阅读说明：本技术 基因组dna的扩增邻近性保留文库片段的基于时间的簇成像 (Time-based cluster imaging of amplified proximity-preserving library fragments of genomic DNA ) 是由 N·莫雷尔 A·斯莱特 V·汤姆森 于 2020-12-01 设计创作，主要内容包括：在一种示例性方法中,针对来自基因组样品的多个文库片段生成一系列基于时间的簇生成图像。该系列中的每个基于时间的簇生成图像按顺序生成。为了生成该系列中的每个基于时间的簇生成图像：i)将相应样品引入流通池中,该相应样品包括该多个文库片段中的邻近性保留文库片段,其中该邻近性保留文库片段附接到固体支持物或彼此附接；ii)将该邻近性保留文库片段从该固体支持物或从彼此释放；iii)扩增该邻近性保留文库片段以生成多个相应的模板链；iv)将该相应的模板链染色；以及v)将该相应的模板链成像。(In one exemplary method, a series of time-based cluster-generated images are generated for a plurality of library fragments from a genomic sample. Each time-based cluster generation image in the series is generated in sequence. To generate each time-based cluster generation image in the series: i) introducing a respective sample into a flow cell, the respective sample comprising proximity-preserving library fragments of the plurality of library fragments, wherein the proximity-preserving library fragments are attached to a solid support or to each other; ii) releasing the proximity-preserving library fragments from the solid support or from each other; iii) amplifying the proximity-preserving library fragments to generate a plurality of corresponding template strands; iv) staining the corresponding template strand; and v) imaging the corresponding template strand.)

1. A method, the 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 generated sequentially by:

introducing a respective sample comprising some of the proximity-preserving library fragments to a flow cell, wherein the some of the proximity-preserving library fragments are attached to a solid support or to each other;

(ii) priming release of said some of said proximity-preserving library fragments from said solid support or from each other;

amplifying the some of the proximity-preserving library fragments to generate a plurality of corresponding template strands;

staining the corresponding template strand; and

imaging the corresponding template strand.

2. The method of claim 1, further comprising assigning a time record for each time-based cluster generation image in the series of the introduction of the respective sample comprising the some of the proximity-preserving library fragments.

3. The method of claim 2, wherein the time record is a time stamp or a number of steps in a sequence.

4. The method of one of claims 1 to 3, further comprising generating resolved clustering images for each of the respective samples using image subtraction, wherein each resolved clustering image records a spatial position and orientation of the respective template strand associated with a different one of the samples.

5. The method of claim 4, further comprising storing the resolved cluster generation image.

6. The method of claim 4 or 5, further comprising:

performing a sequencing operation on the flow cell, the flow cell comprising the respective template strand for each library fragment of the plurality of library fragments; and

combining sequencing reads together in different sets based on the resolved cluster generation images.

7. The method of claim 6, further comprising connecting the different group to each of the one of the samples based on the resolved cluster generation image.

8. The method according to one of claims 1 to 6, wherein:

prior to generating the series of time-based cluster generation images, the method further comprises:

adding a liquid carrier to the plurality of proximity-preserving library fragments to form a mixture; and

diluting the mixture with the liquid carrier to generate a predetermined number of diluted samples to be introduced into the flow cell; and is

The introducing the respective samples involves fluidically directing one of the diluted samples to the flow cell.

9. The method of claim 8, wherein the mixture comprising the plurality of proximity-preserving library fragments is diluted to a predetermined volume based on i) a volume of the flow cell as a limiting dilution and ii) the predetermined number of diluted samples.

10. The method according to one of claims 1 to 9, wherein:

attaching each proximity-preserving library fragment to the solid support; and is

The releasing involves heating.

11. The method according to one of claims 1 to 9, wherein:

each of the proximity-preserving library fragments comprises a first sequence portion at a first end that hybridizes to a first primer sequence on a surface of the flow cell; and is

Prior to amplification, the method further comprises attaching a second sequence portion to each of the hybridization proximity-preserving library fragments at a second end opposite the first end, the second sequence portion being identical to a second primer sequence on the surface of the flow cell.

12. The method according to one of claims 1 to 9, wherein:

attaching each proximity-preserving library fragment to the solid support;

the solid support has a plurality of linkers attached thereto; and is

The method further comprises preparing the proximity-preserving library fragments attached to the solid support by:

labeling the genomic sample in the presence of the solid support and a plurality of L-linkers, each L-linker comprising a transferred strand and a non-transferred strand, thereby generating a plurality of sample fragments, whereby the respective transferred strand is incorporated into the 5' -end of each sample fragment and the respective non-transferred strand is hybridized to a portion of each linker;

connecting the respective transfer strand to a respective one of the plurality of linkers;

digesting the non-transferred strand using a 5'-3' exonuclease; and

attaching a partial Y-linker to each of the transfer strands.

13. The method of claim 12, wherein ligation and digestion occur in a single, single pot protocol.

14. A method, the method comprising:

preparing a mixture comprising a plurality of proximity-preserving library fragments of a genomic sample, the plurality of proximity-preserving library fragments attached to a solid support or to each other;

diluting the mixture to generate a predetermined amount of diluted sample to be introduced into the 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 sample comprising some of the proximity-preserving library fragments into the flow cell;

(ii) priming release of said some of said proximity-preserving library fragments from said solid support or from each other;

amplifying the some of the proximity-preserving library fragments to generate a plurality of template strands;

staining the plurality of template strands; and

imaging the plurality of template strands.

15. The method of claim 14, wherein the mixture is diluted to a predetermined volume based on i) a volume of the flow cell as an ultimate dilution and ii) the predetermined amount of diluted sample to be introduced into the flow cell.

16. The method of claim 14 or 15, further comprising:

generating a second time-based cluster generation image for some other of the proximity-preserving library fragments by:

introducing a second sample of the diluted sample comprising the some other of the proximity-preserving library fragments into the flow-through cell;

(ii) priming release of the some other of the proximity-preserving library fragments from the solid support or from each other;

amplifying the some other of the proximity-preserving library fragments to generate a second plurality of template strands;

staining the second plurality of template strands; and

imaging the second plurality of template strands.

17. The method of claim 16, further comprising generating a predetermined number of time-based cluster-generated images for a predetermined number of the proximity-preserving library fragments by repeating the introducing, the priming releasing, the amplifying, the staining, and the imaging for each other dilution sample of the predetermined number of dilution samples.

18. The method of claim 17, further comprising assigning a time record of the introduction of the respective diluted sample to each of the predetermined number of time-based cluster generation images.

19. The method of claim 18, wherein the time record is a time stamp or a number of steps in a sequence.

20. The method of one of claims 17 to 19, further comprising generating a resolved cluster image for each of the diluted samples introduced into the flow cell using image subtraction, wherein each resolved cluster image records the spatial position and orientation of the plurality of template strands associated with a different one of the diluted samples.

21. The method according to one of claims 14 to 20, wherein:

attaching each proximity-preserving library fragment to the solid support;

the solid support has a plurality of linkers attached thereto; and is

The method further comprises preparing the proximity-preserving library fragments attached to the solid support by:

labeling the genomic sample in the presence of the solid support and a plurality of L-linkers, each L-linker comprising a transferred strand and a non-transferred strand, thereby generating a plurality of sample fragments, whereby the respective transferred strand is incorporated into the 5' -end of each sample fragment and the respective non-transferred strand is hybridized to a portion of each linker;

connecting the respective transfer strand to a respective one of the plurality of linkers;

digesting the non-transferred strand using a 5'-3' exonuclease; and

attaching a partial Y-linker to each of the transfer strands.

22. The method of claim 21, wherein ligation and digestion occur in a single, single pot protocol.

23. A system, the system comprising:

a flow cell receptacle;

a fluid control system comprising a fluid delivery device to deliver a diluted sample and a stain, respectively, to a flow cell positioned in the flow cell receptacle;

a lighting 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 illumination system, and the detection system, the controller:

causing the fluid deliverer to introduce the diluted sample 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 fluid delivery device to introduce the staining agent into the flow cell positioned in the flow cell receptacle;

causing the illumination system to illuminate the staining template strand in the flow cell positioned in the flow cell receptacle; and

causing the detection system to image the illuminated template strand of staining in the flow cell positioned in the flow cell receptacle.

24. The system of claim 23, wherein the flow cell receptacle is part of a sequencer that includes a heater, wherein the controller is to:

causing the heater to initiate release of the some of the proximity-preserving library fragments from a solid support or from each other;

causing the heater to run a thermal cycle to amplify the some of the proximity-preserving library fragments to generate the template strand.

25. The system according to one of claims 23 or 24, further comprising:

a first cartridge comprising the diluted sample; and

a second cartridge containing the colorant.

26. The system according to one of claims 23 to 25, further comprising an electronic storage component to store the image.

27. The system of claim 23, wherein the image is a time-based cluster generation image in 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 generated in order.

Background

There are a variety of methods and applications in which it is desirable to generate libraries of fragmented and tagged deoxyribonucleic acid (DNA) molecules from double-stranded DNA (dsdna) molecules. Generally, the goal is to generate smaller DNA molecules (e.g., DNA fragments) from larger dsDNA molecules for use as templates in DNA sequencing reactions. These templates may enable short read lengths to be obtained. Short sequence reads typically overlap with multiple other short sequence reads to provide redundant coverage over different portions of the overall longer sequence. During data analysis, longer sequence information can be stitched together using overlapping sequences from multiple reads. Thus, overlapping short sequence reads can be aligned to reconstruct longer nucleic acid sequences. In some cases, pre-sequencing steps (such as barcoding specific nucleic acid molecules) can be used to simplify data analysis.

Disclosure of Invention

A 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 generated sequentially by: introducing a respective sample comprising 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; (ii) priming release of said some of the proximity-preserving library fragments from the solid support or from each other; amplifying the ones of the proximity-preserving library fragments to generate a plurality of corresponding template strands; staining the corresponding template strand; and imaging the corresponding template strand.

A second aspect disclosed herein is a method comprising preparing a mixture comprising a plurality of proximity-preserving library fragments of a genomic sample attached to a solid support or to each other; diluting the mixture to generate a predetermined amount of diluted sample to be introduced into the 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 sample comprising some of the proximity-preserving library fragments into a flow-through cell; (ii) priming release of said 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 template strands; dyeing the plurality of template strands; and imaging the plurality of template strands.

A third aspect disclosed herein is a system comprising: a flow cell receptacle; a fluid control system including a fluid delivery device to deliver the diluted sample and the stain, respectively, to a flow cell positioned in the flow cell receptacle; a lighting system positioned to illuminate a flow cell positioned in the flow cell receptacle; a detection system positioned to capture an image of a flow-through cell positioned in the flow-through cell receptacle; and a controller in operable communication 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 a flow cell positioned in the flow cell receptacle; after generating template strands from the proximity-preserving library fragments present in the diluted sample in a flow-through cell positioned in the flow-through cell receptacle, causing a fluid deliverer to introduce a staining agent into the flow-through cell positioned in the flow-through cell receptacle; causing the illumination system to illuminate a chain of staining templates in a flow cell positioned in the flow cell receptacle; and causing the detection system to image the illuminated dyed template strand positioned in the flow cell receptacle.

It should be understood that any features of the first and/or second methods and/or systems 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 described as the present disclosure, including, for example, using parsed cluster images to identify a particular set of template chains.

Drawings

Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, but possibly different, components. For the sake of brevity, reference numerals or features having a previously described function may or may not be described in connection with other drawings in which they appear.

FIG. 1 is a schematic diagram of an example of a method for preparing complexes comprising proximity-preserving library fragments attached to a solid support;

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

FIG. 3 is a schematic illustration of the ligation and digestion of the processes of FIGS. 2A-2C occurring in a single, single pot reaction;

FIG. 4 is a schematic diagram of an example of a portion of a library preparation process to generate attached proximity-preserving library fragments;

FIG. 5A is a top view of an example of a flow cell;

FIG. 5B is an enlarged partial cross-sectional view of an example of a flow channel of a flow cell;

FIG. 5C is an enlarged partial cross-sectional view of another example of a flow channel of a flow cell;

6A-6D are schematic diagrams of several steps in a method for generating a series of time-based cluster-generated images;

FIG. 7 is a schematic of an image taken after cluster generation is performed on different samples (on the left side of FIG. 7), and is a schematic of a resolved cluster image generated for each of the samples (on the right side of FIG. 7);

FIG. 8 is a schematic diagram of an example of a portion of a library preparation process that occurs on a flow cell utilizing the attached proximity-preserving library fragments of FIG. 4;

FIGS. 9A-9C are schematic diagrams of several steps in a method for generating a series of time-based cluster-generated images;

FIG. 10 is an original color screen shot from an Integrated Genomics Viewer (IGV) browser rendered in black and white depicting the results for the AT-rich region of the INTS4P1 gene when the non-transfer strand is removed using heat denaturation and when the non-transfer strand is removed using T7 exonuclease conditions; and is

FIG. 11 is a graph depicting fluorescence (Y-axis, fluorescence units) versus size (X-axis, base pairs) of PCR amplified library fragments generated by the methods shown in FIGS. 2A-2C and 3.

Detailed Description

Library fragments are pieces of deoxyribonucleic acid (DNA) of similar size (e.g., <1000bp) of 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 segments of different lengths may be desirable in order to achieve sub-haploid genomic content within each compartment of the synthetic long reads. Synthesis of long reads (or linked short reads) is enabled when multiple short fragments can be combined together based on the identification of the compartment from which the long DNA fragment originated. Compartmentalization has been physically achieved, for example, using wells, beads, droplets, or other physical compartments.

The common principle for all these compartmentalization methods is: barcode sequences or index sequences are used to identify the compartments from which long DNA fragments originate. The barcode sequence attached to each of the shorter fragments can be unique to a particular long DNA fragment, and thus can facilitate labeling of different compartments during library preparation. It is a barcode sequence that is used to combine short reads together to form a synthetic long read based on the assumption that the short reads all originate from the same compartment.

The exemplary methods disclosed herein enable compartmentalization of different library fragments without the need to incorporate unique barcode sequences. The method utilizes proximity-preserving library fragments and imaging to form a series of time-based cluster-generated images. Each time-based cluster generation image can be used to identify the samples (compartments) that generate a particular set of template chains. In addition, each sequence read can be combined using time-based cluster generation images. This combination allows read linkage, thereby enabling the reconstruction of long DNA fragments.

Definition of

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

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.

Reference throughout this specification to "one example," "another example," "an example," etc., means that a particular element (e.g., feature, structure, composition, configuration, and/or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. Furthermore, it should be understood that the elements described for any example may be combined in any suitable manner in the various examples, unless the context clearly dictates otherwise.

The terms "substantially" and "about" are used throughout this disclosure (including the claims) to describe and describe small fluctuations, such as due to variations in processing. For example, these terms may refer to less than or equal to ± 5% of the stated value, such as less than or equal to ± 2% of the stated value, such as less than or equal to ± 1% of the stated value, such as less than or equal to ± 0.5% of the stated value, such as less than or equal to ± 0.2% of the stated value, such as less than or equal to ± 0.1% of the stated value, such as less than or equal to ± 0.05% of the stated value.

And (3) jointing: the linear oligonucleotide sequence fused to the nucleic acid molecule may be, for example, linked or labeled. Suitable linker lengths may range from about 10 nucleotides to about 100 nucleotides or from about 12 nucleotides to about 60 nucleotides or from about 15 nucleotides to about 50 nucleotides. The linker may comprise any combination of nucleotides and/or nucleic acids. In some examples, the linker can include a sequence that is complementary to at least a portion of a primer (e.g., a primer that includes a universal nucleotide sequence, such as a P5 or P7 sequence). For example, the adapter at one end of the fragment comprises a sequence complementary to at least a portion of the first flow-through pool primer, and the adapter at the other end of the fragment comprises a sequence identical to at least a portion of the second flow-through pool primer. A complementary linker can hybridize to the first flowcell primer and the same linker is a template for its complementary copy, which can hybridize to the second flowcell primer during cluster generation. In some examples, the linker may comprise a sequencing primer sequence or a sequencing binding site. Combinations of different linkers can be incorporated into a nucleic acid molecule (such as a DNA fragment).

Capture site: a portion of the flow cell surface that has been physically modified and/or modified with a chemical property that allows for the localization of the complex. In one example, the capture site can include a chemical capture agent (i.e., a material, molecule, or moiety capable of attaching, retaining, or binding to a target molecule (e.g., a complex)). One 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 linking moiety attached to a target molecule). Yet another example of a chemical capture agent is a chemical agent capable of forming an electrostatic interaction, hydrogen bond, or covalent bond with the complex (e.g., thiol-disulfide exchange, click chemistry, Diels-Alder, etc.).

The compound is as follows: a carrier, such as a solid support, and ready-to-use sequencing nucleic acid fragments attached to the carrier. The vector may also comprise one member of a binding pair, the other member of which is part of the capture site.

Fragment (b): a portion or piece of genetic material (e.g., 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 manner (e.g., by beads, with transposomes, etc.).

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

A "template" nucleic acid molecule (or strand) can refer to a sequence to be analyzed.

Nucleotides in a nucleic acid sample may include naturally occurring nucleic acids and functional analogs thereof. Examples of functional analogs can hybridize to nucleic acids in a sequence-specific manner or can serve as templates for replicating a particular nucleotide sequence. Naturally occurring nucleotides typically have a backbone containing phosphodiester linkages. Similar structures may have alternate backbone linkages, including any of a variety of backbone linkages known in the art. Naturally occurring nucleotides typically have a deoxyribose sugar (e.g., present in DNA) or a ribose sugar (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 unnatural bases. The native DNA may include one or more of adenine, thymine, cytosine, and/or guanine, and the native RNA may include one or more of adenine, uracil, cytosine, and/or guanine. Any non-natural base may be used, such as Locked Nucleic Acid (LNA) and Bridged Nucleic Acid (BNA).

Primer: a nucleic acid molecule that can hybridize to the target sequence, such as a linker attached to a proximity-preserving library fragment. For example, amplification primers can be used as a starting point for template amplification and cluster generation. As another example, a synthesized nucleic acid (template) strand can include a site to which a primer (e.g., a sequencing primer) can hybridize in order to prime synthesis of a new strand complementary to the synthesized nucleic acid (template) strand. Any primer may include any combination of nucleotides or analogs thereof. In some examples, the primer is a single stranded oligonucleotide or polynucleotide. The primer length can be any number of bases in length and can include a variety of natural and/or non-natural nucleotides. In one example, the sequencing primer is a short strand ranging from 10 to 60 bases, or 20 to 40 bases.

I.e. sequencing nucleic acid fragments: the genetic material has a portion of the linker at the 3 'and 5' ends (e.g., a proximity-preserving library fragment). In a ready-to-sequence nucleic acid fragment, 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. That is, the sequencing nucleic acid fragments can be bound via insertion of 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.

Solid support: small bodies made of rigid or semi-rigid material, the shape of which is characterized by, for example, a spherical, ellipsoidal, microspherical or other generally recognized particle shape, whether of regular or irregular size. The solid support can have a sequencing library attached thereto. Exemplary materials that can be used for the solid support include, but are not limited to, glass; plastics, such as acrylic, polystyrene or copolymers of styrene with another material, polypropylene, polyethylene, polybutylene, polyurethane or polytetrafluoroethylene (from The Chemours Co)) (ii) a Polysaccharides or cross-linked polysaccharides, such as agarose or sepharose; nylon; nitrocellulose; a resin; silica or silica-based materials, including silicon and modified silicon; carbon fibers, metals; inorganic glass; fiber optic strands, or a variety of other polymers. Exemplary solid supports include controlled pore glass beads, paramagnetic beads, thoria sol, and the like,Beads (agarose in the form of cross-linked beads, available from Cytivia), nanocrystals and other solid supports known in the art as described, for example, in Microsphere Detection Guide from Bangs Laboratories, Fishers industries.

A base body is rotated: a complex formed between an integrase (e.g., a integrase or transposase) and a transferable strand, a non-transferable strand, or both a transferable strand and a non-transferable strand.

Proximity preserving library fragments

In examples disclosed herein, the library fragments introduced into the flow-through 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, a solid support can be used in library preparation to preserve proximity. In other examples disclosed herein, proximity may be preserved by: fragments attached to each other are formed by the bound transposomes during initial library preparation, the attached fragments are introduced into the flow cell, and library preparation is then completed on the flow cell.

FIG. 1 depicts an example of a method for forming a complex 10 comprising ready-to-use sequencing nucleic acid fragments 12, including fragments 14 from a larger nucleic acid sample, whose proximity is preserved on a solid support 16.

In one exemplary method for forming the complex 10 shown in FIG. 1, the linker sequence 18 is bound to the solid support 16 via one member 20 of a binding pair. In one example, the adapter sequence 18 can include a first sequencing primer sequence (e.g., a 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 (as shown in fig. 5A and 5B). The linker sequence 18 is bound to one member 20 of a binding pair (e.g., biotin) such that it can bind to the surface of the solid support 16 that includes the other member of the binding pair (e.g., avidin, streptavidin, etc.).

As shown in fig. 1, transposome complexes 24 "can also be bound to the solid support 16. Prior to loading transposome complexes 24 "onto the solid support 16, a portion of the Y-linker 25' can be ligated to transposase 32 (although not shown, it is not shown)May include two Tn5 molecules) to form transposome complex 24 ". Part of the Y-linker 25' may comprise two mosaic terminal sequences M which hybridize to each other₁、M₂. One of the mosaic terminal sequences M₁Has free ends that can be attached to fragmented DNA strands during labeling, and is therefore similar to the transferred strand 26 in fig. 2A-2C. Another mosaic terminal sequence M of the mosaic terminal sequences₂Can be attached to a second sequencing primer sequence (e.g., a read 2 sequencing primer sequence) and a second sequence (P7) having a sequence identical to at least a portion of another of the amplification primers (P7) 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 M of the mosaic end sequences₂The second sequencing primer sequence and the second sequence constitute an adapter sequence 22. The linker sequence 22 is not attached to the fragmented DNA strand during labeling and is therefore similar to the non-transferred strand 28 in fig. 2A to 2C.

Loading the transposome complexes 24 "onto the solid support 16 can involve mixing the transposome complexes 24" with the solid support 16 and exposing the mixture to the mosaic terminus M for the portion of the Y-linker 25₁Suitable conditions for ligation to the 3' end of linker sequence 18. As shown in fig. 1, individual transposome complexes 24 "can be attached to each of the linker sequences 18 on the solid support 16.

In this exemplary method to form the composite 10, a labeling process may then be performed. A fluid (e.g., a labeling buffer) comprising a longer nucleic acid sample 30 (e.g., DNA) can be added to the solid support 16 having the linker sequence 18 and transposome complex 24 "bound thereto. When the sample contacts 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 at its 5' end with a mosaic end M of a partial Y-linker 25₁Tagging of the free end of (a).

As shown in FIG. 1, labeling of longer nucleic acid samples 30 results in multiple bridging molecules between transposome complexes 24 ″. The bridging molecule is wrapped around the solid support 16. The transposome complexes 24 "and the linker sequences 18 act as bridging molecules to maintain the proximity of the nucleic acid sample 30, and thus the bridging molecules are the proximity-preserving library fragments 14, 14'.

Transposases can then be removed via Sodium Dodecyl Sulfate (SDS) treatment or heating or proteinase K digestion. Removal of the transposase leaves the proximity-preserving library fragments 14, 14' attached to the solid support 16.

To complete the ready-to-use sequencing fragment, further extension and ligation (indicated by an asterisk in fig. 1) is performed to ensure that fragments 14 and 14' are attached to sequence 22. The resulting composite 10 is shown in fig. 1.

Each proximity-preserving library fragment 14, 14 'is part of a respective ready-to-sequence nucleic acid fragment 12, 12', each of which further includes a respective linker sequence 18 and 22 attached at either end. The adaptor sequences 18 are those that are initially bound to the solid support 16 and include a first sequencing primer sequence and a first sequence that is complementary to one of the flow cell primers. The linker sequence 18 is attached to one member 20 of the binding pair. The linker sequence 22 is from part Y-linker 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 the appropriate linkers for bridge amplification and sequencing, no PCR amplification is performed. These fragments 12, 12' are thus ready-to-use sequenced. In addition, because the proximity-preserving library fragments 14, 14 'are derived from the same longer nucleic acid sample 30, the proximity-preserving library fragments 14, 14' may be suitable for use in the linked long-read applications disclosed herein.

Another exemplary method for forming another exemplary complex 10' (fig. 2C) is depicted in fig. 2A-2C.

In this exemplary method, the linker sequence 18' is bound to the solid support 16 via one member 20 of a binding pair. In one example, the linker sequence 18 'can 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 linker sequence 18' is bound to one member 20 of a binding pair (e.g., biotin) such that it can bind to the surface of the solid support 16 that includes the other member of the binding pair (e.g., avidin, streptavidin, etc.).

As shown in fig. 2A, transposome complexes 24 can also be bound to the solid support 16. Prior to loading transposome complexes 24 onto solid support 16, L-linker 21 can be mixed with transposase 32 (e.g., comprising two Tn5 molecules) to form an illustration of transposome complexes 24. L-linker 21 may comprise two mosaic terminal sequences M hybridized to each other₁、M₂. One of the mosaic terminal sequences M₁Is 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. Another mosaic terminal sequence M of the mosaic terminal sequences₂Is part of the non-transferred strand 28 that is removed after the ligation and labeling process. The mosaic terminal sequence M₂To a complementary hybridizable sequence HC that is complementary to the hybridizable sequence H of the linker sequence 18' attached to the solid support 16. The complementary hybridizable sequence HC enables hybridization of the L-linker 21 to the linker sequence 18'. Thus, the complementary hybridizable sequence HC allows transposome complex 24 to be loaded onto a solid support.

Loading the transposome complexes 24 onto the solid support 16 can involve mixing the transposome complexes 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'. As shown in fig. 2A, each transposome complex 24 can be attached to each of the linker sequences 18' on the solid support 16.

In this exemplary method to form the complex 10', a labeling process is then performed. A fluid (e.g., a labeling buffer) comprising a longer nucleic acid sample (e.g., DNA)30 can be added to the sample having loaded thereonSolid support 16 of transposome complex 24. When the sample 30 contacts the solid support-bound transposome complexes 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 at its 5' end with a mosaic end sequence M of L-linkers 21₁And (4) labeling.

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

Transposase 32 can then be removed via Sodium Dodecyl Sulfate (SDS) treatment or heating or proteinase K digestion.

Ligation can then be performed to free the mosaic terminal sequence M₁Binding to the corresponding linker sequence 18'. In fig. 2A, an asterisk indicates where the connection is performed. In one example, the connection may be initiated by: a buffer containing a suitable ligase is introduced and heated to about a suitable temperature for a suitable time to initiate enzyme activity. Examples of suitable ligases include E.coli (E.coli) DNA ligase, T7 ligase, and the like. In one example, the buffer containing E.coli DNA ligase can also include Nicotinamide Adenine Dinucleotide (NAD)⁺). In this example, heating to about 16 ℃ for about 15 minutes elicits enzyme activity. The resulting structure is shown in fig. 2B.

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

The use of exonuclease to remove the non-transferred strand 28 may be preferable to the use of heat denaturation. The 5'-3' exonuclease 23 effectively digests the nontransferred strand 28, thereby creating a more purified template (than when using heat denaturation) for subsequent hybridization of the attached linker sequence (see reference numeral 22 in fig. 2C). This can improve library yield. In addition, removal of the non-transferred strand 28 via 5'-3' exonuclease 23 can increase library coverage over adenine (a) and thymine (T) rich regions, the loss of which has been observed after thermal denaturation of the non-transferred strand 28 (see fig. 11). Furthermore, digestion via exonuclease does not increase the time of the overall library preparation process.

Referring now to fig. 2C, an example of this method to form the complex 10' involves the introduction of a moiety Y-linker 25. Part of Y-linker 25 comprises a sequence M with a mosaic terminus₁Complementary mosaic terminal sequences M₃And a linker sequence 22. The adapter 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 of the amplification primers on the flow cell (P7). As shown in FIG. 2C, the mosaic terminal sequence M of the partial Y-junction 25₃Mosaic terminal sequence M with transfer strand 26₁Hybridization (now to linker sequence 18') thus attaches a portion of the Y-linker 25 to the solid support 16.

In examples disclosed herein, linker sequences 18, 18', and/or 22 may also include sequencing sample indices or barcode sequences. These sequences can be used as backups or alternatives to the compartmentalization methods disclosed herein.

To generate ready-to-use sequencing fragments, further extension and ligation were performed to ensure that fragments 14 and 14' were attached to mosaic terminal sequence M₃And thus to the sequence 22.

The resulting composite 10' is shown in fig. 2C.

Another exemplary method for forming a composite 10' (shown in fig. 2C) is partially depicted in fig. 3. In this exemplary method, ligation of the transfer strand 26 and digestion of the non-transfer strand 28 are performed as part of a single, single-pot protocol.

In this exemplary method, the linker sequence 18' is bound to the solid support 16 via one member 20 of a binding pair. An adapter sequence 18 'comprising a hybridizable sequence H, a first sequencing primer sequence (e.g., read 1 sequencing primer sequence), and a first sequence (P5) 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) as described with reference to fig. 2A can be used. Also in this exemplary method, transposome complexes 24 are loaded onto the solid support 16, as described with reference to fig. 2A. Briefly, the complementary hybridizable sequence HC of the L-linker 21 of the transposome complex 24 hybridizes to the hybridizable sequence H of the linker sequence 18'.

In this exemplary method to form the complex 10' (fig. 2C), labeling is performed as described with reference to fig. 2A.

Ligation of the transferred strand 26 and digestion of the non-transferred strand 28 may then be performed together. This is schematically illustrated in fig. 3. In order for the ligase and exonuclease to act synergistically, the reagent formulation introduced into the labeled solid support includes buffer, ligase, 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: the reagent formulation was introduced and heated to about 25 ℃ for about 15 minutes to initiate enzyme activity.

Incorporating both ligase and exonuclease into the same reagent formulation may reduce protocol time (e.g., by about 10 minutes compared to the exemplary method shown in fig. 2A-2C), and also reduce the number of washing steps.

This exemplary method then continues with the introduction of a portion of the Y-linker 25, as described with reference to fig. 2C. To generate the ready-to-use sequenced fragments and final complex 10', further extension and ligation is performed to ensure that fragments 14 and 14' are attached to the mosaic terminal sequence M₃And thus to the sequence 22.

The methods for preparing the complexes 10, 10 'described with reference to fig. 1, fig. 2A-fig. 2C, and fig. 3 provide some examples, but it should be understood that other methods may be used as long as they are attached to the solid support 16 with the sequenced nucleic acid fragments 12, 12'.

In other examples disclosed herein, the proximity information may be retained by performing a portion of library preparation from and on the flow cell.

In this example, library preparation can be initiated outside the flow cell using a label, as schematically shown in fig. 4.

In the example shown, a fluid (e.g., a labeling buffer) comprising a longer nucleic acid sample 30 (e.g., double-stranded DNA) may be mixed with the transposome complexes 24'. In the example shown in fig. 4, each transposome complex 24' is a dimer, including a transfer strand 26', a non-transfer strand 28', and two transposases (collectively referred to as "32" in fig. 4). In other examples, different transposome complexes may be used, e.g., one of the transposome complexes includes transposase 32 and transfer strand 26', and another of the transposome complexes includes transposase 32 and non-transfer strand 28'.

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

In this example, the non-transferred strand 28' is a linker such that: which is not incorporated into the fragments 14, 14 'during labeling, but can subsequently be ligated to the other end of each fragment 14, 14'. As shown in fig. 4, the non-transferred strand 28 'may be attached to the transferred strand 26' during labeling via at least partial base pairing. In this example, the non-transferred strand 28 'is an adapter that 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 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).

As shown in fig. 4, in a fluid, transposomes 24' fragment longer nucleic acid samples 30 into fragments 14, 14' and ligate transfer strands 26' to the 5' ends of each fragment 14, 14 '. In one example, the transfer 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 'may be attached to the transferred strand 26' via base pairing.

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

As mentioned herein, the attachment fragment 34 can be introduced into a flow cell where additional processing can be performed to complete library preparation. This is schematically illustrated in fig. 8 and will be further described with reference to the methods disclosed herein.

Flow cell

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

The substrate 38 may be a single layer/material. Examples of suitable single layer substrates include: epoxysiloxanes, glass, modified or functionalized glass, plastics (including acrylic, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene (such as that available from Chemours)) Cycloolefin/cycloolefin polymers (COP) (such as those from Zeon)) Polyimide, etc.), nylon (polyamide), ceramic/ceramic oxide, silicon dioxide, fused or silicon dioxide-based materials, aluminum silicate, silicon and modified silicon (e.g., boron doped p + silicon), silicon nitride(Si₃N₄) Silicon oxide (SiO)₂) Tantalum pentoxide (Ta)₂O₅) Or other tantalum oxide (TaO)_x) Hafnium oxide (HaO)₂) Carbon, metal, inorganic glass, and the like. The 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) having a patterned resin thereon. Other examples of multilayer substrates may include silicon-on-insulator (SOI) substrates.

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

In the example shown in fig. 5A, the flow cell 36 includes a flow channel 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 the substrate 36 and a cover or between two substrates 36) that may have a fluid (e.g., those described herein) introduced therein and removed therefrom. Each flow channel 40 may be separated from each other flow channel 40 such that fluid introduced into any particular flow channel 40 does not flow into any adjacent flow channel 40. Some examples of fluids introduced into flow channel 40 may introduce reaction components (e.g., proximity-preserving library fragments 14, 14' (e.g., located on solid support 16 or attached to each other), polymerases, sequencing primers, nucleotides, etc.), wash solutions, deblocking agents, etc.

The flow channels 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 channels 40 are etched into the glass substrate 38. In another example, flow channels 40 may be patterned into the resin of multilayer substrate 38 using photolithography, nanoimprint lithography, and the like. 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.

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, such that the portion of the substrate surface surrounding the flow channel 40 may be used for attachment to a cover (not shown) or another substrate 38. In some cases, the width of each flow channel 40 may be at least about 1mm, at least about 2.5mm, at least about 5mm, at least about 7mm, at least about 10mm, or greater. In some cases, the length of each channel/flow channel 40 may be at least about 10mm, at least about 25mm, at least about 50mm, at least about 100mm, or greater. The width and/or length of each flow channel 40 may be greater than, less than, or intermediate to the values specified above. In another example, the flow channels 40 are square (e.g., 10mm by 10 mm).

When microcontact, aerosol or inkjet printing is used to deposit the individual materials defining the flow channel walls, the depth of each flow channel 40 may be as small as a monolayer thick. The depth may be greater when a separate material (not shown) is used to bond the cover to the base 38 or to bond two bases 38 together. For other examples, the depth of each flow channel 40 may be about 1 μm, about 10 μm, about 50 μm, about 100 μm, or greater. In one example, the depth may be in a range of about 10 μm to about 100 μm. In another example, the depth may be in a 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 intermediate to the values specified above.

Different examples of architectures within the flow channel 40 of the flow cell 36 are shown in fig. 5B and 5C.

In the example shown in fig. 5B, 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.

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-azidoacetamidylpentyl) acrylamide-co-acrylamide) -PAZAM. Some other forms of PAZAM and acrylamide copolymers are represented by the following structure (I):

wherein:

R^Aselected 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;

R^Bis H or optionally substituted alkyl;

R^C、R^Dand R^EEach independently selected from H and optionally substituted alkyl;

-(CH₂)_p-each of which may be optionally substituted;

p is an integer in the range of 1 to 50;

n is an integer ranging from 1 to 50,000; and

m is an integer ranging from 1 to 100,000.

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

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

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 crosslinked polymers.

In other examples, the polymer hydrogel 42 may be a variation of structure (I). In one example, the acrylamide units may be N, N-dimethylacrylamideAnd (6) replacing. In this example, the acrylamide units in Structure (I) are usefulIn the alternative, wherein R^D、R^EAnd R^FEach is H or C1-C6 alkyl, and R^GAnd R^HEach being a C1-C6 alkyl group (instead of 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, N-dimethylacrylamide may be used in addition to the acrylamide units. In this example, structure (I) may include, in addition to the recurring "n" and "m" featuresWherein R is^D、R^EAnd R^FEach is H or C1-C6 alkyl, and R^GAnd R^HEach is a C1-C6 alkyl group. In this example, q may be an integer in the range of 1 to 100,000.

As another example of an initial polymeric hydrogel, the recurring "n" feature in structure (I) may be replaced with a monomer comprising a heterocycloazide group having structure (II):

wherein R is¹Is H or C1-C6 alkyl; r₂Is H or C1-C6 alkyl; l is a linking group comprising a straight chain having 2 to 20 atoms selected from carbon, oxygen and nitrogen and 10 optional atoms on carbon and any nitrogen atom in the chainA substituent group; e is a straight chain comprising 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atom 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 fused structure.

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

wherein R is^1a、R^2a、R^1bAnd R^2bEach of which is independently selected from hydrogen, optionally substituted alkyl or optionally substituted phenyl; r^3aAnd R^3bEach of which is independently selected from hydrogen, optionally substituted alkyl, optionally substituted phenyl or optionally substituted C7-C14 aralkyl; and each L¹And L²Independently selected from an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker.

It should be understood that other molecules may be used to form the polymer hydrogel 42, so long as they are functionalized to graft the oligonucleotide primers 44, 46 thereto. Other examples of suitable polymer layers include those having a colloidal structure, such as agarose; or those having a polymer network structure, such as gelatin; or those having a cross-linked polymer structure, such as polyacrylamide polymers and copolymers, silane-free acrylamide (SFA), or an azide version 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] cycloaddition reaction. Still other examples of suitable polymer hydrogels 42 include mixed copolymers of acrylamide and acrylate. A variety of polymer architectures containing acrylic monomers (e.g., acrylamides, acrylates, and the like) can be used in the examples disclosed herein, such as branched polymers, including star polymers, star or star block polymers, dendrimers, and the like. For example, monomers (e.g., acrylamide, catalyst-containing acrylamide, etc.) can be incorporated into the branches (arms) of the star polymer in a random or block fashion.

To introduce the polymer hydrogel 42 into the flow channel 40, a mixture of the polymer hydrogel 42 may be created 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., with water or with ethanol and water). The mixture may then be applied to the substrate surface (including in the flow channels 40) using spin coating, dipping or dip coating, spray coating, material flow at positive or negative pressure, or another suitable technique. These types of techniques deposit the polymer hydrogel 42 blanket over the substrate 38A (e.g., in the flow channels 40 and over the interstitial regions 48 surrounding the flow channels 40). Other selective deposition techniques (e.g., involving masks, controlled printing techniques, etc.) may be used to specifically deposit the polymer hydrogel 42 in the flow channel 40 rather than on the interstitial regions 48.

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

Depending on the polymer hydrogel 42, the applied mixture may be exposed to a curing process. In one example, curing may be performed at a temperature in the range of room temperature (e.g., about 25 ℃) to about 95 ℃ for a time in the range of about 1 millisecond to about several days.

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

Flow cell 36 also includes amplification primers 44, 46.

A grafting process may be performed to graft the amplification primers 44, 46 to the polymer hydrogel 42 in the flow channel 40. In one example, the amplification primers 44, 46 may be immobilized to the polymer hydrogel 42 by a single point covalent attachment at or near the 5' ends of the primers 44, 46. This attachment leaves i) the linker-specific portion of the primers 44, 46 free to anneal to their cognate nucleic acid fragments (e.g., the portion of P5' attached to the fragments 14, 14 '), and ii) the 3' hydroxyl group free for primer extension. Any suitable covalent attachment may be used for this purpose. Examples of blocked primers that can be used include alkyne-blocked primers that can attach 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 a commercial flow cell sold by Illumina inc^TM、HISEQX^TM、MISEQ^TM、MISEQDX^TM、MINISEQ^TM、NEXTSEQ^TM、NEXTSEQDX^TM、NOVASEQ^TM、GENOME ANALYZER^TM、ISEQ^TMAnd other instrument platforms.

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 the 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, which may include primers 44, 46; water; a buffer solution; and a catalyst. With either of the grafting methods, the primers 44, 46 react with the 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 selectively graft to the polymer hydrogel 42 in the flow channel 40.

In the example shown in fig. 5C, flow cell 38 includes a multilayer substrate 38B that includes a support 52 and a patterned material 50 positioned on support 52. Patterned material 50 defines recesses 54 separated by gap regions 48. A recess 54 is positioned within each of the flow channels 40.

In the example shown in fig. 5C, patterned material 50 is positioned on support 52. It should be understood that any material that may be selectively deposited or that may be deposited and patterned to form recesses 54 and gap regions 48 may be used as patterning material 50.

For example, the inorganic oxide may be selectively applied to support 52 via vapor deposition, aerosol printing, or inkjet printing. Examples of suitable inorganic oxides include tantalum oxide (e.g., Ta)₂O₅) Alumina (e.g., Al)₂O₃) Silicon oxide (e.g. SiO)₂) Hafnium oxide (e.g., HfO)₂) And the like.

As another example, the resin may be applied to support 52 and then patterned. Suitable deposition techniques include chemical vapor deposition, dip coating, bubble coating, spin coating, spray coating, whipped dispensing, ultrasonic spray coating, knife coating, aerosol printing, screen printing, microcontact printing, and the like. Suitable patterning techniques include photolithography, nanoimprint lithography (NIL), imprint techniques, embossing techniques, molding techniques, microetching techniques, printing techniques, and the like. Some examples of suitable resins include polyhedral oligomeric silsesquioxane resin (POSS) -based resins, 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., from Bellex)) And combinations thereof.

As used herein, the term "polyhedral oligomeric silsesquioxane" (POSS) refers to a silicon dioxide (SiO)₂) And organosilicon (R)₂SiO) between two or more different hybridization intermediates (e.g., RSiO)_1.5) The chemical composition of (1). An example of a POSS may be the POSS described by Kehagias et al in Microelectronic Engineering 86(2009), pp 776-778, which is incorporated by reference in its entirety. In one example, the composition is a compositionHas the chemical formula [ RSiO_3/2]_nWherein the R groups may be the same or different. Exemplary R groups of POSS include epoxy, azide/azide, thiol, poly (ethylene glycol), norbornene, tetrazine, acrylate and/or methacrylate, or additionally, for example, alkyl, aryl, alkoxy, and/or haloalkyl groups. The resin compositions disclosed herein may include one or more different cage or core structures as monomeric units. The polyhedron structure may be T₈Structures, such as:and is represented by the following formula:the monomer unit generally has eight functional arms R₁To R₈。

The monomer unit may have a cage structure with 10 silicon atoms and 10R groups, referred to as T₁₀Such as:or the monomer unit may have a cage structure with 12 silicon atoms and 12R groups, referred to as T₁₂Such as:POSS-based materials can alternatively include T₆、T₁₄Or T₁₆A 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.

As shown in fig. 5C, patterned material 50 includes recesses 54 defined therein and void regions 48 separating adjacent recesses 54. Many different layouts of the recesses 54 are contemplated, including regular, repeating, and non-regular patterns. In one example, the indentations 54 are arranged in a hexagonal grid for tight packing and improved density. Other layouts may include, for example, rectilinear (rectangular) layouts, triangular layouts, and the like. In some examples, the layout or pattern may be in an x-y format with recesses 54 in rows and columns. In some other examples, the layout or pattern may be a repeating arrangement of recesses 54 and/or interstitial regions 48. In still other examples, the layout or pattern may be a random arrangement of recesses 54 and/or interstitial regions 48. The pattern may include dots, pads, bars, swirls, lines, triangles, rectangles, circles, arcs, checkers, lattices, diagonals, arrows, squares, and/or cross-hatching.

The layout or pattern of the recesses 54 may be characterized relative to the density of the recesses 54 (the number of recesses 54) in a defined area. For example, the indentations 54 may be approximately 2 million/mm²The density of (a) exists. The density can be adjusted to different densities, including, for example, about 100/mm²About 1,000 pieces/mm²About 0.1 million/mm²About 1 million/mm²About 2 million/mm²About 5 million/mm²About 1 million/mm²About 5 million/mm²Or a greater or lesser density. It should also be understood that the density of recesses 54 in patterned material 50 may be between one value selected from the lower and one value selected from the upper values of the ranges described above. For example, a high density array may be characterized as having indentations 54 spaced less than about 100nm apart, a medium density array may be characterized as having indentations 54 spaced from about 400nm to about 1 μm apart, and a low density array may be characterized as having indentations 54 spaced greater than about 1 μm apart. While exemplary densities have been provided, it should be understood that any suitable density may be used. The density of the recesses 54 may depend in part on the depth of the recesses 54. In some cases, it is desirable that the spacing between the recesses 54 be even greater than the examples listed herein.

The layout or pattern of the recesses 54 may also or alternatively be based on an average pitch or spacing from the center of a recess 54 to the center of an adjacent recess 54 (center-to-center spacing) or from the edge of one recess 54 to the edge of an adjacent recess 54 (edge-to-edge spacing). The pattern may be regular such that the coefficient of variation around the average pitch is small, or the pattern may be irregular, in which case the coefficient of variation may be relatively large. In either case, the average pitch can be, for example, about 50nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm or greater or less. The average pitch of the specific pattern of the concave portions 54 may be between one value selected from the lower limit values and one value selected from the upper limit values of the above ranges. In one example, the recesses 54 have a pitch (center-to-center spacing) of about 1.5 μm. While an exemplary average pitch value has been provided, it should be understood that other average pitch values may be used.

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

Each recess 54 may have any volume capable of confining a fluid. The minimum or maximum volume may be selected, for example, to accommodate the desired throughput (e.g., multiplexing degree), resolution, nucleotide or analyte reactivity for use downstream of the flow cell 38. For example, the volume can be at least about 1X 10^-3μm³At least about 1X 10^-2μm³At least about 0.1 μm³At least about 1 μm³At least about 10 μm³At least about 100 μm³Or larger. Alternatively or additionally, the volume may be up to about 1 x 10⁴μm³Up to about 1X 10³μm³Up to about 100 μm³Up to about 10 μm³Up to about 1 μm³Up to about 0.1 μm³Or smaller.

The area occupied by each recess opening may be selected based on criteria similar to those described above for volume. For example, the area of each recess opening may be at least about 1 × 10^-3μm²At least about 1X 10^-2μm²At least about 0.1 μm²At least about 1 μm²At least about 10 μm²At least about 100 μm²Or larger. Alternatively or additionally, the area may be up to about 1 × 10³μm²Up to about 100 μm²Up to about 10 μm²Up to about 1 μm²Up to about 0.1 μm²Up to about 1X 10^-2μm²Or smaller. Each timeThe area occupied by the individual recess openings may be greater than, less than, or intermediate to the values specified above.

The depth of each depression 54 may 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 greater. Alternatively or additionally, the depth may be up to about 1 × 10³μ m, up to about 100 μm, up to about 10 μm, or less. In some examples, the depth is about 0.4 μm. The depth of each recess 54 may be greater than, less than, or intermediate to the values specified above.

In some cases, the diameter or length and width of each depression 54 may be at least about 50nm, 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 additionally, the diameter or length and width may be up to about 1 × 10³μ m, up to about 100 μm, up to about 10 μm, up to about 1 μm, up to about 0.5 μm, up to about 0.1 μm, or less (e.g., about 50 nm). In some examples, the diameter or length and width is about 0.4 μm. The diameter or length and width of each recess 54 may be greater than, less than, or intermediate to the values specified above.

In the example shown in fig. 5C, the polymer hydrogel 42 is positioned within each of the recesses 54. The polymer hydrogel 42 may be applied as described with reference to fig. 5B such that the polymer hydrogel 42 is present in the indentations 54 and not on the surrounding interstitial regions 48.

Although not shown in fig. 5A, 5B, or 5C, it should be understood that the flow cell 36 may also include a lid 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.).

The cover may 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, or the like. Is suitable forA commercially available example of borosilicate glass of (a) is D available from Schott North America, incA commercially available example of a suitable plastic material (i.e., a cyclic olefin polymer) is available from Zeon Chemicals l.pAnd (5) producing the product.

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

In other examples, flow cell 36 may also include additional patterned or unpatterned substrates 38 attached to substrate 38. The substrate 38 may be bonded as described herein.

Method

The method generally includes generating a series of time-based cluster generation images for a plurality of proximity-preserving library fragments 14, 14' from a genomic sample. Each time-based cluster generation image in the series is sequentially generated by: introducing a respective sample comprising some of the proximity-preserving library fragments 14, 14 'to the flow cell 36, wherein the some of the proximity-preserving library fragments 14, 14' are attached to the solid support 16 (fig. 1) or to each other (e.g., the attached fragments 34 shown in fig. 3); priming release of the proximity-preserving library fragments 14, 14' from the solid support 16 or from each other 34; amplifying the proximity-preserving library fragments 14, 14' to generate a plurality of corresponding template strands; staining the corresponding template strand; and imaging the corresponding template strand.

Proximity-preserving library fragments 14, 14' are used in combination with sequential amplification and imaging using the methods disclosed herein to generate the series of time-based cluster-generated images. Each time-based cluster generation image records the spatial position and orientation of the template strand generated using a particular sample. Thus, these images can be used to identify the location of clusters (template strands) that originate from a particular sample introduced into the flow cell at a particular time.

The method may vary somewhat depending on whether the composite 10 is introduced into the flow cell 36 or whether the attachment segments 34 are introduced into the flow cell. Various examples will now be described.

Methods of using the composites 10 or 10

In this example, a genomic sample (e.g., sample 30) is fragmented to form a plurality of proximity-preserving fragments 14, 14', each of which is attached to a solid support 16. It will be appreciated that not all proximity-retaining fragments 14, 14' from the genomic sample 30 may be attached to the same solid support 16; rather, the proximity-retaining fragments 14, 14' associated with a particular portion of the genomic sample 30 may be attached to a respective solid support 16. Thus, the genomic sample 30 is fragmented to form multiple complexes 10 or 10'. This can be achieved as described with reference to fig. 1, 2A-2C, 3, or using any other proximity preservation method that incorporates linkers 18, 22 or 18', 22 into each of the fragments 14, 14' to make them ready-to-use sequenced fragments 12, 12 '.

The complex 10 or 10' formed using the genomic sample 30 may be incorporated into a mixture. Thus, each of the plurality of proximity-retaining 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.

A liquid carrier may be added to the plurality of proximity retention fragments 14, 14 '(e.g., present in the complex 10 or 10') to initially form a mixture, and then the mixture may be diluted with additional liquid carrier to generate a predetermined number of diluted samples that will be separately introduced into the flow-through cell 36 (or its individual channels 40).

The final volume of the mixture produced, and thus the dilution of the mixture, can be controlled in any desired manner. In some cases, the dilution may depend on the volume of the flow cell 36 (or, for example, each channel 40 of a multi-channel flow cell) and the desired amount of sample to be introduced into the flow cell 36. In one example, the volume of the flow cell 36 (or its channels 40) may be used as the limiting dilution. Thus, in some examples, a carrier liquid may be added to dilute the mixture to a predetermined volume, where the predetermined volume is based on i) the volume of the flow cell 36 as the limiting dilution and ii) a predetermined amount of the diluted sample to be introduced into the flow cell 36. For example, the flow cell 36 or one channel 40 of the flow cell 36 may have a volume of about 50 μ Ι _, and the desired number of samples may be 200. In this example, the mixture may be diluted to about 10,000 μ L. As another example, the flow cell 36 or one channel 40 of the flow cell 36 may have a volume of about 100 μ Ι _ and the desired number of samples may be 384. In this example, the mixture may be diluted to about 38,400 μ L.

The desired amount of diluted sample may depend on, for example, the volume of the flow cell 36 and the desired resolution of each template strand in the corresponding time-based cluster-generated image. When using a smaller volume flow cell 38, it may be desirable to have a more diluted mixture such that each individual diluted sample to be introduced into the flow cell 36 contains less proximity-preserving library fragments 14, 14' (as compared to if a less diluted mixture is used). In this type of flow cell 36, less proximity-preserving library fragments 14, 14' will result in fewer template strands, which may improve the resolution of the template strands in the corresponding time-based cluster-generated images. In one example, the desired number of samples may range from 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 on the number of diluted samples may depend in part on the desired time frame for performing the overall method.

The mixture may then be divided into a predetermined number of diluted samples. In one example, the diluted mixture may be split such that all diluted samples are generated simultaneously. In another example, when a predetermined volume of any one of the diluted samples is introduced into the flow cell 36, the sample may be separated from the bulk mixture.

Fig. 6A-6D illustrate examples of an unpatterned flow cell 36 (e.g., as shown in fig. 5B) from a top view during different stages of generating time-based cluster-generated images.

As schematically shown in fig. 6A-6D, the flow cell 36 may be incorporated into a system comprising a flow cell receptacle 35; a fluid control system 37 including a fluid delivery 39 to deliver the diluted sample 56A and a stain (not shown) to the flow cell 36 positioned in the flow cell receptacle 35, respectively; a lighting system 62 positioned to illuminate the flow cell 36 positioned in the flow cell receptacle 35; a detection system 64 positioned to capture an image of the flow cell 36 positioned in the flow cell receptacle 35; and a controller 41 in operable communication with the fluid control system 37, the illumination system 62, and the detection system 64, the controller 41: causing the fluid deliverer 39 to introduce the diluted sample 56A into the flow cell 36 positioned in the flow cell receptacle 35; after generating the template strand 58A from the proximity-preserving library fragments present in the diluted sample 56A in the flow-through cell 36 positioned in the flow-through cell receptacle 35, causing the fluid delivery device 39 to introduce the staining agent into the flow-through cell 36 positioned in the flow-through cell receptacle 35; causing the illumination system 62 to illuminate the chain of staining templates in the flow cell 36 positioned in the flow cell receptacle 35; and causes the detection system 64 to image the illuminated dyed template strand positioned in the flow cell 36 in the flow cell receptacle 35.

When in place, the flow cell 36 is in fluid communication with a fluid control system 39 (e.g., a pump, a valve, etc.) and is in optical communication with an illumination system 62 and a detection system 64.

In fig. 6A, a first diluted sample (including some of complexes 10 or 10', shown as 10A in fig. 6A) of the diluted sample 56A is introduced into the flow cell 36. Introducing any of the respective diluted samples 56A (or 56B in fig. 6C, for example) involves fluidically directing one of the diluted samples 56A, 56B to the flow cell 36. The diluted sample 56A may be introduced, for example, into the cartridge 45, and the fluid control system 37 may fluidly deliver 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.).

Considering the concentration of the complex 10A in the diluted sample 56A, most, if not all, of the complex 10A will settle onto the polymer hydrogel 42 and any primers 44, 46 thereon (which are not shown in fig. 6A-6D). In some examples, due to the depth of the flow channels 40 or recesses 54, the composite 10A may settle and remain in the flow channels 40 or recesses 54. In other examples, the flow channel 40 or recess 54 may include a capture site to which the complex 10A attaches.

It should be understood that some of the complexes 10A may not settle and that these complexes 10A will be removed from the flow cell 36 prior to further processing. Thus, some examples of methods then include washing the uncaptured complex 10A from the flow cell 36. Washing may involve introducing a fluid into the flow cell 36. This flow may push any complexes 10A that have not yet settled and/or adhered through the outlet of the flow cell 36.

This example of the method then includes triggering release of the proximity-preserving library fragments 14, 14' from the respective solid supports 16 to which they are attached. In this example, sequenced nucleic acid fragments 12, 12' (including proximity-preserving library fragments 14, 14' and linkers 18, 22 or 18', 22 attached thereto) are used to release from the respective solid supports 16. In FIG. 6A, the release of ready-to-use sequencing nucleic acid fragments 12, 12' from solid supports 16 is represented by the arrows pointing outward from each solid support 16.

The release of the ready-to-sequence nucleic acid fragments 12, 12' can be triggered in several different ways. In one example, initiating release involves heating the flow cell 36. In this example, the system may include a heater 43. The controller 41 can cause the heater 43 to initiate release of some of the proximity-preserving library fragments 12, 12' from the solid support 16 or from each other. For example, a temperature above 70 ℃ may be used to at least partially break the bonds, thereby initiating the release of the ready-to-sequence nucleic acid fragments 12, 12'. In another example, initiating release involves introducing a lysing agent into the flow cell 36. The fluid control system 37 may be used to deliver a lysing agent. The cleavage agent can initiate 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 lysing agent, and heating to about 92 ℃ can be used to induce release of the biotin-oligonucleotide from the solid support 16.

The released ready-to-use sequencing nucleic acid fragments 12, 12' are transported from the solid support 16 and seeded onto the polymeric hydrogel 42. More specifically, the amplification primers 46, 48 inoculate the released, ready-to-use sequencing nucleic acid fragments 12, 12' in a relatively limited manner. In one example, the seeding is accomplished by hybridization of the first or second sequence of the fragments 12, 12' to complementary primers in the primers 46, 48 on the polymer hydrogel 42 in the flow cell 36. The inoculation can be carried out at a hybridization temperature suitable for the fragment, i.e.the sequencing nucleic acid fragments 12, 12' and the primers 46, 48. The heater 43 may be controlled to bring the flow cell 36 to the inoculation temperature.

A washing process may be performed to remove the beads.

The inoculated, ready-to-use sequenced nucleic acid fragments 12, 12' can then be amplified using any suitable method, such as cluster generation. In one example of cluster generation, the released ready-to-use sequencing nucleic acid fragments 12, 12 'are replicated from the hybridization primers 46, 48 by 3' extension using high fidelity DNA polymerase. The original ready-to-use sequencing nucleic acid fragments 12, 12' are denatured, thereby immobilizing these copies within the flow channel 40 or some of the recesses 54. Any clonal amplification procedure can be used. In one example, isothermal bridge amplification can be used to amplify the immobilized copies. For example, the replicated template loops back to hybridize to the adjacent complementary primers 46, 48, and the polymerase replicates the replicated template to form double-stranded bridges, denaturing these double-stranded bridges to form two single strands. The two strands loop back and hybridize to adjacent complementary primers 46, 48 and are extended again to form two new double-stranded loops. This process was repeated for each template copy through isothermal denaturation and amplification cycles to generate dense clonal clusters. Each cluster of the double-stranded bridge is denatured. In one example, the antisense strand is removed by specific base cleavage, leaving a forward template polynucleotide strand. It should be understood that clustering results in the formation of several template strands 58A in some of the flow channels 40 or recesses 54. In some examples, the controller 41 causes the heater 43 to run thermal cycling to amplify the inoculated, ready-to-use sequencing nucleic acid fragments 12, 12'.

FIG. 6B shows a cluster 60A of template strands 58A generated from a complex 10A of a first diluted sample 56A (compartment). For clarity, the cluster 60A in FIG. 6B is summarized. Although four clusters 60A are shown in fig. 6B, it will be appreciated 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. Cluster 60A is generated from each of the released ready-to-use sequencing nucleic acid fragments 12, 12'. Furthermore, the released ready-to-use sequencing nucleic acid fragments 12, 12' may diffuse across the entire flow cell surface, and thus may generate clusters 60A across the entire flow cell surface.

After the cluster 60A is generated for the first diluted sample 56A, a stain is introduced into the flow cell 36. Any fluorescent dye capable of staining the template strand 58A may be used. Examples of suitable fluorescent stains include those available from Molecular Probes, incDye family (e.g. dye)Green、Gold、Safe et al), ethidium bromide, propidium iodide, crystal violet,Dyes (from Biotium), DAPI (4', 6-diamidino-2-phenylindole), and the like. The staining agent 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.

The illumination system 62 can then be used to illuminate the dyed template strand 58A in the flow cell 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 component may be, for example, a reflector, dichroic mirror, beam splitter, collimator, lens, filter, wedge mirror, prism, mirror, detector, or the like. The illumination system may be operably positioned to direct excitation light to the flow cell surface corresponding to the stain used.

The detection system 64 can be used to capture an image I of the fluorescent template strand 58A₁. The image I₁Is a time-based cluster generation image for the diluted sample 56A because it depicts the spatial position and orientation of the template strand 58A associated with the diluted sample 56A. Any suitable camera may be used to capture an image I of a cluster 60A on the flow cell 36₁。

Image I₁May be electronically stored for later retrieval and use. Thus, some examples of systems include a system to store image I₁The electronic storage component 47. In electronic recording, image I₁May be attached to the diluted sample 56A. Or may be an image I₁A time record is assigned. Thus, some examples of methods include generating an image for each time-based cluster in the series₁Assigning a time record of introduction of the respective sample comprising some of the proximity-preserving library fragments. 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., sample 1 of 200, sample 2 of 200,.. sample X of 200), or a combination thereof.

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

The process shown and described with reference to fig. 6A and 6B is then repeated with a second diluted sample 56B (shown in fig. 6C).

In fig. 6C, a second diluted sample 56B is introduced into the flow cell 36. Complex 10B (which may be complex 10 or 10') settles and/or adheres to the flow cell surface.

As shown in fig. 6C, the method then includes priming the release of the proximity-preserving library fragments 14, 14' from the respective solid supports 16 to which they are attached. In this example, sequenced nucleic acid fragments 12, 12' (including proximity-preserving library fragments 14, 14' and linkers 18, 22 or 18', 22 attached thereto) are used to release from the respective solid supports 16.

The released ready-to-use sequencing nucleic acid fragments 12, 12' are transported from the solid support 16 and seeded onto the polymeric hydrogel 42. The inoculated, ready-to-use sequenced nucleic acid fragments 12, 12' can then be amplified using any suitable method, such as cluster generation. It should be understood that the wheels are clustered such that several more template strands 58B are formed in some of the flow channels 40 or recesses 54.

FIG. 6D shows clusters 60B of template strands 58B generated from the corresponding complexes 10B of the second diluted sample 56B (compartment). For clarity, clusters 60A and 60B in FIG. 6D are summarized. Although three clusters 60B are shown in fig. 6D, 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 ready-to-sequence nucleic acid fragments 12, 12' released from each solid support 16.

After cluster 60B is generated for second diluted sample 56B, the stain is reintroduced into flow-through cell 36. The same staining agent used to stain the template strand 58A may be used to stain the template strand 58B and any subsequently generated template strands.

The illumination system 62 can then be used to illuminate the dyed template strands 58A and 58B in the flow cell 36. The detection system 64 can be used to capture images I of the fluorescent template strands 58A and 58B in the respective clusters 60A and 60B₂。

Image I₂And may also be stored electronically for later retrieval and use. In electronic recording, image I₂Can be connected withTo diluted sample 56B. Or may be an image I₂A time record is assigned. The time record may include a timestamp indicating when the diluted sample 56B was imaged, the number of steps in the sequence (e.g., sample 2 of 200), or a combination thereof.

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.

The process shown and described with reference to fig. 6A and 6B can then be repeated, for example, using the system, to obtain the amount of diluted sample derived from the original mixture. Each additional image I₃、I₄、…I_xNew clusters 60C, 60D, … 60X of template strands 58C, 58D, … 58X generated by introducing the respective diluted samples 56C, 56D, … 56X will be depicted. All images I obtained for the respective diluted samples 56A, 56B, … 56X₁、I₂、…I_xAre each associated with a particular mixture and thus a particular longer nucleic acid molecule 30.

Because each sequential image I₂、I₃、I₄、…I_xDepicts with respect to the immediately preceding image I₁、I₂、I₃、…I_xNewly formed clusters 60B, 60C, 60D, … 60X, 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_xShown in fig. 6.

FIG. 7 depicts images I taken for six different diluted samples 56A, 56B, 56C, 56D, 56E, 56F introduced, amplified, stained, and imaged sequentially as described with reference to FIGS. 6A and 6B₁、I₂、I₃、I₄、I₅、I₆. As depicted, 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.

Because of the image I for the first sample 56A₁Including clusters 60A from one sample, there is no need to generate a resolved cluster image because the original imageI₁May be used for spatial identification of the cluster 60A. For each sample introduced after the first sample 56A, for the other images I₂、I₃、I₄、I₅、I₆Generates a resolved cluster image RI₂、RI₃、RI₄、RI₅、RI₆. For example, from image I₂(delineating the template strand 58A in cluster 60A and the template strand 58B in cluster 60B) minus the image I₁(delineating the template strand 58A in the cluster 60A) to generate a resolved cluster image RI for the second sample 56B₂. The parsed cluster image RI₂The spatial position and orientation of the template strand 58B in the cluster 60B associated with the second sample 56B is depicted. As another example, from image I₃(which depicts template strand 58A in cluster 60A, template strand 58B in cluster 60B, and template strand 58C in cluster 60C) minus image I₁(template strand 58A in rendering cluster 60A) and image I₂(template strand 58A in cluster 60A and template strand 58B in cluster 60B) to generate a resolved cluster image RI for a third sample (e.g., 56C)₃. The parsed cluster image RI₃The spatial position and orientation of the template strand 58C in the cluster 60C associated with the third sample 56C is depicted. Parsed cluster image RI₄、RI₅And RI₆May be generated in a similar manner by subtracting any of the aforementioned images.

It should be understood that a series I may be paired₁、I₂、I₃、…I_xImage subtraction is performed by either. The resulting resolved cluster image RI for any given diluted sample 56X_xThe spatial position and orientation of the template strand 58X associated with the diluted sample 56X is depicted.

The parsed cluster images can be stored for subsequent analysis.

Method of using attachment segments 34

In this example, the genomic sample is fragmented to form a plurality of proximity-preserving fragments 14, 14', which are attached to one another, e.g., as attachment fragments 34. It will be appreciated that not all of the proximity-retaining fragments 14, 14' from the genomic sample may be attached to each other; rather, the process shown in fig. 4 may result in the formation of several attachment segments 34.

The attached fragments 34 formed using the genomic sample can be incorporated into the mixture. Thus, each of the plurality of proximity-retaining 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.

A liquid carrier may be added to the plurality of attachment segments 34 to initially form a mixture, and then the mixture may be diluted with additional liquid carrier to generate a predetermined number of diluted samples that will be separately introduced into the flow-through cell 36 (or its individual channels 40).

The final volume of the resulting mixture, and thus the dilution of the mixture, can be controlled in any desired manner and as described herein.

The mixture may then be divided into a predetermined number of diluted samples. In one example, the diluted mixture may be split such that all diluted samples are generated simultaneously. In another example, when a predetermined volume of any one of the diluted samples is introduced into the flow cell 36, the sample may be separated from the bulk mixture.

In this exemplary method, the attachment fragment 34 shown in fig. 4 is introduced into the flow channel as part of one of the diluted samples. An example of this diluted sample 66A is depicted in fig. 8.

As shown in fig. 8, within flowthrough cell 36, transposase 32 is removed from attachment fragments 34. This can be achieved, for example, using SDS or a protease. Removal of the transposase 32 releases the corresponding proximity-retaining fragment 14, 14 '(and any strand 26', 28 'directly or indirectly attached thereto) from the adjacent proximity-retaining fragment 14, 14'. In other words, the sub-segment 72 of the attachment segment 34 is released and can be seeded onto the polymeric hydrogel 42 via the transfer strand 26'. As schematically shown in FIG. 8, the transfer strand 26' hybridizes to a corresponding and complementary primer 46 on the surface of the flow-through cell 36. In some cases, heat may be applied during hybridization. The application of heat may depend on the melting temperature of the transfer chain 26'. For example, the P5 'portion of the transfer strand 26' hybridizes to the complementary P5 amplification primer 46 attached to the polymer hydrogel 42.

The 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 transposases. A second wash solution such as TRIS or hybridization wash buffer may be used to rinse the flow cell 36.

Prior to amplification, the method example further includes introducing a second sequence portion (e.g., non-transferred strand 28') into each of the hybrid proximity-preserving library fragments 14, 14' at a terminus opposite the hybridizing end. Thus, in some examples of the method, each of the proximity-preserving library fragments 14, 14' comprises a first sequence portion at a first end that hybridizes to a first primer sequence 46 on the surface of the flow cell 26; and prior to amplification, the method further comprises attaching a second sequence portion to each of the hybridization proximity retention library fragments 14, 14' at a second end opposite the first end, the second sequence portion being identical to the second primer sequence 48 on the surface of the flow cell 36 such that a copy of the second sequence portion can hybridize to the second primer sequence 48.

The introduction of the second sequence portion may be performed using an extension linkage. In one example, an extension ligation (as represented by arrow 74 in fig. 8) can be initiated to join the non-transferred strand 28 'to the corresponding fragment 14, 14'. In one example, the extension ligation may be initiated by introducing the extension ligation mixture into the flow cell 36 and heating to a temperature suitable for enzymatic activity (e.g., in the range of about 37 ℃ to about 50 ℃).

The extension ligation mixture may include a ligase (e.g., a DNA ligase) that catalyzes the formation of a bond between the non-transferred strand 28 'and its corresponding fragment 14 or 14'. As described with reference to fig. 4, 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 one of the amplification primers 48(P7) on the flow cell surface. This second sequence enables a complementary copy (e.g., P7') to be generated during amplification, which can hybridize to the amplification primer 48(P7) on the flow cell surface during cluster generation. Thus, ligation results in the formation of ready-to-use sequencing library fragments 12, 12' attached to the flow cell surface.

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

When the resulting fragments 12, 12 'are attached to each other, heat can be used to dissociate the fragments 12' from the fragments 12. Fragments 12' that are not hybridized to the primer 46 can be removed from the flow cell 36 by washing.

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

Fig. 9A shows a cluster 70A of template strands 68A generated from the attached fragments 34 of the diluted sample 66A (shown in fig. 8). For clarity, the cluster 70A in fig. 9A is summarized. Although fig. 9A shows four clusters 70A, it is to 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 ready-to-sequence nucleic acid fragments 12, 12' released from the attached fragments 34.

After the cluster 70A is generated for the first diluted sample 66A, a stain is introduced into the flow-through cell 36, as described with reference to fig. 6B. The staining agent 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.

The illumination system 62 can then be used to illuminate the stained template strand 68A in the flow cell 36, and the detection system 64 can be used to capture an image I of the fluorescent template strand 68A₁. The image I₁Is directed to diluting sample 66A based on timeThe cluster generates an image because it depicts the spatial position and orientation of the template strand 68A associated with the diluted sample 66A.

Image I₁May be electronically stored for later retrieval and use. In electronic recording, image I₁May be attached to the diluted sample 66A. Or may be an image I₁A time record is assigned. The time record may include a timestamp indicating when the diluted sample 66A was introduced and/or imaged, the number of steps in the introduction and/or imaging sequence (e.g., sample 1 of 200, sample 2 of 200,.. sample X of 200), or a combination thereof.

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

The process shown and described with reference to fig. 8 and 9A is then repeated with a second diluted sample 66B (shown in fig. 9B).

In fig. 9B, a second diluted sample 66B is introduced into the flow cell 36. The attachment segment 34 may be broken down into sub-segments 72 as described with reference to fig. 8. One transfer strand 26 of at least some of the sub-fragments 72 will hybridize to the complementary amplification primer 46 on the flow cell 36. Extension ligation and other processes as described with reference to figure 8 may then be performed, which results in ready-to-use attachment of the sequenced nucleic acid fragments 12 to the flow cell surface.

Cluster generation may be performed as described herein with reference to fig. 6A.

Fig. 9C shows a cluster 70B of template strands 68B generated from the attached fragments 34 of the second diluted sample 66B. For clarity, clusters 70A and 70B in FIG. 9C are summarized. Although fig. 9C shows two clusters 70B, it is to 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 ready-to-sequence nucleic acid fragments 12, 12' released from the attached fragments 34.

After the cluster 70B is generated for the second diluted sample 66B, the stain is again introduced into the flow-through cell 36. The same stain used to stain template strand 68A may be used to stain template strand 68B and any subsequently generated template strands.

The illumination system 62 can then be used to illuminate the dyed template strands 68A and 68B in the flow cell 36.Detection system 64 can be used to capture images I of fluorescent template strands 68A and 68B in respective clusters 70A and 70B₂。

Image I₂And may also be stored electronically for later retrieval and use. In electronic recording, image I₂May be attached to the diluted sample 66B. Or may be an image I₂A time record is assigned. The time record may include a timestamp indicating when the diluted sample 56B was imaged and/or introduced, the number of steps in the sequence (e.g., 1000 samples 2), or a combination thereof.

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.

The process shown and described with reference to fig. 9B and 9C may then be repeated 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 respective diluted samples 66C, 66D, … 66X will be depicted. All images I obtained for the corresponding diluted samples 66A, 66B, … 66X₁、I₂、…I_xAre each associated with a particular mixture and thus a particular longer nucleic acid molecule 30.

Because each sequential image I₂、I₃、I₄、…I_xDepicts with respect to the immediately preceding image I₁、I₂、I₃、…I_xNewly formed clusters 70B, 70C, 70D, … 70X, image subtraction can be used to generate a resolved cluster image for each sample introduced after the first sample 66A. The parsed cluster image RI may be generated as described with reference to fig. 7_x。

It should be understood that a series I may be paired₁、I₂、I₃、…I_xImage subtraction is performed by either. The resulting resolved cluster image RI for any given diluted sample 66X_xThe spatial position and orientation of the template strand 68X associated with the diluted sample 66X is depicted.

The parsed cluster images can be stored for subsequent analysis.

Other methods

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

Thus, some examples of methods include generating a time-based cluster generation image for each of the limiting dilution samples introduced into the flow cell 36 by: controlling the diffusion of the mixture into the flow cell such that one of the limiting dilution samples is introduced into the flow cell 36 at a time; triggering release of the proximity-preserving library fragments 14, 14' from the solid support 16 or from each other (e.g., from the attached fragments 34) in one of the limiting dilution samples in the flow cell 36; amplifying the proximity-preserving library fragments 14, 14' to generate a plurality of corresponding template strands 58A, 68A; staining the respective template strands 58A, 68A; and imaging the corresponding template strand 58A, 68A.

Sequencing and analysis

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

For example, Sequencing By Synthesis (SBS) reactions can be performed in a sequence such as HISEQ^TM、HISEQX^TM、MISEQ^TM、MISEQDX^TM、MINISEQ^TM、NOVASEQ^TM、NEXTSEQDX^TM、ISEQ^TM、NEXTSEQ^TMOr other sequencer system from Illumina (san diego, california). In SBS, extension of sequencing primers along the template strands 58A, 58B, … 58X is monitored to determine the sequence of nucleotides in the template. Primers 46, 48 that block binding of template strands 58A, 58B, … 58X to any flow cell (not attached)To the 3' end of the template strand 58A, 58B, … 58X) to prevent interference with the sequencing reaction, and in particular, to prevent undesired priming.

Sequencing primers that hybridize to complementary sequences on the template strands 58A, 58B, … 58X can be introduced. The sequencing primers prepare the template strands 58A, 58B, … 58X for sequencing.

The underlying chemical process may be polymerization (e.g., catalyzed by a polymerase) or ligation (e.g., catalyzed by a ligase). In certain polymerase-based SBS procedures, fluorescently labeled nucleotides are added to the sequencing primer in a template-dependent manner, such that detection of the order and type of nucleotides added to the sequencing primer can be used to determine the sequence of the template. For example, to initiate the first SBS cycle, one or more labeled nucleotides, DNA polymerase, etc., can be delivered to/through the flow cell 36, etc., where sequencing primer extension causes the labeled nucleotides to be incorporated. This incorporation can be detected by an imaging event. During an imaging event, the illumination system 62 may provide excitation light to the flow cell 36.

In some examples, the fluorescently labeled nucleotides can further include a reversible termination property that terminates further primer extension upon addition of the nucleotide to the template. For example, a nucleotide analog having a reversible terminator moiety can be added to the template such that subsequent extension does not occur until the deblocking agent is delivered to remove the moiety. Thus, for examples using reversible termination, the deblocking agent may be delivered to the flow cell 36 or the like (after detection occurs).

Washing may occur between various fluid delivery steps. The SBS cycle can then be repeated n times to extend the template n nucleotides, thereby detecting sequences of length n.

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

Can be based on the resolved cluster image RI₂、RI₃、…RI_xWill be obtained during the sequencing operationThe resulting sequencing reads were combined. Can then be based on the parsed cluster image RI₂、RI₃、…RI_xThe combined sequencing reads are ligated to a respective one of the diluted samples. It can be concluded that the combined and linked sequencing reads originate from the same longer nucleic acid sample 30. Thus, some examples of methods include performing a sequencing operation on a flow cell 26 that includes a respective template strand for each library fragment of a plurality of library fragments; and generating an image RI, RI based on the parsed cluster₂、RI₃、…RI_xSequencing reads were combined together in different sets.

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

Examples

Example 1

Barcode oligonucleotides (e.g., linker 18) were attached to M280 streptavidin beads (thermolfisher) via biotin linkers to form a bead pool. The universal transposomes are hybridized to complementary sequences at the ends of the oligonucleotides to form bead-linked transposomes (BLTs). The BLT was then washed in wash buffer, resuspended in working buffer, and accessory proteins (single-stranded binding protein (thermoleisher) and double-stranded binding protein (Illumina)) were added.

High molecular weight NA12878 DNA (using Qiagen)HMW DNA extraction protocol was extracted from cultured cells) was added to the BLT mixture and the tubes were gently inverted to mix well. The tube was then incubated at room temperature for about 15 minutes to wrap the DNA around the beads. Labeling buffer (containing magnesium chloride and tris acetate) was then added to each tube and the samples were incubated at about 55 ℃ for about 10 minutes. During this step, transposome-tagged DNA and tagged DNA are attached to the BLT. After the labeling reaction, Sodium Dodecyl Sulfate (SDS) was added and the sample was left at room temperatureThe transposase was denatured by incubating for about 5 minutes. The tube was then placed on a magnet and the supernatant removed. The beads were washed with wash buffer. After the last wash, the beads were resuspended in a ligase mixture (containing T7 ligase and its associated buffer-T7 ligase buffer from NEB). The samples were then mixed and incubated at room temperature for about 45 minutes. During this time, gaps in the transferred strand (where the transposomes first hybridized) are ligated, thereby physically attaching the tagged DNA to the beads. The sample was then placed on a magnet, the supernatant removed, and the beads washed again in the wash buffer.

The tagged beads are then divided into two groups for removal of non-transfer strands and introduction of sample indices (e.g., linker 22). One group (comparison group) was exposed to a comparison workflow in which heating was used to remove non-transferred chains. The other set (example set) was exposed to an exemplary workflow in which exonuclease was used to remove non-transferred strands.

The comparative group was resuspended in wash buffer and heated to about 80 ℃ for about 5 minutes to denature the non-transferred strands. The tube containing the comparative set was then placed on a magnet, the supernatant removed, and the beads washed. The sample index diluted in wash buffer was then added to the beads of the comparative group and the mixture was incubated at about 80 ℃ for about 1 minute, after which the temperature was allowed to slowly drop.

The exemplified panel was suspended in a T7 exonuclease cocktail (containing T7 exonuclease and NEB buffer 4) and allowed to incubate for about 10 minutes at room temperature. The 5 'to 3' exonuclease activity of the T7 exonuclease digests the nontransferred strand. The tube containing the example set was then placed on a magnet, the supernatant removed, and the beads washed. The sample index diluted in wash buffer was then added to the exemplary set of beads and the mixture was incubated at about 55 ℃ for about 5 minutes to allow the sample index to anneal.

The tubes containing the comparative and exemplary sets, respectively, were placed on a magnet and the supernatant removed. The extension ligation mixture was added and the sample was incubated at about 37 ℃ for about 5 minutes. The tubes were again placed on the magnet, the supernatant was removed, and the beads of the comparative and example groups were washed in the wash buffer. After the last wash, the beads were resuspended in wash buffer.

Some beads of the comparative and some beads of the example sets were subsampled and used in the PCR reaction. Each PCR mix consisted of Illumina PCR mix EPM and P5 and P7 oligonucleotides, except for the corresponding beads. Each sample was amplified using PCR.

Following PCR, subsamples of PCR supernatants of each of the comparative and exemplary groups were transferred to new tubes and subjected to 0.50 x-0.62 x size selection (solid phase reversible immobilization, SPRI) using sample purification beads. The resulting library was eluted in resuspension buffer. These libraries were in individual HISEQ^TMSequencing on a 2500Rapid flow cell (using standard 2X 101 cycling read lengths). After Fastq generation, the samples were aligned to the human genome (hg38) and the data were imported into IGVs.

Figure 10 shows the coverage obtained using libraries from each of the comparative (labeled as heat denaturation) and exemplary (labeled as T7 exonuclease) sets of AT-rich regions of the human genome that are known to be negatively affected by high temperature steps in the library preparation protocol. The results for the comparative set of library fragments whose non-transfer strands were removed via thermal denaturation are shown at the top of fig. 10, and the results for the exemplary set of library fragments whose non-transfer strands were removed via exonuclease digestion are shown at the bottom of fig. 10. As shown, there is full coverage in the AT-rich region for the example set of library fragments, while there is only partial coverage in the same region for the comparative set of library fragments. These results indicate that using the enzymatic digestion methods disclosed herein increases library coverage and improves sequencing on AT-rich regions of the genome when compared to thermal denaturation.

Example 2

Complexes were prepared as described in the methods of fig. 2A to 2C (multi-step ligation and digestion) and fig. 3 (single pot ligation and digestion).

BLT production, DNA binding, labeling and exposure to SDS were performed as described in example 1. After removal of transposase and washing associated therewith, the tagged beads were then divided into two groups to remove non-transferred strands via multi-step ligation and digestion (referred to as example group 2) or via single pot ligation and digestion (referred to as example group 3).

Example group 2 was resuspended in an e.coli DNA ligase mixture that included e.coli DNA ligase and its associated buffer, both from NEB. The example group 2 samples were then mixed and incubated at about 16 ℃ for about 15 minutes. The tube containing the sample of example set 2 was then placed on a magnet, the supernatant removed, and the example set 2 beads washed in a wash buffer. After washing, the example group 2 beads were resuspended in a mixture containing T7 exonuclease and NEB buffer 4 and incubated at about 25 ℃ for about 10 minutes.

Example set 3 was resuspended in a combined ligase and exonuclease cocktail (including E.coli DNA ligase, T7 exonuclease, NAD) at about 25 ℃⁺And CUTSMAT^TMBuffer (from NEB)) for about 15 minutes.

The tubes containing example set 2 and example 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 each example set of beads and the mixtures were incubated at about 55 ℃ for about 5 minutes to allow the sample index to anneal.

The tubes containing example set 2 and example set 3, respectively, were placed on a magnet and the supernatant removed. The extension ligation mixture was added and the sample was incubated at about 37 ℃ for about 5 minutes. The tubes were again placed on the magnet, the supernatant was removed, and the beads of the comparative and example groups were washed in the wash buffer. After the last wash, the beads were resuspended in wash buffer.

Some beads of example set 2 and some beads of example set 3 were subsampled and used in a PCR reaction. Each PCR mix consisted of Illumina PCR mix EPM and P5 and P7 oligonucleotides, except for the corresponding beads. Each sample was amplified using PCR.

After PCR, a sub-sample of the PCR supernatant of each of example set 2 and example 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 the trace in FIG. 11 was obtained. The results show that the size distribution and yield of the library 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 transfer chains.

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 methods described with reference to fig. 6A-6D (including cluster generation, staining, and imaging) can then be performed using each of the diluted samples to generate a series of time-based cluster-generated images. When all diluted samples are amplified and imaged, the flow cell is ready for sequencing operations. The library preparation techniques disclosed herein and time-based imaging techniques can be used together to efficiently and reliably reconstitute long DNA fragments.

Additional description

Further, it should be understood that the ranges provided herein include the stated range and any value or subrange within the stated range, as if they were explicitly listed. For example, a range expressed as from about 2mm to about 300mm should be interpreted to include not only the explicitly recited limits of about 2mm to about 300mm, but also include individual values such as about 15mm, 22.5mm, 245mm, and the like, as well as sub-ranges such as from about 20mm to about 225mm, and the like.

It should be understood that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It is also to be understood that the terms explicitly employed herein, which may also appear in any disclosure incorporated by reference, are to be accorded the most consistent meanings with the specific concepts disclosed herein.

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