are wrapped around the solid support 16. The transposome complexes 24" and the adapter sequences 18 act as bridging molecules to maintain the proximity of the nucleic acid sample 30, and thus the bridging molecules are proximity-preserving library fragments 14, 14'.

[0050] The transposase may then be removed via sodium dodecyl sulfate (SDS) treatment or heat or proteinase K digestion. Removal of the transposase leaves the proximity-retaining library fragments 14 , 14 ′ attached to the solid support 16 .

[0051] To complete the ready-to-sequence fragments, further extension and ligation (in Figure 1 (indicated by an asterisk in the figure) to ensure that fragments 14 and 14' are attached to sequence 22. The resulting complex 10 is Figure 1 Shown in.

[0052] Each proximity retention library fragment 14, 14' is a portion of a corresponding ready-to-sequence nucleic acid fragment 12, 12', each of which also includes a corresponding adapter sequence 18 and 22 attached at either end. The adapter sequence 18 is those initially bound to the solid support 16, and includes a first sequencing primer sequence and a first sequence complementary to one of the flow cell primers. The adapter sequence 18 is attached to one member 20 of the binding pair. The adapter sequence 22 is from a partial Y-adapter 25' and includes a second sequence identical to another flow cell primer and a second sequencing primer sequence. Because each ready-to-sequence nucleic acid fragment 14, 14' includes a suitable adapter for bridge amplification and sequencing, PCR amplification is not performed. Therefore, these fragments 12, 12' are ready-to-sequence. In addition, because the proximity retention library fragments 14, 14' come from the same longer nucleic acid sample 30, the proximity retention library fragments 14, 14' can be applied to the linked long read applications disclosed herein.

[0053] For forming another exemplary composite 10′ ( Figure 2C Another exemplary method of FIG. 2A to FIG. 2C middle.

[0054] In this exemplary method, the adapter sequence 18' is bound to the solid support 16 through one member 20 of the binding pair. In one example, the adapter sequence 18' may include a hybridizable sequence H, a first sequencing primer sequence (e.g., a read 1 sequencing primer sequence), and a first sequence (e.g., P5) that is identical to at least a portion of one of the amplification primers (e.g., P5) on the flow cell, such that its copy (e.g., P5') is complementary to the amplification primer (P5). The adapter sequence 18' is bound to one member 20 of the binding pair (e.g., biotin) so that it can be bound to the surface of the solid support 16, which includes another member of the binding pair (e.g., avidin, streptavidin, etc.).

[0055] like Figure 2AAs shown, the transposome complex 24 may also be bound to the solid support 16. Before the transposome complex 24 is loaded on the solid support 16, the L-linker 21 may be mixed with a transposase 32 (e.g., including two Tn5 molecules) to form an example of the transposome complex 24. The L-linker 21 may include two mosaic end sequences M1 and M2 that hybridize to each other. One mosaic end sequence M1 in the mosaic end sequence is a transfer strand 26 that is added to one end of each fragment 14, 14' during the ligation process that occurs after the labeling process. The other mosaic end sequence M2 in the mosaic end sequence is a portion of the non-transferred strand 28 that is removed after the ligation and labeling process. The mosaic end sequence M2 is attached to a complementary hybridizable sequence HC that is complementary to a hybridizable sequence H of an adapter sequence 18' attached to the solid support 16. The complementary hybridizable sequence HC enables the L-linker 21 to hybridize with the adapter sequence 18'. Therefore, the complementary hybridizable sequence HC enables the transposome complex 24 to be loaded onto the solid support.

[0056] 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-linker 21 with the hybridizable sequence H of the linker sequence 18′. Figure 2A As shown, individual transposome complexes 24 may be attached to each of the adapter sequences 18 ′ on the solid support 16 .

[0057] In this exemplary method for forming the complex 10', a labeling process is then performed. A fluid (e.g., labeling buffer) including a longer nucleic acid sample (e.g., DNA) 30 may be added to a solid support 16 having a transposome complex 24 loaded thereon. When the sample 30 contacts the solid support-bound transposome complex 24, the longer nucleic acid sample 30 is labeled. During this labeling example, the sample 30 is fragmented into fragments 14, 14', and each of the fragments 14, 14' is tagged at its 5' end with a mosaic end sequence M1 of the L-adapter 21.

[0058] like Figure 2A As shown, labeling of the longer nucleic acid sample 30 results in multiple bridging molecules between adjacent transposome complexes 24 and thus adjacent adapter sequences 18'. The bridging molecules are wrapped around the solid support 16. The transposome complexes 24 and adapter sequences 18' act as bridging molecules to maintain the proximity of the nucleic acid sample 30, and thus the bridging molecules are proximity-preserving library fragments 14, 14'.

[0059] The transposase 32 can then be removed via sodium dodecyl sulfate (SDS) treatment or heat or proteinase K digestion.

[0060] A ligation can then be performed to join the free mosaic end sequence M1 to the corresponding adapter sequence 18'. Figure 2A In the example, the asterisk indicates where the ligation is performed. In one example, the ligation can be initiated by introducing a buffer containing a suitable ligase and heating it to about a suitable temperature for a suitable time to initiate enzyme activity. Examples of suitable ligases include E. coli DNA ligase, T7 ligase, etc. In one example, the buffer containing E. coli DNA ligase can also include nicotinamide adenine dinucleotide (NAD + ). In this example, heating to about 16°C for about 15 minutes triggers enzyme activity. The resulting structure is Figure 2B Shown in.

[0061] The non-transferred strands 28 of the L-adapter 21 can then be removed. In this example, each of the non-transferred strands 28 is removed using any suitable 5'-3' exonuclease 23 (such as T7 exonuclease). In one example, the removal of the non-transferred strands can be initiated by introducing a buffer containing the 5'-3' exonuclease 23 and waiting for a predetermined time. The 5'-3' exonuclease 23 is capable of digesting the non-transferred strands 28 at room temperature (e.g., about 22° C. to about 25° C.), so no additional heating is used. The digested non-transferred strands 28 can then be washed away.

[0062] Using an exonuclease to remove the non-transferred strand 28 may be preferable to using heat denaturation. 5′-3′ exonuclease 23 efficiently digests the non-transferred strand 28, thereby producing a more purified template (compared to when heat denaturation is used) for subsequent attachment of the adapter sequence (see Figure 2C 22 in the figure). This can increase the library yield. In addition, the removal of the non-transferred strand 28 by the 5'-3' exonuclease 23 can increase the library coverage on the adenine (A) and thymine (T) rich regions, the loss of which has been observed after heat denaturation of the non-transferred strand 28 (see Fig.11 ). In addition, digestion by exonuclease does not increase the time of the overall library preparation process.

[0063] See now Figure 2C , this example of the method for forming complex 10' involves introducing a partial Y-linker 25. The partial Y-linker 25 includes a mosaic end sequence M3 complementary to the mosaic end sequence M1 and a linker sequence 22. The linker sequence 22 may include a second sequencing primer sequence (e.g., a read 2 sequencing primer sequence) and a second sequence (P7') that is complementary to another amplification primer (P7) in the amplification primers on the flow cell. Figure 2CAs shown, the mosaic end sequence M3 of the partial Y-linker 25 hybridizes to the mosaic end sequence M1 of the transferred strand 26 (now attached to the linker sequence 18 ′), thereby attaching the partial Y-linker 25 to the solid support 16 .

[0064] In the examples disclosed herein, the adapter sequences 18, 18' and / or 22 may also include a sequencing sample index or barcode sequence. These sequences may be used as a backup or alternative to the compartmentalization methods disclosed herein.

[0065] To generate sequencing ready fragments, further extension and ligation are performed to ensure that fragments 14 and 14 ′ are attached to mosaic end sequence M3 and thus to sequence 22 .

[0066] The resulting complex 10′ Figure 2C Shown in.

[0067] For forming the composite 10′ (in Figure 2C Another exemplary method of the invention is partially depicted in Figure 3 In this exemplary method, ligation of transferred strands 26 and digestion of non-transferred strands 28 are performed as part of a single, one-pot protocol.

[0068] In this exemplary method, the linker sequence 18' is bound to the solid support 16 via one member 20 of the binding pair. Figure 2A The adapter sequence 18′ includes a hybridizable sequence H, a first sequencing primer sequence (e.g., a read 1 sequencing primer sequence), and a first sequence (P5), which is identical to at least a portion of one of the amplification primers (e.g., P5) on the flow cell, such that its copy (e.g., P5′) is complementary to the amplification primer (P5). Also in this exemplary method, the transposome complex 24 is loaded on the solid support 16, as described in reference Figure 2A Briefly, the complementary hybridizable sequence HC of the L-linker 21 of the transposome complex 24 hybridizes with the hybridizable sequence H of the linker sequence 18'.

[0069] In this exemplary method for forming composite 10' ( Figure 2C ), as referenced Figure 2A Perform labeling as described.

[0070] The ligation of the transferred strand 26 and the digestion of the non-transferred strand 28 can then be performed together. Figure 3 In order to make the ligase and the exonuclease work synergistically, the reagent formula introduced into the labeled solid support includes a buffer, a ligase, a 5′-3′ exonuclease 23 (e.g., T7 exonuclease), and any other components required by the corresponding enzyme (e.g., cofactors, such as NAD +In one example, ligation and digestion can be initiated by introducing the reagent formulation and heating to about 25°C for about 15 minutes to initiate enzyme activity.

[0071] Incorporating the ligase and exonuclease into the same reagent formulation can shorten protocol time (e.g., FIG. 2A to FIG. 2C The exemplary method shown is shortened by about 10 minutes) and also reduces the number of washing steps.

[0072] The method example then continues with the introduction of a partial Y-joint 25, as shown in reference Figure 2C To generate ready-to-sequence fragments and final complex 10 ′, further extension and ligation are performed to ensure that fragments 14 and 14 ′ are attached to mosaic end sequence M3 and thus to sequence 22 .

[0073] refer to Figure 1 , Figure 2A-2C and Figure 3 The methods described for preparing the complexes 10 , 10 ′ provide some examples, but it should be understood that other methods may be used as long as the ready-to-sequence nucleic acid fragments 12 , 12 ′ are attached to the solid support 16 .

[0074] In other examples disclosed herein, proximity information can be preserved by performing a portion of the library preparation from a flow cell and a portion of the library preparation on a flow cell.

[0075] In this example, a tag can be used to prime the library preparation outside the flow cell, such as Figure 4 It is schematically shown in FIG.

[0076] In the example shown, a fluid (e.g., labeling buffer) including a longer nucleic acid sample 30 (e.g., double-stranded DNA) can be mixed with the transposome complex 24'. Figure 4 In the example shown, each transposome complex 24' is a dimer, including a transferred strand 26', a non-transferred strand 28' and two transposases (which are in Figure 4 In other examples, different transposome complexes may be used, for example, one of the transposome complexes includes the transposase 32 and the transferred strand 26', and another of the transposome complexes includes the transposase 32 and the non-transferred strand 28'.

[0077] In this example, the transferred strand 26' is an adapter added to one end of each fragment 14, 14' during the labeling process. In one example, each transferred strand 26 is an adapter that includes a first sequencing primer sequence (e.g., the read 1 sequencing primer sequence) and a first sequence (P5') that is complementary to at least a portion of one of the amplification primers (e.g., P5) on the flow cell.

[0078] In this example, the non-transferred strand 28' is an adapter that is not incorporated into the fragments 14, 14' during labeling, but can be subsequently attached to the other end of each fragment 14, 14'. Figure 4 As shown, the non-transferred strand 28' can be attached to the transferred strand 26' via at least partial base pairing during labeling. In this example, the non-transferred strand 28' is a linker including a second sequencing primer sequence (e.g., the read 2 sequencing primer sequence) and a second sequence (P7) that is identical to at least a portion of one of the amplification primers (e.g., P7) on the flow cell, such that its copy (e.g., P7') is complementary to the amplification primer (P7).

[0079] like Figure 4 As shown, within the fluid, the transpososome 24′ fragments the longer nucleic acid sample 30 into fragments 14, 14′ and connects the transferred strand 26′ to the 5′ end of each fragment 14, 14′. In one example, the transferred strand 26′ is incorporated into the 5′-end of each fragment 14, 14′ of the longer nucleic acid sample 30 by single-sided transposition. The non-transferred strand 28′ can be attached to the transferred strand 26′ via base pairing.

[0080] This exemplary labeling method maintains the proximity of the longer nucleic acid sample 30 because the generated fragments 14, 14' (and any transferred strands 26' and non-transferred strands 28' attached directly or indirectly thereto) remain attached to each other by the transposase 32. The attached proximity-preserving library fragments 14, 14' are referred to herein as attached fragments 34.

[0081] As mentioned herein, the attached fragments 34 can be introduced into a flow cell where additional processing can be performed to complete library preparation. Figure 8 Schematically shown in FIG. 1 and will be further described with reference to the methods disclosed herein.

[0082] Flow cell

[0083] The methods disclosed herein may utilize a flow cell 36, an example of which is depicted in FIG. Figure 5A The flow cell 36 includes a substrate 38 that at least partially defines a channel or flow channel 40.

[0084] Substrate 38 may be a single layer / single material. Examples of suitable single layer substrates include epoxysilicone, glass, modified or functionalized glass, plastics (including acrylic, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene (such as available from Chemours), polytetrafluoroethylene (such as polytetrafluoroethylene ... ), cycloolefins / cycloolefin polymers (COP) (such as those available from Zeon ), polyimide, etc.), nylon (polyamide), ceramic / ceramic oxides, silicon dioxide, fused silicon dioxide or silicon dioxide-based materials, aluminum silicate, silicon and modified silicon (e.g., boron-doped p+ silicon), silicon nitride (Si3N4), silicon oxide (SiO2), tantalum pentoxide (Ta2O5) or other tantalum oxides (TaO x ), hafnium oxide (HaO2), carbon, metal, inorganic glass, etc. Substrate 38 may also be a multilayer structure. Some examples of multilayer structures include glass or silicon with a coating of tantalum oxide or another ceramic oxide at the surface. Other examples of multilayer structures include a base support (e.g., glass or silicon) with a patterned resin thereon. Other examples of multilayer substrates may include silicon on insulator (SOI) substrates.

[0085] In one example, substrate 38 may have a diameter in the range of about 2 mm to about 300 mm, or may be a rectangular sheet or panel with a maximum dimension of up to about 10 feet (about 3 meters). In one example, substrate 38 is a wafer with a diameter in the range of about 200 mm to about 300 mm. In another example, substrate 38 is a die with a width in the range of about 0.1 mm to about 10 mm. Although exemplary dimensions have been provided, it should be understood that substrate 38 with any suitable dimensions may be used. For another example, a panel may be used as a rectangular support having a larger surface area than a 300 mm round wafer.

[0086] exist Figure 5A In the example shown, the flow cell 36 includes flow channels 40. Although several flow channels 40 are shown, it should be understood that any number of channels 40 (e.g., a single channel 40, four channels 40, etc.) may be included in the flow cell 36. Each flow channel 40 is an area defined between two bonding components (e.g., between a substrate 36 and a cover or between two substrates 36) that may have fluids (e.g., those described herein) introduced therein and removed therefrom. Each flow channel 40 may be separated from each other flow channel 40 so that a fluid introduced into any particular flow channel 40 does not flow into any adjacent flow channel 40. Some examples of fluids introduced into the flow channels 40 may introduce reaction components (e.g., proximity retention library fragments 14, 14' (e.g., located on a solid support 16 or attached to each other), polymerases, sequencing primers, nucleotides, etc.), washing solutions, deblocking agents, etc.

[0087] The flow channel 40 may be defined in the substrate 38 using any suitable technique depending in part on the material 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 may be patterned into the resin of the multilayer substrate 38 using photolithography, nanoimprint lithography, etc. In yet another example, a separate material (not shown) may 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.

[0088] In one example, the flow channel 40 has a straight configuration. The length and width of the flow channel 40 may be less than the length and width of the substrate 38, respectively, so that the portion of the substrate surface surrounding the flow channel 40 can be used to attach to a cover (not shown) or another substrate 38. In some cases, the width of each flow channel 40 can be at least about 1mm, at least about 2.5mm, at least about 5mm, at least about 7mm, at least about 10mm or more. In some cases, the length of each channel / flow channel 40 can be at least about 10mm, at least about 25mm, at least about 50mm, at least about 100mm or more. The width and / or length of each flow channel 40 can be greater than, less than, or between the values ​​specified above. In another example, the flow channel 40 is a square (e.g., 10mm×10mm).

[0089] When microcontact, aerosol or inkjet printing is used to deposit a separate material that defines the flow channel wall, the depth of each flow channel 40 can be as small as a single layer thick. When a separate material (not shown) is used to bond the cover to the substrate 38 or to bond two substrates 38 together, the depth can be greater. For other examples, the depth of each flow channel 40 can be about 1 μm, about 10 μm, about 50 μm, about 100 μm or more. In one example, the depth may be in the range of about 10 μm to about 100 μm. In another example, the depth may be in the range of about 10 μm to about 30 μm. In yet another example, the depth is about 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.

[0090] Different examples of the architecture within the flow channel 40 of the flow cell 36 are described in Figure 5B and Figure 5C Shown in.

[0091] exist Figure 5B In the example shown, the flow cell 36 includes a single-layer substrate 38A and a flow channel 40 at least partially defined in the single-layer substrate 38A.

[0092] A polymer hydrogel 42 is present in the flow channel 40. Examples of the polymer hydrogel 42 include acrylamide copolymers, such as poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide)-PAZAM. Some other forms of PAZAM and acrylamide copolymers are represented by the following structure (I):

[0093]

[0094] in:

[0095] R A is selected from the group consisting of azido, optionally substituted amino, optionally substituted alkenyl, optionally substituted alkyne, halogen, optionally substituted hydrazone, optionally substituted hydrazine, carboxyl, hydroxyl, optionally substituted tetrazole, optionally substituted tetrazine, nitrile oxide, nitrone, sulfate, and thiol;

[0096] R B is H or optionally substituted alkyl;

[0097] R C , R D and R E are each independently selected from H and optionally substituted alkyl;

[0098] -(CH2) p - each of which may be optionally substituted;

[0099] p is an integer ranging from 1 to 50;

[0100] n is an integer in the range of 1 to 50,000; and

[0101] m is an integer in the range of 1 to 100,000.

[0102] One of ordinary skill in the art will recognize that the arrangement of recurring "n" and "m" features in structure (I) is representative and that the monomer subunits may be present in the polymer structure in any order (e.g., random, block, patterned, or a combination thereof).

[0103] Other forms of acrylamide copolymers and PAZAM may have a molecular weight in the range of about 5 kDa to about 1500 kDa, or about 10 kDa to about 1000 kDa, or in one specific example may be about 312 kDa.

[0104] In some examples, other forms of acrylamide copolymers and PAZAM are linear polymers. In some other examples, other forms of acrylamide copolymers and PAZAM are lightly cross-linked polymers.

[0105] In other examples, the polymer hydrogel 42 can be a variation of structure (I). In one example, the acrylamide unit can be N,N-dimethylacrylamide. In this example, the acrylamide unit in structure (I) can be replaced by Replace, where R D , RE and RF are each H or C1-C6 alkyl, and R G and RH are each C1-C6 alkyl (rather than H, as in the case of acrylamide). In this example, q may be an integer in the range of 1 to 100,000. In another example, in addition to the acrylamide units, N,N-dimethylacrylamide may be used. In this example, in addition to the recurring "n" and "m" features, structure (I) may also include wherein RD, RE and RF are each H or C1-C6 alkyl, and R G and R H Each is a C1-C6 alkyl group. In this example, q can be an integer in the range of 1 to 100,000.

[0106] As another example of an initial polymer hydrogel, the recurring "n" feature in structure (I) can be replaced with a monomer including a heterocyclic azido group having structure (II):

[0107]

[0108] Where R 1 R2 is H or C1-C6 alkyl; L is a linking group including a straight chain having 2 to 20 atoms selected from carbon, oxygen and nitrogen and 10 optional substituents on the carbon and any nitrogen atoms in the chain; E is a straight 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 atoms in the chain; A is an N-substituted amide having H or C1-C4 alkyl attached 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.

[0109] As another example, polymer hydrogel 42 may include recurring units of each of structures (III) and (IV):

[0110]

[0111] Where R 1a , R 2a , R 1b and R 2b Each of R is independently selected from hydrogen, optionally substituted alkyl or optionally substituted phenyl; 3a and R 3bEach of L is independently selected from hydrogen, optionally substituted alkyl, optionally substituted phenyl or optionally substituted C7-C14 aralkyl; and each L 1 and L 2 are independently selected from an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker.

[0112] It should be understood that other molecules can be used to form the polymer hydrogel 42, as long as they are functionalized to graft the oligonucleotide primers 44, 46 thereto. Other examples of suitable polymer layers include those with colloidal structures, such as agarose; or those with polymer mesh structures, such as gelatin; or those with cross-linked polymer structures, such as polyacrylamide polymers and copolymers, silane-free acrylamide (SFA) or azidated versions of SFA. Examples of suitable polyacrylamide polymers can be synthesized from acrylamide and acrylic acid or acrylic acid containing vinyl groups, or from monomers that form a [2+2] photocycloaddition reaction. Other examples of suitable polymer hydrogels 42 include mixed copolymers of acrylamide and acrylates. A variety of polymer architectures containing acrylic monomers (e.g., acrylamide, acrylates, etc.) can be used in the examples disclosed herein, such as branched polymers, including star polymers, star or star block polymers, dendrimers, etc. For example, monomers (e.g., acrylamide, acrylamide containing a catalyst, etc.) can be incorporated into the branches (arms) of star polymers in a random or block manner.

[0113] In order to introduce the polymer hydrogel 42 into the flow channel 40, a mixture of the polymer hydrogel 42 can be generated and then applied to the substrate 38A (having the flow channel 40 at least partially defined therein). In one example, the polymer hydrogel 42 may be present in a mixture (e.g., a mixture with water or a mixture with ethanol and water). The mixture can then be applied to the substrate surface (including in the flow channel 40) using spin coating, dipping or dip coating, spraying, a material flow under positive or negative pressure, or another suitable technique. These types of techniques blanket deposit the polymer hydrogel 42 on the substrate 38A (e.g., deposited in the flow channel 40 and deposited on the gap region 48 surrounding the flow channel 40). Other selective deposition techniques (e.g., involving masks, controlled printing techniques, etc.) can be used to specifically deposit the polymer hydrogel 42 in the flow channel 40 rather than on the gap region 48.

[0114] In some examples, the substrate surface (including the portion exposed in flow channel 40) can be activated and then the mixture (including polymer hydrogel 42) can be applied thereto. In one example, silane or silane derivatives (e.g., norbornene silane) can be deposited on the substrate surface using vapor deposition, spin coating, or other deposition methods. In another example, the substrate surface can be exposed to plasma ashing to produce a surfactant (e.g., -OH groups) that can attach to polymer hydrogel 42.

[0115] The applied mixture may be exposed to a curing process depending on the polymer hydrogel 42. In one example, curing may be performed at a temperature ranging from room temperature (eg, about 25°C) to about 95°C for a time ranging from about 1 millisecond to about several days.

[0116] In some embodiments, polishing may then be performed to remove the polymer hydrogel 42 from the void regions 48 at the perimeter of the flow channel 40 while leaving the polymer hydrogel 42 on the surface at least substantially intact in the flow channel 40 .

[0117] The flow cell 36 also includes amplification primers 44 , 46 .

[0118] A grafting process can be performed to graft amplification primers 44, 46 to the polymer hydrogel 42 in the flow channel 40. In one example, the amplification primers 44, 46 can be fixed to the polymer hydrogel 42 by a single point covalent attachment at or near the 5' end of the primers 44, 46. This attachment allows i) the adapter-specific portion of the primers 44, 46 to anneal freely to their cognate nucleic acid fragments (e.g., attached to the P5' portion of the fragments 14, 14'), and ii) the 3' hydroxyl group to be free for primer extension. Any suitable covalent attachment can be used for this purpose. Examples of capped primers that can be used include alkyne-terminated primers that can be attached to the azide portion of the polymer hydrogel 42. Specific examples of suitable primers 44, 46 include the use of P5 and P7 primers on the surface of commercial flow cells sold by Illumina Inc. to TM HISEQX TM MISEQ TM MISEQDX TM MINISEQ TM 、NEXTSEQ TM 、NEXTSEQDX TM 、NOVASEQ TM 、GENOME ANALYZER TM 、ISEQ TM and other instrument platforms.

[0119] In one example, grafting may involve flow-through deposition (e.g., using a temporarily bonded or permanently bonded cap), bubble coating, spray coating, whipped dispensing, or by another suitable method of attaching primers 44, 46 to the polymer hydrogel 42 in the flow channel 42. Each of these exemplary techniques may utilize a primer solution or mixture that may include primers 44, 46; water; a buffer; 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. Thus, the primers 44, 46 are selectively grafted to the polymer hydrogel 42 in the flow channel 40.

[0120] exist Figure 5C In the example shown, flow cell 38 includes a multilayer substrate 38B including a support 52 and a patterned material 50 positioned on support 52. Patterned material 50 defines recessed portions 54 separated by interstitial regions 48. Recessed portions 54 are positioned within each of flow channels 40 in the flow channels.

[0121] exist Figure 5C In the example shown, patterned material 50 is positioned on support 52. It should be understood that any material that can be selectively deposited or can be deposited and patterned to form recessed portions 54 and interstitial regions 48 can be used as patterned material 50.

[0122] For example, the inorganic oxide may be selectively applied to the support 52 via vapor deposition, aerosol printing, or inkjet printing. Examples of suitable inorganic oxides include tantalum oxide (e.g., Ta2O5), aluminum oxide (e.g., Al2O3), silicon oxide (e.g., SiO2), hafnium oxide (e.g., HfO2), and the like.

[0123] As another example, the resin can be applied to the support 52 and then patterned. Suitable deposition techniques include chemical vapor deposition, dip coating, bubble coating, spin coating, spray coating, whipping dispensing, ultrasonic spray coating, doctor blade coating, aerosol printing, screen printing, microcontact printing, etc. Suitable patterning techniques include photolithography, nanoimprint lithography (NIL), imprinting technology, embossing technology, molding technology, microetching technology, printing technology, etc. Some examples of suitable resins include resins based on polyhedral oligomeric silsesquioxane resins (POSS), non-POSS epoxy resins, poly (ethylene glycol) resins, polyether resins (e.g., ring-opened epoxides), acrylic resins, acrylate resins, methacrylate resins, amorphous fluoropolymer resins (e.g., available from Bellex) ) and their combinations.

[0124] As used herein, the term "polyhedral oligomeric silsesquioxane" (POSS) refers to a hybrid intermediate between silicon dioxide (SiO2) and organosilicon (R2SiO2) (e.g., RSiO2). 1.5 ). An example of POSS may be the POSS described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated by reference in its entirety. In one example, the composition is a chemical composition having the formula [RSiO 3 / 2 ] n The organosilicon compound of POSS, wherein the R groups can be the same or different. Exemplary R groups of POSS include epoxy, azide / azido, thiol, poly(ethylene glycol), norbornene, tetrazine, acrylate and / or methacrylate, or additionally, for example, alkyl, aryl, alkoxy and / or haloalkyl groups. The resin composition disclosed herein may include one or more different cage or core structures as monomer units. The polyhedral structure may be a T8 structure, such as: And it is expressed by the following formula: The monomer unit typically has eight functional arms R1 to R8.

[0125] The monomer unit may have a cage structure with 10 silicon atoms and 10 R groups, referred to as T 10 , such as: Alternatively, the monomer unit may have a cage structure with 12 silicon atoms and 12 R groups, referred to as T 12 , such as: POSS-based materials may alternatively include T6, T 14 or T 16 Cage Structure. The average cage content can be adjusted during synthesis and / or controlled by purification methods, and the cage size distribution of the monomer units can be used in the examples disclosed herein.

[0126] like Figure 5CAs shown, the patterned material 50 includes a recessed portion 54 defined therein and a gap region 48 separating adjacent recessed portions 54. Many different layouts of the recessed portion 54 can be envisioned, including regular, repeated and irregular patterns. In one example, the recessed portion 54 is arranged in a hexagonal grid so as to be closely packed and improve density. Other layouts may include, for example, a straight (rectangular) layout, a triangular layout, etc. In some examples, the layout or pattern may be an xy format of recessed portions 54 in the form of rows and columns. In some other examples, the layout or pattern may be a repeated arrangement of recessed portions 54 and / or gap regions 48. In other examples, the layout or pattern may be a random arrangement of recessed portions 54 and / or gap regions 48. The pattern may include dots, pads, stripes, swirls, lines, triangles, rectangles, circles, arcs, plaids, lattices, diagonal lines, arrows, squares and / or cross hatches.

[0127] The layout or pattern of the recesses 54 can be characterized with respect to the density (number of recesses 54) of the recesses 54 in a defined area. For example, the recesses 54 can be approximately 2 million / mm 2 The density can be adjusted to different densities, including, for example, about 100 / mm 2 , about 1,000 pieces / mm 2 , about 0.1 million / mm 2 , about 1 million pieces / mm 2 , about 2 million pieces / mm 2 , about 5 million pieces / mm 2 , about 10 million / mm 2 , about 50 million / mm 2 Or a greater or lesser density. It should also be understood that the density of the recesses 54 in the patterned material 50 can be between a value selected from the lower limit value and a value selected from the upper limit value of the above range. For example, a high-density array can be characterized as having recesses 54 spaced less than about 100 nm, a medium-density array can be characterized as having recesses 54 spaced from about 400 nm to about 1 μm, and a low-density array can be characterized as having recesses 54 spaced greater than about 1 μm. Although exemplary densities have been provided, it should be understood that any suitable density can be used. The density of the recesses 54 can depend in part on the depth of the recesses 54. In some cases, it is desirable that the spacing between the recesses 54 is even greater than the examples listed herein.

[0128] The layout or pattern of the recessed portion 54 may also be based on or alternatively based on the average pitch or spacing (center-to-center spacing) from the center of the recessed portion 54 to the center of the adjacent recessed portion 54 or the average pitch or spacing (edge-to-edge spacing) from the edge of one recessed portion 54 to the edge of the adjacent recessed portion 54. The pattern may be regular so 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, about 50 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm, or more or less. The average pitch of a particular pattern of the recessed portion 54 may be between a value selected from the lower limit value of the above range and a value in the upper limit value. In one example, the recessed portion 54 has a pitch (center-to-center spacing) of about 1.5 μm. Although exemplary average pitch values ​​have been provided, it should be understood that other average pitch values ​​may be used.

[0129] The size of each recess 54 may be characterized by its volume, open area, depth, and / or diameter.

[0130] Each recess 54 can have any volume capable of confining a fluid. The minimum or maximum volume can be selected, for example, to accommodate the desired throughput (e.g., multiplexing), resolution, nucleotide or analyte reactivity for downstream use of the flow cell 38. For example, the volume can be at least about 1×10 -3 μm 3 , at least about 1×10 -2 μm 3 , at least about 0.1 μm 3 , at least about 1 μm 3 , at least about 10 μm 3 , at least about 100 μm 3 Alternatively or in addition, the volume may be at most about 1×10 4 μm 3 , at most about 1×10 3 μm 3 , up to about 100μm 3 , up to about 10μm 3 , up to about 1μm 3 , up to about 0.1μm 3 or smaller.

[0131] The area occupied by each recess opening can be selected based on criteria similar to those described above for volume. For example, the area of ​​each recess opening can be at least about 1×10 -3 μm 2 , at least about 1×10 -2 μm 2 , at least about 0.1 μm 2, at least about 1 μm 2 , at least about 10 μm 2 , at least about 100 μm 2 Alternatively or in addition, the area may be at most about 1×10 3 μm 2 , up to about 100μm 2 , up to about 10μm 2 , up to about 1μm 2 , up to about 0.1μm 2 , at most about 1×10 -2 μm 2 The area occupied by each recess opening may be greater than, less than, or in between the values ​​specified above.

[0132] The depth of each recess 54 can be large enough to accommodate a portion of the polymer hydrogel 42. In one example, the depth can be at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, at least about 100 μm, or more. Alternatively or in addition, the depth can be at most about 1×10 3 um, at most about 100 μm, at most about 10 μm, or less. In some examples, the depth is about 0.4 μm. The depth of each recess 54 can be greater than, less than, or between the values ​​specified above.

[0133] In some cases, the diameter or length and width of each recess 54 can be at least about 50 nm, at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, at least about 100 μm, or more. Alternatively or in addition, the diameter or length and width can be at most about 1×10 3 In some examples, the diameter or length and width is about 0.4 μm. The diameter or length and width of each recess 54 can be greater than, less than, or between the values ​​specified above.

[0134] exist Figure 5C In the example shown, the polymer hydrogel 42 is positioned within each of the recesses 54. The polymer hydrogel 42 may be as described with reference to Figure 5B It is applied in such a way that the polymer hydrogel 42 is present in the depressions 54 and not in the surrounding gap region 48 .

[0135] Although in Figure 5A , Figure 5B or Figure 5C38, but it should be understood that the flow cell 36 may also include a cover attached to the base 38. In one example, the cover may be bonded to at least a portion of the base 38, for example, at some of the gap regions 48. The bond formed between the cover and the base 38 may be a chemical bond or a mechanical bond (e.g., using fasteners, etc.).

[0136] The cover can be any material that is transparent to the excitation light directed to the substrate 38. For example, the cover can be glass (e.g., borosilicate, fused silica, etc.), plastic, etc. A commercially available example of a suitable borosilicate glass is D Commercially available examples of suitable plastic materials (ie, cycloolefin polymers) are available from Zeon Chemicals LP. product.

[0137] The cover can be bonded to the 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 spacer layer can be used to bond the cover to the substrate 38. The spacer layer can be any material that seals at least a portion of the substrate 38 and the cover together. In some examples, the spacer layer can be a radiation absorbing material that helps bond the substrate 38 and the cover.

[0138] In other examples, the flow cell 36 may also include an additional patterned or non-patterned substrate 38 attached to the substrate 38. The substrate 38 may be bonded as described herein.

[0139] method

[0140] The method generally includes generating a series of time-based clustering images for a plurality of proximity-preserving library fragments 14, 14' from a genomic sample. Each time-based clustering image in the series is sequentially generated by introducing a corresponding sample including some of the proximity-preserving library fragments 14, 14' to a flow cell 36, wherein the some of the proximity-preserving library fragments 14, 14' are attached to a solid support 16 ( Figure 1 ) or attached to each other (e.g. Figure 3 14 ′ from the solid support 16 or from each other 34; amplifying the proximity-retaining library fragments 14 , 14 ′ to generate a plurality of corresponding template strands; staining the corresponding template strands; and imaging the corresponding template strands.

[0141] Using the methods disclosed herein, proximity-preserving library fragments 14, 14' are used in combination with sequential amplification and imaging to generate the series of time-based clustering images. Each time-based clustering image records the spatial position and orientation of the template strands generated using a specific sample. Therefore, these images can be used to identify cluster (template strand) positions, where the cluster originates from a specific sample introduced into the flow cell at a specific time.

[0142] The method may vary slightly depending on whether the composite 10 is introduced into the flow cell 36 or whether the attachment fragment 34 is introduced into the flow cell. Various examples will now be described.

[0143] Methods of using the composite 10 or 10'

[0144] In this example, a genomic sample (e.g., sample 30) is fragmented to form a plurality of proximity-retaining fragments 14, 14', each of which is attached to a solid support 16. It should be understood that not all proximity-retaining fragments 14, 14' from a genomic sample 30 may be attached to the same solid support 16; rather, proximity-retaining fragments 14, 14' associated with a particular portion of a genomic sample 30 may be attached to a corresponding solid support 16. Thus, a genomic sample 30 is fragmented to form a plurality of complexes 10 or 10'. This may be described in detail with reference to Figure 1 , FIG. 2A to FIG. 2C , Figure 3 As described, or using any other proximity preserving method that incorporates adapters 18, 22 or 18', 22 into each of the fragments 14, 14' to render them ready-to-sequence fragments 12, 12'.

[0145] The complex 10 or 10' formed using the genomic sample 30 can be incorporated into the mixture. Therefore, each of the plurality of proximity retention fragments 14, 14' is also incorporated into the mixture. The liquid carrier of the mixture can be a buffer, such as Tris-HCl buffer or 0.5× sodium citrate saline (SSC) buffer.

[0146] A liquid carrier can be added to multiple adjacent retention fragments 14, 14' (for example, present in complex 10 or 10') to initially form a mixture, and then the mixture can be diluted with additional liquid carrier to generate a predetermined number of diluted samples, which will be introduced separately into the circulation pool 36 (or its separate channels 40).

[0147] The final volume of the generated mixture can be controlled in any desired manner, thereby controlling the dilution of the mixture. In some cases, the dilution may depend on the volume of the circulation cell 36 (or, for example, each channel 40 of the multichannel circulation cell) and the desired number of samples to be introduced into the circulation cell 36. In one example, the volume of the circulation cell 36 (or its channel 40) can be used as a limiting dilution. Therefore, in some examples, a carrier liquid can be added to dilute the mixture to a predetermined volume, wherein the predetermined volume is based on i) the volume of the circulation cell 36 as the limiting dilution and ii) the predetermined number of diluted samples to be introduced into the circulation cell 36. For example, a circulation cell 36 or a channel 40 of the circulation cell 36 may have a volume of about 50 μL, and the desired number of samples may be 200. In this example, the mixture may be diluted to about 10,000 μL. For another example, a circulation cell 36 or a channel 40 of the circulation cell 36 may have a volume of about 100 μL, and the desired number of samples may be 384. In this example, the mixture may be diluted to about 38,400 μL.

[0148] The desired number of diluted samples may depend, for example, on the volume of the circulation pool 36 and the desired resolution of each template chain in the corresponding time-based cluster generation image. When a smaller volume circulation pool 38 is used, it may be desirable to have a more diluted mixture so that each individual diluted sample to be introduced into the circulation pool 36 contains fewer proximity-retained library fragments 14, 14' (compared to if a less diluted mixture is used). In this type of circulation pool 36, fewer proximity-retained library fragments 14, 14' will result in fewer template chains, which can improve the resolution of the template chains in the corresponding time-based cluster generation image. In one example, the desired number of samples may be in the range of about 100 samples to about 1000 samples. In other examples, the desired number of diluted samples to be prepared from the mixture may exceed 1000. The upper limit of the number of diluted samples may depend in part on the desired time range for performing the overall method.

[0149] The mixture can then be divided into a predetermined number of diluted samples. In one example, the diluted mixture can be separated so that all diluted samples are generated simultaneously. In another example, when any one of the diluted samples of a predetermined volume is introduced into the circulation pool 36, the sample can be separated from the bulk mixture.

[0150] FIG. 6A to FIG. 6D FIG. 3 shows a non-patterned flow cell 36 (eg, as shown in FIG. 1 ) viewed from a top view during different stages of generating a time-based cluster generation image. Figure 5B ) as shown.

[0151] like FIG. 6A to FIG. 6DAs schematically shown, the flow cell 36 can be introduced into such a system, which includes a flow cell receptacle 35; a fluid control system 37, which includes a fluid delivery device 39 for delivering the diluted sample 56A and the stain (not shown) to the flow cell 36 positioned in the flow cell receptacle 35, respectively; an illumination system 62, which is positioned to illuminate the flow cell 36 positioned in the flow cell receptacle 35; a detection system 64, which is positioned to capture an image of the flow cell 36 positioned in the flow cell receptacle 35; and a controller 41, which is connected to the fluid control system 37, the illumination system 62 and the detection system 64. Operably communicating, the controller 41: causes the fluid delivery device 39 to introduce the diluted sample 56A into the flow cell 36 positioned in the flow cell receptacle 35; causes the fluid delivery device 39 to introduce a stain into the flow cell 36 positioned in the flow cell receptacle 35 after generating the template chain 58A from the proximity-retained library fragments present in the diluted sample 56A in the flow cell 36 positioned in the flow cell receptacle 35; causes the illumination system 62 to illuminate the stained template chain in the flow cell 36 positioned in the flow cell receptacle 35; and causes the detection system 64 to image the illuminated stained template chain in the flow cell 36 positioned in the flow cell receptacle 35.

[0152] When in the proper position, the flow cell 36 is in fluid communication with a fluid control system 39 (eg, pumps, valves, etc.) and in optical communication with an illumination system 62 and a detection system 64 .

[0153] exist Fig. 6A In the example of the embodiment of the present invention, the first diluted sample in the diluted sample 56A (including some of the complexes in the complex 10 or 10') is diluted to Fig. 6A 10A) is introduced into the flow cell 36. A corresponding diluted sample 56A (or for example Figure 6C Any diluted sample 56A, 56B) in the flow cell 36 involves fluidly directing one of the diluted samples 56A, 56B to the flow cell 36. The diluted sample 56A can be introduced into, for example, a cartridge 45, and the fluid control system 37 can fluidly transport the diluted sample 56A from the cartridge 45 to the flow channel 40 of the flow cell 36 using a fluid delivery device 39 (e.g., a pump, a valve, etc.).

[0154] Given the concentration of complex 10A in diluted sample 56A, most, if not all, of complex 10A will settle onto polymer hydrogel 42 and any primers 44, 46 thereon (which are present in the FIG. 6A to FIG. 6D In some examples, the complex 10A may settle and remain in the flow channel 40 or the recess 54 due to the depth of the flow channel 40 or the recess 54. In other examples, the flow channel 40 or the recess 54 may include a capture site to which the complex 10A is attached.

[0155] It should be understood that some complexes 10A may not settle, and these complexes 10A will be removed from the flow cell 36 before further processing. Therefore, some examples of methods then include washing the uncaptured complexes 10A from the flow cell 36. Washing can involve introducing a fluid into the flow cell 36. The flow can push any complexes 10A that have not settled and / or attached through the outlet of the flow cell 36.

[0156] This example of the method then includes initiating the release of the proximity-preserving library fragments 14, 14' from the respective solid supports 16 to which they are attached. In this example, the sequencing nucleic acid fragments 12, 12' (including the proximity-preserving library fragments 14, 14' and the adapters 18, 22 or 18', 22 attached thereto) are released from the respective solid supports 16. Fig. 6A , the release of the sequencing nucleic acid fragments 12 , 12 ′ from the solid supports 16 is indicated by arrows pointing outward from each solid support 16 .

[0157] The release of the ready-to-sequence nucleic acid fragments 12, 12' can be triggered in several different ways. In one example, triggering the release involves heating the circulation pool 36. In this example, the system may include a heater 43. The controller 41 can cause the heater 43 to trigger the release of some of the proximity retention library fragments 12, 12' from the solid support 16 or from each other. For example, a temperature above 70°C can be used to at least partially break the bond, thereby triggering the release of the ready-to-sequence nucleic acid fragments 12, 12'. In another example, triggering the release involves introducing a cleavage agent into the circulation pool 36. The fluid control system 37 can be used to deliver the cleavage agent. The cleavage agent can trigger the chemical, enzymatic or photochemical release of the ready-to-sequence nucleic acid fragments 12, 12' from the solid support 16. In these examples, another stimulus (such as heat or light) can trigger the cleavage agent to release the ready-to-sequence nucleic acid fragments 12, 12' from the solid support 16. For example, free biotin can be introduced as a cleavage agent, and heating to about 92°C can be used to induce the release of biotin-oligonucleotides from the solid support 16.

[0158] The released ready-to-sequence nucleic acid fragments 12, 12' are transported from the solid support 16 and seeded onto the polymer hydrogel 42. More specifically, the amplification primers 46, 48 seed the released ready-to-sequence nucleic acid fragments 12, 12' in a relatively confined manner. In one example, seeding is accomplished by hybridization between the first sequence or the second sequence of the fragments 12, 12' and the complementary primers in the primers 46, 48 on the polymer hydrogel 42 in the flow cell 36. The seeding can be performed at a hybridization temperature suitable for the fragments ready-to-sequence nucleic acid fragments 12, 12' and the primers 46, 48. The heater 43 can be controlled to bring the flow cell 36 to the seeding temperature.

[0159] A washing process may be performed to remove the beads.

[0160] Then any suitable method (such as cluster generation) can be used to amplify the inoculated ready-to-use sequencing nucleic acid fragments 12, 12 '. In one example of cluster generation, a high-fidelity DNA polymerase is used to replicate the released ready-to-use sequencing nucleic acid fragments 12, 12 ' from the hybridization primers 46, 48 by 3 'extension. The original ready-to-use sequencing nucleic acid fragments 12, 12 ' are denatured so that these copies are fixed in some recesses in the flow channel 40 or the recess 54. Any clonal amplification process can be used. In one example, isothermal bridge amplification can be used to amplify fixed copies. For example, the replicated template loops back to hybridize with adjacent complementary primers 46, 48, and the polymerase replicates the replicated template to form a double-stranded bridge, and these double-stranded bridges are denatured to form two single strands. These two chains loop back 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 by isothermal denaturation and amplification cycles to produce dense clonal clusters. Each cluster of double-stranded bridges is denatured. In one example, the antisense strand is removed by specific base cleavage, leaving the forward template polynucleotide strand. It should be understood that clustering results in the formation of several template strands 58A in some of the recesses in the flow channel 40 or the recesses 54. In some examples, the controller 41 causes the heater 43 to run a thermal cycle to amplify the seeded, ready-to-sequence nucleic acid fragments 12, 12'.

[0161] Figure 6B Cluster 60A of template strands 58A generated from complex 10A of first dilution sample 56A (compartment) is shown. For clarity, the Figure 6B Cluster 60A in. Although Figure 6B Four clusters 60A are shown, but it should be understood that the number of clusters 60A will depend on the number of complexes 10A introduced into the sample 56A and the number of ready-to-sequence nucleic acid fragments 12, 12' released from each solid support 16. Clusters 60A are generated from each of the released ready-to-sequence nucleic acid fragments 12, 12'. In addition, the released ready-to-sequence nucleic acid fragments 12, 12' can diffuse over the entire flow cell surface, so clusters 60A can be generated over the entire flow cell surface.

[0162] After clusters 60A are generated for the first diluted sample 56A, a stain is introduced into the flow cell 36. Any fluorescent stain capable of staining the template strand 58A may be used. Examples of suitable fluorescent stains include those available from Molecular Probes, Inc. Dye families (e.g. Green, Gold, Safe, etc.), ethidium bromide, propidium iodide, crystal violet, Dye (from Biotium), DAPI (4′,6-diamidino-2-phenylindole), etc. The stain is introduced into the flow cell 36 , for example from a second cartridge (not shown), allowed to incubate for a suitable period of time to stain the template strand 58A, and then drained from the flow cell 36 .

[0163] The illumination system 62 can then be used to illuminate the dye template strand 58A in the circulation pool 36. The illumination system may include a light source and a plurality of optical components. Examples of light sources may include lasers, arc lamps, LEDs, or laser diodes. The optical components may be, for example, reflectors, dichroic mirrors, beam splitters, collimators, lenses, filters, wedges, prisms, mirrors, detectors, etc. The illumination system may be operably positioned to direct the excitation light to the circulation pool surface corresponding to the dye used.

[0164] The detection system 64 can be used to capture an image I1 of the fluorescent template strands 58A. The image I1 is a time-based cluster generation image for the diluted sample 56A because it depicts the spatial position and orientation of the template strands 58A associated with the diluted sample 56A. Any suitable camera can be used to capture the image I1 of the clusters 60A on the flow cell 36.

[0165] Image I1 can be stored electronically for subsequent retrieval and use. Therefore, some examples of the system include an electronic storage component 47 for storing image I1. In the electronic record, image I1 can be connected to diluted sample 56A. A time record can also be assigned to image I1. Therefore, some examples of the method include assigning a time record of the introduction of a corresponding sample including some proximity-retained library fragments in the proximity-retained library fragments to each time-based cluster generation image 1 in the series. The time record may include a timestamp indicating when the diluted sample 56A was introduced and / or imaged, the number of steps in the introduction and / or imaging sequence (e.g., 200 of sample 1, 200 of sample 2, ... 200 of sample X), or a combination thereof.

[0166] Washing may be performed after imaging of clusters 60A on flow cell 36. Water, buffer, or another mild washing solution may be used.

[0167] Then the second diluted sample 56B ( Figure 6C ) Repeated reference Fig. 6A and Figure 6B Process shown and described.

[0168] exist Figure 6C In the flow cell, the second diluted sample 56B is introduced into the flow cell 36. The complex 10B (which may be the complex 10 or 10') settles and / or adheres to the flow cell surface.

[0169] like Figure 6CAs shown, the method then includes initiating the release of the proximity-preserving library fragments 14, 14' from the respective solid supports 16 to which they are attached. In this example, the sequencing nucleic acid fragments 12, 12' (including the proximity-preserving library fragments 14, 14' and the adapters 18, 22 or 18', 22 attached thereto) are released from the respective solid supports 16.

[0170] The released sequencing-ready nucleic acid fragments 12, 12' 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 round of clustering results in the formation of several more template strands 58B in some of the recesses in the flow channel 40 or the recesses 54.

[0171] Fig.6D Cluster 60B of template strands 58B generated from corresponding complex 10B of second diluted sample 56B (compartment) is shown. For clarity, the Fig.6D Clusters 60A and 60B in FIG. Fig.6D Three clusters 60B are shown, but it will be appreciated that the number of clusters 60B will depend on the number of complexes 10B introduced into the sample 56B and the number of sequencing-ready nucleic acid fragments 12 , 12 ′ released from each solid support 16 .

[0172] After clusters 60B are generated for second diluted sample 56B, the stain is reintroduced into flow cell 36. The same stain used to stain template strand 58A can be used to stain template strand 58B and any subsequently generated template strands.

[0173] Illumination system 62 may then be used to illuminate stained template strands 58A and 58B in flow cell 36. Detection system 64 may be used to capture image 12 of fluorescent template strands 58A and 58B in respective clusters 60A and 60B.

[0174] Image 12 may also be stored electronically for subsequent retrieval and use. In the electronic record, image 12 may be linked to diluted sample 56B. A time record may also be assigned to image 12. The time record may include a timestamp indicating when diluted sample 56B was imaged, a step number in the sequence (e.g., sample 2 of 200), or a combination thereof.

[0175] Washing may be performed after imaging of the clusters 60A, 60B on the flow cell 36. Water, buffer, or another mild washing solution may be used.

[0176] The system can then be used, for example, to repeatedly refer to Fig. 6A and Figure 6BThe process shown and described above is used to obtain the number of diluted samples derived from the original mixture. Each additional image I3, I4, ... I x New clusters 60C, 60D, ... 60X of template strands 58C, 58D, ... 58X generated by the introduction of the corresponding diluted samples 56C, 56D, ... 56X will be depicted. All images I1, I2, ... I2 obtained for the corresponding diluted samples 56A, 56B, ... 56X x All are associated with a specific mixture and therefore with a specific longer nucleic acid molecule 30.

[0177] Because each sequential image I2, I3, I4, ... I x Depicts the image I1, I2, I3, ... I x Newly formed clusters 60B, 60C, 60D, ... 60X, so image subtraction can be used to generate a resolved cluster image for each sample introduced after the first sample 56A. Some exemplary resolved cluster images RI x Shown in Figure 6.

[0178] Figure 7 Depicted for reference Fig. 6A and Figure 6B The images I1, I2, I3, I4, I5, I6 of the six different diluted samples 56A, 56B, 56C, 56D, 56E, 56F that were sequentially introduced, amplified, stained, and imaged. As depicted in the figure, new clusters 60A, 60B, 60C, 60D, 60E, and 60F are generated for each of the newly introduced and processed diluted samples 56A, 56B, 56C, 56D, 56E, 56F, respectively.

[0179] Because the image I1 for the first sample 56A includes the cluster 60A from one sample, it is not necessary to generate a resolved cluster image because the original image I1 can be used for spatial identification of the cluster 60A. For each sample introduced after the first sample 56A, a resolved cluster image RI2, RI3, RI4, RI5, RI6 is generated for each of the other images I2, I3, I4, I5, I6. For example, the image I1 (depicting the template chain 58A in the cluster 60A) can be subtracted from the image I2 (depicting the template chain 58A in the cluster 60A and the template chain 58B in the cluster 60B) to generate a resolved cluster image RI2 for the second sample 56B. The resolved cluster image RI2 depicts the spatial position and orientation of the template chain 58B in the cluster 60B associated with the second sample 56B. As another example, image I1 (depicting template strand 58A in cluster 60A) and image I2 (depicting template strand 58A in cluster 60A and template strand 58B in cluster 60B) can be subtracted from image I3 (depicting template strand 58A in cluster 60A, template strand 58B in cluster 60B, and template strand 58C in cluster 60C) to generate a resolved cluster image RI3 for a third sample (e.g., 56C). The resolved cluster image RI3 depicts the spatial position and orientation of template strand 58C in cluster 60C associated with the third sample 56C. Resolved cluster images RI4, RI5, and RI6 can be generated in a similar manner by subtracting any of the aforementioned images.

[0180] It should be understood that a series of I1, I2, I3, ... I x The resulting resolved cluster image RI for any given dilution sample 56X x The spatial position and orientation of the template strand 58X associated with the diluted sample 56X is depicted.

[0181] The parsed cluster images may be stored for subsequent analysis.

[0182] Method using attached fragment 34

[0183] In this example, the genomic sample is fragmented to form a plurality of proximity-preserved fragments 14, 14' that are attached to one another, for example as attachment fragments 34. It should be understood that not all proximity-preserved fragments 14, 14' from the genomic sample may be attached to one another; Figure 4 The process shown may result in the formation of several attached segments 34 .

[0184] The attachment fragment 34 formed using the genomic sample can be incorporated into the mixture. Therefore, each of the plurality of proximity retention fragments 14, 14' is also incorporated into the mixture. The liquid carrier of the mixture can be a buffer, such as Tris-HCl buffer or 0.5× sodium citrate saline (SSC) buffer.

[0185] A liquid carrier may be added to the plurality of attached segments 34 to initially form a mixture, which may then be diluted with additional liquid carrier to generate a predetermined number of diluted samples to be individually introduced into the flow cell 36 (or individual channels 40 thereof).

[0186] The final volume of the resulting mixture, thereby controlling the dilution of the mixture, can be controlled in any desired manner and as described herein.

[0187] The mixture can then be divided into a predetermined number of diluted samples. In one example, the diluted mixture can be separated so that all diluted samples are generated simultaneously. In another example, when any one of the diluted samples of a predetermined volume is introduced into the circulation pool 36, the sample can be separated from the bulk mixture.

[0188] In this exemplary method, Figure 4 The attached segment 34 is shown as being introduced into the flow channel as part of one of the diluted samples. An example of the diluted sample 66A is depicted in FIG. Figure 8 middle.

[0189] like Figure 8 As shown, within the flow cell 36, the transposase 32 is removed from the attachment segment 34. This can be achieved, for example, using SDS or a protease. The removal of the transposase 32 releases the corresponding proximity retention segment 14, 14' (and any strands 26', 28' directly or indirectly attached thereto) from the adjacent proximity retention segment 14, 14'. In other words, the sub-segments 72 of the attachment segment 34 are released and can be seeded onto the polymer hydrogel 42 via the transferred strands 26'. As shown in FIG. Figure 8 Schematically shown, the transferred strand 26' hybridizes with the corresponding and complementary primer 46 on the surface of the flow cell 36. In some cases, heat can be applied during hybridization. The application of heat can depend on the melting temperature of the transferred strand 26'. For example, the P5' portion of the transferred strand 26' hybridizes with the complementary P5 amplification primer 46 attached to the polymer hydrogel 42.

[0190] A wash solution may flow through the flow channel of the flow cell 36 to remove the transposase 32 from the flow cell 36. Examples of suitable wash solutions include SDS, which can remove the transposase. A second wash solution such as TRIS or a hybridization wash buffer may be used to rinse the flow cell 36.

[0191] Prior to amplification, the method example also includes introducing a second sequence portion (e.g., non-transferred strand 28′) into each of the hybridized proximity retention library fragments 14, 14′ at the end opposite to the hybridized end. Thus, in some examples of the method, each of the proximity retention library fragments 14, 14′ includes a first sequence portion at a first end that hybridizes with a first primer sequence 46 on the surface of the flow cell 26; and prior to amplification, the method also includes attaching a second sequence portion to each of the hybridized proximity retention library fragments 14, 14′ at a second end opposite to the first end, the second sequence portion being identical to a second primer sequence 48 on the surface of the flow cell 36, so that a copy of the second sequence portion can hybridize with the second primer sequence 48.

[0192] The introduction of the second sequence portion can be carried out using an extended ligation. In one example, an extended ligation (e.g. Figure 8 74 in the figure) to join the non-transferred strand 28' to the corresponding fragment 14, 14'. In one example, the extension ligation can be initiated by introducing the extension ligation mixture into the flow cell 36 and heating it to a temperature suitable for enzyme activity (e.g., in the range of about 37°C to about 50°C).

[0193] The extension ligation mixture may include a ligase (eg, DNA ligase) that catalyzes the formation of a bond between the non-transferred strand 28' and its corresponding fragment 14 or 14'. Figure 4 As described, the non-transferred strand 28′ includes a second sequencing primer sequence (e.g., a read 2 sequencing primer sequence) and a second sequence (P7) that is identical to at least a portion of another amplification primer 48 (P7) among the amplification primers on the surface of the flow cell. The second sequence enables a complementary copy (e.g., P7′) to be generated during amplification, which can hybridize with the amplification primer 48 (P7) on the surface of the flow cell during cluster generation. Thus, the connection results in the formation of ready-to-use sequencing library fragments 12, 12′ attached to the surface of the flow cell.

[0194] The extension ligation mixture may also include blocking groups attached to the exposed ends of primers 46 to prevent undesired extension at these primers 46. Alternatively, primers 46 may be grafted to a surface having a blocking group (e.g., a 3' phosphate group) attached thereto. In other examples, no blocking group may be used.

[0195] When the resulting fragments 12, 12' are attached to each other, heating may be used to dissociate the fragment 12' from the fragment 12. The fragments 12' that have not hybridized to the primer 46 may be removed from the flow cell 36 by washing.

[0196] When used, any blocked primers 46 can then be deblocked (e.g., using a kinase or another suitable deblocking agent) so that amplification can proceed. In this example, amplification can be performed using any suitable method such as cluster generation. Cluster generation can be described herein with reference to Fig. 6A Reference Fig. 6A The system described.

[0197] Fig.9A The dilution of sample 66A ( Figure 8 Cluster 70A of template strand 68A generated by attachment fragment 34 of FIG. 1 (shown in FIG. 1 ). For clarity, the Fig.9A Cluster 70A in. Although Fig.9A Four clusters 70A are shown, but it should be understood that the number of clusters 70A will depend on the number of attached fragments 34 introduced into the sample 66A and the number of sequencing-ready nucleic acid fragments 12 , 12 ′ released from the attached fragments 34 .

[0198] After clusters 70A are generated for the first diluted sample 66A, a stain is introduced into the flow cell 36 as shown in FIG. Figure 6B The stain is introduced into the flow cell 36 , allowed to incubate for a suitable period of time to stain the template strand 68A, and then drained from the flow cell 36 .

[0199] The illumination system 62 can then be used to illuminate the stained template strands 68A in the flow cell 36, and the detection system 64 can be used to capture an image I1 of the fluorescent template strands 68A. This image I1 is a time-based clustering image for the diluted sample 66A because it depicts the spatial position and orientation of the template strands 68A associated with the diluted sample 66A.

[0200] Image I1 may be stored electronically for subsequent retrieval and use. In the electronic record, image I1 may be linked to diluted sample 66A. A time record may also be assigned to image I1. The time record may include a timestamp indicating when diluted sample 66A was introduced and / or imaged, the number of steps in the introduction and / or imaging sequence (e.g., sample 1 at 200, sample 2 at 200, ... sample X at 200), or a combination thereof.

[0201] Washing may be performed after imaging of clusters 70A on flow cell 36. Water, buffer, or another mild washing solution may be used.

[0202] Then the second diluted sample 66B ( Fig. 9B ) Repeated reference Figure 8 and Fig.9A Process shown and described.

[0203] exist Fig. 9BIn the embodiment of the present invention, the second diluted sample 66B is introduced into the flow cell 36. The attached fragment 34 can be decomposed into sub-fragments 72, as shown in FIG. Figure 8 A transferred strand 26 of at least some of the subfragments 72 will hybridize with a complementary amplification primer 46 on the flow cell 36. A reference may then be performed. Figure 8 The described extension, ligation and other processes result in the attachment of the ready-to-sequence nucleic acid fragments 12 to the flow cell surface.

[0204] Cluster generation can be done as described in this article Fig. 6A Execute as described.

[0205] Fig. 9C Cluster 70B of template strands 68B generated by attached fragments 34 of second diluted sample 66B is shown. For clarity, the Fig. 9C Clusters 70A and 70B in FIG. Fig. 9C Two clusters 70B are shown, but it should be understood that the number of clusters 70B will depend on the number of attached fragments 34 introduced into the sample 66B and the number of sequencing-ready nucleic acid fragments 12 , 12 ′ released from the attached fragments 34 .

[0206] After clusters 70B are generated for second diluted sample 66B, stain is reintroduced into flow cell 36. The same stain used to stain template strand 68A can be used to stain template strand 68B and any subsequently generated template strands.

[0207] Illumination system 62 may then be used to illuminate stained template strands 68A and 68B in flow cell 36. Detection system 64 may be used to capture image 12 of fluorescent template strands 68A and 68B in corresponding clusters 70A and 70B.

[0208] Image 12 may also be stored electronically for subsequent retrieval and use. In the electronic record, image 12 may be linked to diluted sample 66B. A time record may also be assigned to image 12. The time record may include a timestamp indicating when diluted sample 56B was imaged and / or introduced, a step number in the sequence (e.g., Sample 2 of 1000), or a combination thereof.

[0209] Washing may be performed after imaging of the clusters 70A, 70B on the flow cell 36. Water, buffer, or another mild washing solution may be used.

[0210] Then you can refer to Fig. 9B and Fig. 9C The process shown and described above is used to obtain the number of diluted samples derived from the original mixture. Each additional image I3, I4, ... I xNew clusters 70C, 70D, ... 70X of template strands 68C, 68D, ... 68X generated by the introduction of the corresponding diluted samples 66C, 66D, ... 66X will be depicted. All images I1, I2, ... I2 obtained for the corresponding diluted samples 66A, 66B, ... 66X x All are associated with a specific mixture and therefore with a specific longer nucleic acid molecule 30.

[0211] Because each sequential image I2, I3, I4, ... I x Depicts the image I1, I2, I3, ... I x Newly formed clusters 70B, 70C, 70D, ... 70X can therefore be image subtracted to generate resolved cluster images for each sample introduced after the first sample 66A. Figure 7 The generated analyzed cluster image RI x .

[0212] It should be understood that a series of I1, I2, I3, ... I x The resulting resolved cluster image RI for any given dilution sample 66X x The spatial position and orientation of the template strand 68X associated with the diluted sample 66X is depicted.

[0213] The parsed cluster images may be stored for subsequent analysis.

[0214] Other methods

[0215] Rather than introducing a separate diluted sample 56A, 66A, the diluted mixture can be diffused into the flow cell in predetermined volumes, and processing on the flow cell 38 can be performed as described herein to amplify, stain, and record images of the generated template strands 58X, 68X. Diffusion can be controlled so that one predetermined volume is introduced at a time.

[0216] Thus, some examples of the method include generating a time-based cluster generation image for each of the limiting dilution samples introduced into the circulation pool 36 by: controlling the diffusion of the mixture into the circulation pool so that one of the limiting dilution samples in the circulation pool 36 is introduced at a time; triggering the release of proximity-retained library fragments 14, 14′ from the solid support 16 or from each other (e.g., from the attached fragment 34) in one of the limiting dilution samples in the circulation pool 36; amplifying the proximity-retained library fragments 14, 14′ to generate multiple corresponding template chains 58A, 68A; staining the corresponding template chains 58A, 68A; and imaging the corresponding template chains 58A, 68A.

[0217] Sequencing and analysis

[0218] When all diluted samples from the mixture are amplified and imaged as described herein, flow cell 36 is ready for sequencing operations. A variety of sequencing methods or techniques can be used, including those generally referred to as sequencing by synthesis (SBS), cycle array sequencing, sequencing by ligation, pyrosequencing, and the like.

[0219] For example, a synthesis sequencing (SBS) reaction can be performed in a system such as HISEQ TM HISEQX TM MISEQ TM MISEQDX TM MINISEQ TM 、NOVASEQ TM 、NEXTSEQDX TM 、ISEQ TM 、NEXTSEQ TM or other sequencer systems available from Illumina (San Diego, CA). In SBS, the extension of sequencing primers along template strands 58A, 58B, ... 58X is monitored to determine the sequence of the nucleotides in the template. The 3′ ends of template strands 58A, 58B, ... 58X and any flow cell-bound primers 46, 48 (not attached to template strands 58A, 58B, ... 58X) may be blocked to prevent interference with the sequencing reaction, and in particular, to prevent undesired priming.

[0220] A sequencing primer can be introduced that hybridizes to a complementary sequence on template strands 58A, 58B, ... 58X. The sequencing primer renders template strands 58A, 58B, ... 58X ready for sequencing.

[0221] The basic chemical process can be polymerization (e.g., catalyzed by a polymerase) or connection (e.g., catalyzed by a ligase). In a specific polymerase-based SBS process, fluorescently labeled nucleotides are added to the sequencing primer in a template-dependent manner so that the sequence of the template can be determined using detection of the order and type of nucleotides added to the sequencing primer. For example, in order to trigger the first SBS cycle, one or more labeled nucleotides, DNA polymerase, etc. can be delivered to / through the circulation cell 36, etc., in which the sequencing primer extension causes the labeled nucleotides to be incorporated. This incorporation can be detected by an imaging event. During the imaging event, the illumination system 62 can provide excitation light to the circulation cell 36.

[0222] In some examples, the fluorescently labeled nucleotides may further include a reversible termination property that terminates further primer extension once the nucleotides are added to the template. For example, a nucleotide analog having a reversible terminator portion may be added to the template such that subsequent extension does not occur until a deblocking agent is delivered to remove the portion. Thus, for examples using reversible termination, a deblocking agent may be delivered to a flow cell 36, etc. (after detection occurs).

[0223] Washing may occur between the various fluid delivery steps.The SBS cycle may then be repeated n times to extend the template by n nucleotides, thereby detecting a sequence of length n.

[0224] Although SBS has been described in detail, it should be understood that the flow cell 36 described herein may be used for genotyping with other sequencing protocols, or in other chemical and / or biological applications.

[0225] Based on the analyzed cluster images RI2, RI3, ... RI x The sequence reads obtained during the sequencing operation are grouped together. Then, the cluster images RI2, RI3, ... RI x The combined sequencing reads are linked to a corresponding one of the diluted samples. It can be inferred that the combined and linked sequencing reads are derived from the same longer nucleic acid sample 30. Therefore, some examples of the method include performing a sequencing operation on a flow cell 26, the flow cell including a corresponding template strand for each of a plurality of library fragments; and generating images RI, RI2, RI3, ... RI based on the resolved clusters. x Sequencing reads were grouped together in different groups.

[0226] In order to further illustrate the present disclosure, examples are given herein. It should be understood that these examples are provided for illustrative purposes and should not be construed as limiting the scope of the present disclosure.

[0227] Reality Examples

[0228] Example 1

[0229] The barcode oligonucleotides (e.g., adapter 18) were attached to M280 streptavidin beads (Thermofisher) via a biotin adapter to form a pool of beads. The universal transposome was hybridized to the complementary sequence at the end of the oligonucleotide to form a bead-linked transposome (BLT). The BLT was then washed in wash buffer, resuspended in working buffer, and auxiliary proteins (single-stranded binding protein (Thermofisher) and double-stranded binding protein (Illumina)) were added.

[0230] High molecular weight NA12878 DNA (using Qiagen HMW DNA extraction protocol (extracted from cultured cells) is added to the BLT mixture, and the tube is gently flipped to mix. The tube is then incubated at room temperature for about 15 minutes to wrap the DNA around the beads. Then labeling buffer (containing magnesium chloride and tris acetate) is added to each tube, and the sample is incubated at about 55°C for about 10 minutes. During this step, the transposome-labeled DNA and the labeled DNA are attached to the BLT. After the labeling reaction, sodium dodecyl sulfate (SDS) is added and the sample is incubated at room temperature for about 5 minutes to denature the transposase. The tube is then placed on a magnet and the supernatant is removed. The beads are washed with washing buffer. After the last wash, the beads are resuspended in a ligase mixture (containing T7 ligase and its associated buffer-T7 ligase buffer from NEB). The samples are then mixed and incubated at room temperature for about 45 minutes. During this period of time, the gap in the transfer chain (the position where the transposome initially hybridizes) is connected, so that the labeled DNA is physically attached to the beads. The samples were then placed on a magnet, the supernatant removed, and the beads washed again in wash buffer.

[0231] The labeled beads are then divided into two groups for removal of non-transferred strands and introduction of sample indexes (e.g., adapter 22). One group (the comparison group) is exposed to a comparison workflow in which heat is used to remove non-transferred strands. The other group (the example group) is exposed to an example workflow in which an exonuclease is used to remove non-transferred strands.

[0232] The comparison group was resuspended in wash buffer and heated to about 80°C for about 5 minutes to denature the non-transferred strands. The tube containing the comparison group was then placed on a magnet, the supernatant was removed, and the beads were washed. The sample index diluted in wash buffer was then added to the beads of the comparison group, and the mixture was incubated at about 80°C for about 1 minute, after which the temperature was slowly decreased.

[0233] The sample set was suspended in a T7 exonuclease mix (containing T7 exonuclease and NEB buffer 4) and allowed to incubate at room temperature for about 10 minutes. The 5' to 3' exonuclease activity of the T7 exonuclease digests the non-transferred strand. The tube containing the sample set was then placed on a magnet, the supernatant was removed, and the beads were washed. The sample index diluted in wash buffer was then added to the beads of the sample set, and the mixture was incubated at about 55°C for about 5 minutes to allow the sample index to anneal.

[0234] The tubes containing the comparative group and the example group were placed on a magnet and the supernatant was removed. The extension ligation mixture was added and the samples were incubated at about 37°C for about 5 minutes. The tubes were placed on a magnet again, the supernatant was removed, and the beads of the comparative group and the example group were washed in wash buffer. After the last wash, the beads were resuspended in wash buffer.

[0235] Some beads of the comparison group and some beads of the example group were subsampled and used in PCR reactions. In addition to the corresponding beads, each PCR mixture consisted of Illumina PCR mixture EPM and P5 and P7 oligonucleotides. Each sample was amplified using PCR.

[0236] After PCR, a subsample of the PCR supernatant from each of the comparison and example groups was transferred to a new tube and subjected to 0.50×-0.62× size selection (solid phase reversible immobilization, SPRI) using sample purification beads. The resulting libraries were eluted in resuspension buffer. These libraries were then cloned in separate HISEQ TM The samples were sequenced on a 2500 Rapid flow cell (using a standard 2×101 cycle read length). After Fastq generation, the samples were aligned to the human genome (hg38) and the data were imported into IGV.

[0237] Fig.10 The coverage obtained using libraries from each of the comparative group (labeled as heat denaturation) and the exemplary group (labeled as T7 exonuclease) for an AT-rich region of the human genome, which is known to be negatively affected by high temperature steps in library preparation protocols, is shown. The results for the comparative group library fragments whose non-transferred strands were removed via heat denaturation are shown in Fig.10 The results for an example set of library fragments whose non-transferred strands were removed via exonuclease digestion are shown at the top of Fig.10 . As shown in the figure, there is complete coverage in the AT-rich region for the example group library fragments, while there is only partial coverage in the same region for the comparison group library fragments. These results show that when compared with thermal denaturation, the enzymatic digestion method disclosed herein increases library coverage and improves sequencing on the AT-rich region of the genome.

[0238] Example 2

[0239] like FIG. 2A to FIG. 2C method (multi-step ligation and digestion) and Figure 3 The complexes were prepared as described in the method of (single-pot ligation and digestion).

[0240] BLT generation, DNA binding, labeling, and exposure to SDS were performed as described in Example 1. After removal of the transposase and the washes associated therewith, the tagged beads were then divided into two groups to remove the non-transferred strand via multiple steps of ligation and digestion (referred to as Example Group 2) or via a single-pot ligation and digestion (referred to as Example Group 3).

[0241] Example Set 2 was resuspended in an E. coli DNA ligase mix, which included E. coli DNA ligase and its associated buffer, both from NEB. The Example Set 2 samples were then mixed and incubated at about 16°C for about 15 minutes. The tube containing the Example Set 2 samples was then placed on a magnet, the supernatant was removed, and the Example Set 2 beads were washed in wash buffer. After washing, the Example Set 2 beads were resuspended in a mixture containing T7 exonuclease and NEB buffer 4 and incubated at about 25°C for about 10 minutes.

[0242] Example 3 was resuspended in a combined ligase and exonuclease mixture (including E. coli DNA ligase, T7 exonuclease, NAD + and CUTSMART TM Buffer (from NEB)) for about 15 minutes.

[0243] The tubes containing sample set 2 and sample set 3 were then placed on a magnet, the supernatant removed, and the corresponding beads washed. The sample index diluted in wash buffer was then added to the beads of each sample set, and the mixtures were incubated at about 55°C for about 5 minutes to allow the sample index to anneal.

[0244] The tubes containing Example Group 2 and Example Group 3, respectively, were placed on a magnet and the supernatant was removed. The extension ligation mixture was added and the samples were incubated at about 37°C for about 5 minutes. The tubes were placed on a magnet again, the supernatant was removed, and the beads of the comparative group and the example group were washed in wash buffer. After the last wash, the beads were resuspended in wash buffer.

[0245] Some beads from Example Set 2 and some beads from Example Set 3 were subsampled and used in PCR reactions. Each PCR mix consisted of Illumina PCR mix EPM and P5 and P7 oligonucleotides in addition to the corresponding beads. Each sample was amplified using PCR.

[0246] After PCR, a subsample of the PCR supernatant from each of sample set 2 and sample set 3 was transferred to a new tube. Sample purification beads were added and 2.5× SPRI was performed, where the resulting library was eluted in resuspension buffer. The purified library was then run on a Bioanalyzer 2100 High Sensitivity Chip and a PCR result was obtained. Fig.11The results show that the size distribution and yield of the libraries are comparable for multi-step ligation and digestion and for single-pot ligation and digestion. These results indicate that the combined reagent formulation does not adversely affect ligation and does not result in digestion of fragments or transferred strands.

[0247] The exemplary bead-bound library fragments (complexes) obtained using the library preparation methods described in Examples 1 and 2 can be divided into subsamples and diluted to form a plurality of diluted samples as described herein. The reference FIG. 6A to FIG. 6D The method described (including cluster generation, staining and imaging) is used to generate a series of time-based cluster generation images. When all diluted samples are amplified and imaged, the flow cell is ready for sequencing operations. The library preparation technology disclosed herein and the time-based imaging technology can be used together to efficiently and reliably reconstruct long DNA fragments.

[0248] Additional Notes

[0249] In addition, it should be understood that the ranges provided herein include the specified range and any value or sub-range within the specified range, as if they were explicitly listed. For example, a range expressed by about 2 mm to about 300 mm should be interpreted as including not only the explicitly listed limit values ​​of about 2 mm to about 300 mm, but also single values ​​such as about 15 mm, 22.5 mm, 245 mm, etc., and sub-ranges such as about 20 mm to about 225 mm, etc.

[0250] It should be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below (assuming such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. Specifically, all combinations of the claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be understood that terminology expressly employed herein that may also appear in any disclosure incorporated by reference should be given the meaning most consistent with the specific concepts disclosed herein.

[0251] While several examples have been described in detail, it is to be understood that modifications may be made to the disclosed examples. Therefore, the above description should be considered non-limiting.

Claims

1. A system, comprising: Flow cell receptacle; a fluid control system comprising a fluid delivery device for delivering a diluted sample and a stain to a flow cell positioned in the flow cell receptacle, respectively; 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 operable communication with the fluid control system, the lighting system, and the detection system, the controller: causing the fluid delivery device to introduce the diluted sample into the flow cell positioned in the flow cell receptacle; causing the fluid delivery device to introduce the stain into the flow cell positioned in the flow cell receptacle after generating template strands from proximity-preserving library fragments present in the diluted sample in the flow cell positioned in the flow cell receptacle; causing the illumination system to illuminate the dye-template strand in the flow cell positioned in the flow cell receptacle; as well as The detection system is caused to image the illuminated stained template strand in the flow cell positioned in the flow cell receptacle.

2. The system of claim 1, wherein the flow cell receptacle is part of a sequencer comprising a heater, wherein the controller is configured to: causing the heater to trigger release of the some of the proximity-preserving library fragments from a solid support or from each other; The heater is caused to run a thermal cycle to amplify the some of the proximity-preserved library fragments to generate the template strand.

3. The system according to claim 1 , further comprising: a first cartridge, the first cartridge containing the diluted sample; and A second cartridge, wherein the second cartridge contains the coloring agent.

4. System according to one of claims 1 to 3, further comprising electronic storage means for storing the image.

5. The system of claim 4, wherein the image is a time-based clustering image in a series of time-based clustering images for a plurality of proximity-preserving library fragments from a genomic sample, wherein each time-based clustering image in the series is generated sequentially.

6. A method comprising: preparing a mixture comprising a plurality of proximity-preserving library fragments of a genomic sample, the plurality of proximity-preserving library fragments being attached to a solid support or to each other by a transposase and comprising a first sequence portion at a first end; diluting the mixture to generate a predetermined amount of diluted sample to be introduced into the flow cell; as well as Generating a time-based clustering image of proximity-preserving library fragments for a first sample in the predetermined number of diluted samples by: controlling diffusion of the first sample in the predetermined amount of diluted sample into the flow cell; Inducing the release of the proximity retention library fragments from the solid support or from each other in the first sample of the predetermined number of diluted samples, whereby the first sequence portions of the proximity retention library fragments in the first sample of the predetermined number of diluted samples hybridize with first primer sequences on the polymer hydrogel on the surface of the flow cell, respectively, thereby seeding the proximity retention library fragments to the polymer hydrogel; amplifying the proximity-preserving library fragments to generate a plurality of corresponding template strands on the surface of the flow cell; staining the corresponding template strand; as well as The corresponding template strand is imaged.

7. The method according to claim 6, further comprising: Generating a time-based clustering image of proximity-preserving library fragments for a second sample in the predetermined number of diluted samples by: controlling diffusion of the second sample in the predetermined amount of diluted samples into the circulation cell and diffusion of the first sample in the predetermined amount of diluted samples out of the circulation cell; Inducing the release of the proximity retention library fragments from the solid support or from each other in the second sample in the predetermined number of diluted samples, whereby the first sequence portions of the proximity retention library fragments in the second sample in the predetermined number of diluted samples hybridize with the first primer sequences on the polymer hydrogel on the surface of the flow cell, respectively, thereby seeding the proximity retention library fragments to the polymer hydrogel; amplifying the proximity-preserving library fragments to generate a second plurality of template strands on the surface of the flow cell; staining the second plurality of template strands; as well as The second plurality of template strands are imaged.

Images