Patent Description:
A first aspect disclosed herein is a method comprising generating a series of time-based clustering images for a plurality of contiguity preserved library fragments from a genome sample, wherein each time-based clustering image in the series is sequentially generated by: introducing, to a flow cell, a respective sample including some of the contiguity preserved library fragments, wherein the some of the contiguity preserved library fragments are attached to a solid support or are attached to each other; initiating release of the some of the contiguity preserved library fragments from the solid support or from each other; amplifying the some of the contiguity preserved library fragments to generate a plurality of respective template strands; staining the respective template strands; and imaging the respective template strands.

A second aspect disclosed herein is a method comprising preparing a mixture including a plurality of contiguity preserved library fragments of a genome sample, the plurality of contiguity preserved library fragments being attached to solid supports or being attached to each other; diluting the mixture to generate a predetermined number of dilution samples to be introduced to a flow cell; and generating a time-based clustering image for at least one of the contiguity preserved library fragments by: introducing a first of the dilution samples including some of the contiguity preserved library fragments to the flow cell; initiating release of the some of the contiguity preserved library fragments from the solid support or from each other; amplifying the some of the contiguity preserved library fragments to generate a plurality of template strands; staining the plurality of template strands; and imaging the plurality of template strands.

A third aspect disclosed herein, but not being part of the present invention, is a system comprising a flow cell receptacle; a fluidic control system including delivery fluidics to respectively deliver a dilution sample and a stain to a flow cell positioned in the flow cell receptacle; an illumination system positioned to illuminate the flow cell positioned in the flow cell receptacle; a detection system positioned to capture an image of the flow cell positioned in the flow cell receptacle; and a controller in operative communication with the fluidic control system, illumination system, and the detection system, the controller to: cause the delivery fluidics to introduce the dilution sample to the flow cell positioned in the flow cell receptacle; cause the delivery fluidics to introduce the stain to the flow cell positioned in the flow cell receptacle after template strands are generated in the flow cell positioned in the flow cell receptacle from contiguity preserved library fragments present in the dilution sample; cause the illumination system to illuminate the stained template strands in the flow cell positioned in the flow cell receptacle; and cause the detection system to image the illuminated, stained template strands in the flow cell positioned in the flow cell receptacle.

It is to be understood that any features of the first method and/or the second method and/or system disclosed herein may be combined together in any desirable manner and/or configuration and/or with any of the examples disclosed herein to achieve the benefits as described in this disclosure, including, for example, the identification of a particular group of template strands using a resolved cluster image.

Library fragments are similarly sized (e.g., < <NUM> bp) deoxyribonucleic acid (DNA) pieces of a larger or longer DNA fragment. Library fragments can be grouped together in sequencing data if a common originating compartment can be identified in which the long DNA fragment originated. Compartmentalization of different long DNA fragments may be desirable in order to achieve sub-haploid genome content within each compartment for synthetic long reads. Synthetic long reads (or linked short reads) are enabled when a plurality of short fragments can be grouped together based on the identification of the compartment in which the long DNA fragment originated. Compartmentalization has been accomplished physically, for example, using wells, beads, droplets, or other physical compartments.

Common to all of these compartmentalization approaches is the principle that a barcode sequence or index sequence is used to identify the compartment in which the long DNA fragment originated. The barcode sequence attached to each of the shorter fragments may be unique to a particular long DNA fragment, and thus can help to mark different compartments during library preparation. It is the barcode sequence which is used to group short reads together to form synthetic long reads based on the assumption that the short reads all originate from the same compartment.

The example methods disclosed herein achieve compartmentalization of different library fragments without having to incorporate a unique barcode sequence. The method utilizes contiguity preserved library fragments and imaging to create a series of time-based clustering images. Each time-based clustering image can be used to identify a sample (compartment) from which a particular set of template strands was generated. Moreover, each sequenced read can be grouped using the time-based clustering images. This grouping allows the reads to the linked, thus enabling the reconstitution of a long DNA fragment.

Terms used herein will be understood to take on their ordinary meaning in the relevant art unless specified otherwise. Several terms used herein and their meanings are set forth below.

As used herein, the singular forms "a," "an," and "the" refer to both the singular as well as plural, unless the context clearly indicates otherwise. The term "comprising" as used herein 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 the specification to "one example," "another example," "an example," and so forth, 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. In addition, it is to be understood that the described elements 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" used throughout this disclosure, including the claims, are used to describe and account for small fluctuations, such as due to variations in processing. For example, these terms can refer to less than or equal to ±<NUM>% from a stated value, such as less than or equal to ±<NUM>% from a stated value, such as less than or equal to ±<NUM>% from a stated value, such as less than or equal to ±<NUM>% from a stated value, such as less than or equal to ±<NUM>% from a stated value, such as less than or equal to ±<NUM>% from a stated value, such as less than or equal to ±<NUM>% from a stated value.

Adapter: A linear oligonucleotide sequence that can be fused to a nucleic acid molecule, for example, by ligation or tagmentation. Suitable adapter lengths may range from about <NUM> nucleotides to about <NUM> nucleotides, or from about <NUM> nucleotides to about <NUM> nucleotides, or from about <NUM> nucleotides to about <NUM> nucleotides. The adapter may include any combination of nucleotides and/or nucleic acids. In some examples, the adapter can include a sequence that is complementary to at least a portion of a primer, for example, a primer including a universal nucleotide sequence (such as a P5 or P7 sequence). As an example, the adapter at one end of a fragment includes a sequence that is complementary to at least a portion of a first flow cell primer, and the adapter at the other end of the fragment includes a sequence that is identical to at least a portion of a second flow cell primer. The complementary adapter can hybridize to the first flow cell primer, and the identical adapter is a template for its complementary copy, which can hybridize to the second flow cell primer during clustering. In some examples, the adapter can include a sequencing primer sequence or sequencing binding site. Combinations of different adapters may be incorporated into a nucleic acid molecule, such as a DNA fragment.

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

Complex: A carrier, such as a solid support, and sequencing-ready nucleic acid fragments attached to the carrier. The carrier may also include one member of a binding pair whose other member is part of the capture site.

Fragment: A portion or piece of genetic material (e.g., DNA, RNA, etc.). Contiguity preserved library fragments are smaller pieces of the longer nucleic acid sample that has been fragmented, where the smaller fragments are held together in some manner (e.g., by a bead, with a transposome, 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) may refer to a sequence that is to be analyzed.

The nucleotides in a nucleic acid sample may include naturally occurring nucleic acids and functional analogs thereof. Examples of functional analogs are capable of hybridizing to a nucleic acid in a sequence specific fashion or capable of being used as a template for replication of a particular nucleotide sequence. Naturally occurring nucleotides generally have a backbone containing phosphodiester bonds. An analog structure can have an alternate backbone linkage including any of a variety known in the art. Naturally occurring nucleotides generally have a deoxyribose sugar (e.g., found in DNA) or a ribose sugar (e.g., found in RNA). An analog structure can have an alternate sugar moiety including any of a variety known in the art. Nucleotides can include native or non-native bases. A native DNA can include one or more of adenine, thymine, cytosine and/or guanine, and a native RNA can include one or more of adenine, uracil, cytosine and/or guanine. Any non-native base may be used, such as a locked nucleic acid (LNA) and a bridged nucleic acid (BNA).

Primer: A nucleic acid molecule that can hybridize to a target sequence, such as an adapter attached to a contiguity preserved library fragment. As one example, an amplification primer can serve as a starting point for template amplification and cluster generation. As another example, a synthesized nucleic acid (template) strand may include a site to which a primer (e.g., a sequencing primer) can hybridize in order to prime synthesis of a new strand that is complementary to the synthesized nucleic acid (template) strand. Any primer can 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 long and can include a variety of natural and/or non-natural nucleotides. In an example, the sequencing primer is a short strand, ranging from <NUM> to <NUM> bases, or from <NUM> to <NUM> bases.

Sequencing-ready nucleic acid fragments: A portion (e.g., contiguity preserved library fragment) of genetic material having adapters at the <NUM>' and <NUM>' ends. In the sequencing-ready 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. A sequencing-ready nucleic acid fragment may be bound via insertion of transposons bound to the surface of a solid support (e.g., bead), or directly immobilized through a binding pair or other cleavable linker.

Solid support: A small body made of a rigid or semi-rigid material having a shape characterized, for example, as a sphere, oval, microsphere, or other recognized particle shape whether having regular or irregular dimensions. The solid support can have a sequencing library attached thereto. Example materials that are useful for the solid support include, without limitation, glass; plastic, such as acrylic, polystyrene or a copolymer of styrene and another material, polypropylene, polyethylene, polybutylene, polyurethane or polytetrafluoroethylene (TEFLON® from The Chemours Co); polysaccharides or cross-linked polysaccharides such as agarose or Sepharose; nylon; nitrocellulose; resin; silica or silica-based materials including silicon and modified silicon; carbon-fiber, metal; inorganic glass; optical fiber bundle, or a variety of other polymers. Example solid supports include controlled pore glass beads, paramagnetic beads, thoria sol, SEPHAROSE® beads (cross-linked beaded form of agarose, available from Cytivia), nanocrystals and others known in the art as described, for example, in Microsphere Detection Guide from Bangs Laboratories, Fishers Ind.

Transposome: A complex formed between an integration enzyme (e.g., an integrase or a transposase) and a transferrable strand, a non-transferrable strand, or both transferrable and non-transferrable strands.

In the examples disclosed herein, the library fragments that are introduced to the flow cell are contiguity preserved library fragments. Contiguity preserved library fragments are smaller pieces of a longer nucleic acid sample that has been fragmented, where the smaller pieces are physically held together in some manner. In some examples disclosed herein, the contiguity may be preserved using a solid support in library preparation. In other examples disclosed herein, the contiguity may be preserved by creating fragments that are attached to one another during initial library preparation through bound transposomes, introducing the attached fragments to the flow cell, and then completing the library preparation on the flow cell.

<FIG> depicts an example of a method for forming a complex <NUM> including sequencing-ready nucleic acid fragments <NUM> including fragments <NUM> from the larger nucleic acid sample, whose contiguity is preserved on a solid support <NUM>.

In one example method to form the complex <NUM> shown in <FIG>, an adapter sequence <NUM> is bound to the solid support <NUM> through one member <NUM> of a binding pair. In an example, this adapter sequence <NUM> may include a first sequencing primer sequence (e.g., a read <NUM> 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 (shown in <FIG>). The adapter sequence <NUM> is bound to the one member <NUM> of the binding pair (e.g., biotin) so that it can be bound to the surface of the solid support <NUM>, which includes the other member (e.g., avidin, streptavidin, etc.) of the binding pair.

As shown in <FIG> a transposome complex <NUM>" may also be bound to the solid support <NUM>. Prior to loading the transposome complex <NUM>" on the solid support <NUM>, a partial Y-adapter <NUM>' may be mixed with a transposase enzyme <NUM> (which, while not shown, may include two Tn5 molecules) to form an example of the transposome complex <NUM>". The partial Y-adapter <NUM>' may include two mosaic end sequences M<NUM>, M<NUM> that are hybridized to each other. One of the mosaic end sequences M<NUM> has a free end that is able to attach to the fragmented DNA strands during tagmentation, and thus is similar to the transferred strand <NUM> in <FIG>. The other of the mosaic end sequences M<NUM> may be attached to a second sequencing primer sequence (e.g., a read <NUM> sequencing primer sequence), and a second sequence (P7) that has the same sequence as at least a portion of another of the amplification primers (P7) on the flow cell, so that its copy is complementary (e.g., P7') to the amplification primer (P7). In this example, the other of the mosaic end sequences M<NUM>, the second sequencing primer sequence and the second sequence make up adapter sequence <NUM>. The adapter sequences <NUM> are not attached to the fragmented DNA strands during tagmentation, and thus are similar to the non-transferred strands <NUM> in <FIG>.

Loading the transposome complex <NUM>" on the solid support <NUM> may involve mixing the transposome complex <NUM>" with the solid support <NUM>, and exposing the mixture to suitable conditions for ligating the mosaic end M<NUM> of the partial Y-adapter <NUM>' to the <NUM>'-end of the adapter sequence <NUM>. As shown in <FIG>, individual transposome complexes <NUM>" may be attached to each of the adapter sequences <NUM> on the solid support <NUM>.

In this example method to form the complex <NUM>, a tagmentation process may then be performed. A fluid (e.g., a tagmentation buffer) including the longer nucleic acid sample <NUM> (e.g., DNA) may be added to the solid support <NUM> having the adapter sequence <NUM> and the transposome complexes <NUM>" bound thereto. As the sample contacts the transposome complexes <NUM>", the longer nucleic acid sample is tagmented. During this example of tagmentation, the sample <NUM> is fragmented into fragments <NUM>, <NUM>', and each of the fragments <NUM>, <NUM>' is tagged, at its <NUM>' end, with the free end of the mosaic end M<NUM> of the partial Y-adapter <NUM>'.

As shown in <FIG>, tagmentation of the longer nucleic acid sample <NUM> results in a plurality of bridged molecules between the transposome complexes <NUM>". The bridged molecules wrap around the solid support <NUM>. The transposome complexes <NUM>" and the adapter sequences <NUM> maintain the contiguity of the nucleic acid sample <NUM> as bridged molecules, and thus the bridged molecules are the contiguity preserved library fragments <NUM>, <NUM>'.

The transposase enzyme may then be removed via sodium dodecyl sulfate (SDS) treatment or heat or proteinase K digestion. Removal of the transposase enzymes leaves the contiguity preserved library fragments <NUM>, <NUM>' attached to the solid support <NUM>.

To complete the sequencing ready fragments, further extension and ligation (denoted by the stars in <FIG>) is undertaken to ensure fragments <NUM> and <NUM>' are attached to sequences <NUM>. The resulting complex <NUM> is shown in <FIG>.

Each contiguity preserved library fragment <NUM>, <NUM>' is part of a respective sequencing-ready nucleic acid fragments <NUM>, <NUM>', each of which also includes respective adapter sequences <NUM> and <NUM> attached at either end. The adapter sequences <NUM> are those initially bound to the solid support <NUM>, and include the first sequencing primer sequence and the first sequence complementary to one of the flow cell primers. The adapter sequences <NUM> are attached to the one member <NUM> of a binding pair. The adapter sequences <NUM> are from the partial Y-adapter <NUM>', and include the second sequence identical to another flow cell primer and the second sequencing primer sequence. Because each sequencing-ready nucleic acid fragment <NUM>, <NUM>' includes suitable adapters for bridge amplification and sequencing, PCR amplification is not performed. These fragments <NUM>, <NUM>' are thus sequencing-ready. Moreover, because the contiguity preserved library fragments <NUM>, <NUM>' are from the same longer nucleic acid sample <NUM>, the contiguity preserved library fragments <NUM>, <NUM>' may be suitable for the linked long read applications disclosed herein.

Another example method for forming another example complex <NUM>' (<FIG>) is depicted in <FIG>.

In this example method, the adapter sequence <NUM>' is bound to the solid support <NUM> through one member <NUM> of a binding pair. In an example, this adapter sequence <NUM>' may include a hybridizable sequence H, a first sequencing primer sequence (e.g., a read <NUM> 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, so that its copy is complementary (e.g., P5') to the amplification primer (P5). The adapter sequence <NUM>' is bound to the one member <NUM> of the binding pair (e.g., biotin) so that it can be bound to the surface of the solid support <NUM>, which includes the other member (e.g., avidin, streptavidin, etc.) of the binding pair.

As shown in <FIG>, a transposome complex <NUM> may also be bound to the solid support <NUM>. Prior to loading the transposome complex <NUM> on the solid support <NUM>, an L-adapter <NUM> may be mixed with a transposase enzyme <NUM> (e.g., including two Tn5 molecules) to form an example of the transposome complex <NUM>. The L-adapter <NUM> may include two mosaic end sequences M<NUM>, M<NUM> that are hybridized to each other. One of the mosaic end sequences M<NUM> is a transferred strand <NUM> that is added to one end of each fragment <NUM>, <NUM>' during a ligation process that takes place after a tagmentation process. The other of the mosaic end sequences M<NUM> is part of a non-transferred strand <NUM> that is removed after the ligation and tagmentation processes. This mosaic end sequence M<NUM> is attached to a complementary hybridizable sequence HC which is complementary to the hybridizable sequence H of the adapter sequence <NUM>' attached to the solid support <NUM>. The complementary hybridizable sequence HC enables the L-adapter <NUM> to hybridize to the adapter sequence <NUM>'. Thus, the complementary hybridizable sequence HC enables the transposome complex <NUM> to be loaded onto the solid support.

Loading the transposome complex <NUM> on the solid support 16_may involve mixing the transposome complex <NUM> with the solid support <NUM>, and exposing the mixture to suitable conditions for the hybridization of the complementary hybridizable sequence HC of the L-adapter <NUM> to the hybridizable sequence H of the adapter sequence <NUM>'. As shown in <FIG>, individual transposome complex <NUM> may be attached to each of the adapter sequences <NUM>' on the solid support <NUM>.

In this example method to form the complex <NUM>', a tagmentation process is then performed. A fluid (e.g., a tagmentation buffer) including the longer nucleic acid sample (e.g., DNA) <NUM> may be added to the solid support <NUM> having the transposome complex <NUM> loaded thereon. As the sample <NUM> contacts the solid support-bound transposome complexes <NUM>, the longer nucleic acid sample <NUM> is tagmented. During this example of tagmentation, the sample <NUM> is fragmented into fragments <NUM>, <NUM>', and each of the fragments <NUM>, <NUM>' is tagged, at its <NUM>'-end, with the mosaic end sequence M<NUM> of the L-adapter <NUM>.

As shown in <FIG>, tagmentation of the longer nucleic acid sample <NUM> results in a plurality of bridged molecules between adjacent transposome complexes <NUM>, and thus adjacent adapter sequences <NUM>'. The bridged molecules wrap around the solid support <NUM>. The transposome complexes <NUM> and adapter sequences <NUM>' maintain the contiguity of the nucleic acid sample <NUM> as bridged molecules, and thus the bridged molecules are the contiguity preserved library fragments <NUM>, <NUM>'.

The transposase enzyme <NUM> may then be removed via sodium dodecyl sulfate (SDS) treatment or heat or proteinase K digestion.

Ligation may then be performed to bind the free mosaic end sequences M<NUM> to the respective adapter sequences <NUM>'. In <FIG>, the stars represent where ligation is performed. In an example, ligation may be initiated by introducing a buffer containing a suitable ligase, and heating to about suitable temperature for a suitable time to initiate enzyme activity. Examples of suitable ligase enzymes include E. Coli DNA ligase, T7 ligase, etc. In an example, the buffer containing E. Coli DNA ligase may also include nicotinamide adenine dinucleotide (NAD+). In this example, heating to about <NUM> for about <NUM> minutes initiates the enzyme activity. The resulting structure is shown in <FIG>.

The non-transferred strand <NUM> of the L-adapter <NUM> may then be removed. In this example, each of the non-transferred strands <NUM> is removed using any suitable <NUM>'-<NUM>' exonuclease enzyme <NUM>, such as T7 exonuclease. In an example, non-transferred strand removal may be initiated by introducing a buffer containing the <NUM>'-<NUM>' exonuclease enzyme <NUM>, and waiting for a predetermined time. The <NUM>'-<NUM>' exonuclease enzyme <NUM> is able to digest the non-transferred strand <NUM> at room temperature (e.g., from about <NUM> to about <NUM>), and thus additional heating is not used. The digested non-transferred strands <NUM> can then be washed away.

Using an exonuclease enzyme to remove the non-transferred strands <NUM> may be more desirable than using heat denaturation. The <NUM>'-<NUM>' exonuclease enzyme <NUM> efficiently digests the non-transferred strands <NUM>, yielding a cleaner template (than when heat denaturation is used) for hybridization of a subsequently attached adapter sequence (see reference numerals <NUM> in <FIG>). This can improve the library yield. Additionally, removing the non-transferred strands <NUM> via <NUM>'-<NUM>' exonuclease enzyme <NUM> can improve library coverage over adenine (A) and thymine (T) rich regions, losses of which have been observed after heat denaturation of the non-transferred strands <NUM> (see <FIG>). Still further, digestion via the exonuclease enzyme does not increase the time of the overall library preparation process.

Referring now to <FIG>, this example of the method to form the complex <NUM>' involves introducing a partial Y-adapter <NUM>. The partial Y-adapter <NUM> includes a mosaic end sequence M<NUM> that is complementary to the mosaic end sequence M<NUM> and an adapter sequence <NUM>. The adapter sequence <NUM> may include a second sequencing primer sequence (e.g., a read <NUM> sequencing primer sequence), and a second sequence (P7') that is complementary to another of the amplification primers (P7) on the flow cell. As shown in <FIG>, the mosaic end sequence M<NUM> of the partial Y-adapter <NUM> hybridizes to the mosaic end sequence M<NUM> of the transferred strand <NUM> (now ligated to the adapter sequence <NUM>'), and thus attaches the partial Y-adapter <NUM> to the solid support <NUM>.

In the examples disclosed herein, the adapter sequences <NUM>, <NUM>' and/or <NUM> may also include a sequencing sample index or a barcode sequence. These sequences may be used as a backup or alternative to the compartmentalization methods disclosed herein.

To generate sequencing ready fragments, further extension and ligation is undertaken to ensure fragments <NUM> and <NUM>' are attached to the mosaic end sequence M<NUM>, and thus the sequences <NUM>.

The resulting complex <NUM>' is shown in <FIG>.

Yet another example method for forming the complex <NUM>' (shown in <FIG>) is partially depicted in <FIG>. In this example method, ligation of the transferred strand <NUM> and digestion of the non-transferred strand <NUM> are performed as part of a single, one-pot protocol.

In this example method, the adapter sequence <NUM>' is bound to the solid support <NUM> through one member <NUM> of a binding pair. The adapter sequence <NUM>' as described in reference to <FIG> may be used, which includes the hybridizable sequence H, the first sequencing primer sequence (e.g., a read <NUM> sequencing primer sequence), and the first sequence (P5) that is identical to at least a portion of one of the amplification primers (e.g., P5) on the flow cell, so that its copy is complementary (e.g., P5') to the amplification primer (P5). Also in this example method, the transposome complex <NUM> is loaded on the solid support <NUM> as described in reference to <FIG>. Briefly, the complementary hybridizable sequence HC of the L-adapter <NUM> of the transposome complex <NUM> is hybridized to the hybridizable sequence H of the adapter sequence <NUM>'.

In this example method to form the complex <NUM>' (<FIG>), tagmentation is performed as described in reference to <FIG>.

Ligation of the transferred strand <NUM> and digestion of the non-transferred strand <NUM> may then be performed together. This is schematically illustrated in <FIG>. For the ligase and the exonuclease enzyme to work synergistically, the reagent formulation that is introduced to the tagmented solid support includes a buffer, the ligase enzyme, the <NUM>'-<NUM>' exonuclease enzyme <NUM> (e.g., T7 exonuclease), and any other component required by the respective enzymes (e.g., a cofactor, such as NAD+). In an example, ligation and digestion may be initiated by introducing the reagent formulation, and heating to about <NUM> for about <NUM> minutes to initiate enzyme activity.

Incorporating the ligase and the exonuclease enzyme into the same reagent formulation can decrease protocol time (e.g., by about <NUM> minutes compared to the example method shown in <FIG>), and also reduces the number of wash steps.

This example of the method then continues with introducing the partial Y-adapter <NUM>, as described in reference to <FIG>. To generate sequencing ready fragments and the final complex <NUM>', further extension and ligation is undertaken to ensure fragments <NUM> and <NUM>' are attached to the mosaic end sequence M<NUM>, and thus the sequences <NUM>.

The methods described in reference to <FIG>, <FIG>, and <FIG> for making the complexes <NUM>, <NUM>' provide a few examples, but it is to be understood that other methods may be used as long as sequencing-ready nucleic acid fragments <NUM>, <NUM>' are attached to the solid support <NUM>.

In other examples disclosed herein, the contiguity information may be preserved by performing a portion of the library preparation off of the flow cell, and performing a portion of the library preparation on the flow cell.

In this example, library preparation may be initiated outside of the flow cell using tagmentation, as shown schematically in <FIG>.

In the example shown, a fluid (e.g., a tagmentation buffer) including the longer nucleic acid sample <NUM> (e.g., double stranded DNA) may be mixed with transposome complexes <NUM>'. In the example shown in <FIG>, each transposome complex <NUM>' is a dimer, including two transposase enzymes (which are collectively shown as "<NUM>" in <FIG>), a transferred strand <NUM>', and a non-transferred strand <NUM>'. In other examples, different transposome complexes may be used, for example, one of which includes the transposase enzymes <NUM> and the transferred strand <NUM>' and another of which includes the transposase enzymes <NUM> and the non-transferred strand <NUM>'.

In this example, the transferred strands <NUM>' are adapters that are added to one end of each fragment <NUM>, <NUM>' during the tagmentation process. In an example, each transferred strand <NUM> is an adapter including a first sequencing primer sequence (e.g., a read <NUM> 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.

In this example, the non-transferred strands <NUM>' are adapters that are not incorporated into the fragment <NUM>, <NUM>' during tagmentation, but rather, may be subsequently ligated to the other end of each fragment <NUM>, <NUM>'. As shown in <FIG>, the non-transferred strands <NUM>' may be attached to the transferred strand <NUM>' via at least partial base-pairing during tagmentation. In this example, the non-transferred strand <NUM>' is an adapter including a second sequencing primer sequence (e.g., a read <NUM> 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, so that its copy is complementary (e.g., P7') to the amplification primer (P7).

As shown in <FIG>, within the fluid, the transposomes <NUM>' fragment the longer nucleic acid sample <NUM> into fragments <NUM>, <NUM>' and ligate the transferred strands <NUM>' to the <NUM>' end of each fragment <NUM>, <NUM>'. In an example, the transferred strand <NUM>' is incorporated to the <NUM>'-end of each fragment <NUM>, <NUM>' of the longer nucleic acid sample <NUM> by one-sided transposition. The non-transferred strands <NUM>' may be attached to the transferred strand <NUM>' via base-pairing.

This example tagmentation process maintains the contiguity of the longer nucleic acid sample <NUM> because the generated fragments <NUM>, <NUM>' (and any transferred strands <NUM>' and non-transferred strands <NUM>' directly or indirectly attached thereto) remain attached to one another via the transposase <NUM>. The attached contiguity preserved library fragments <NUM>, <NUM>' are referred to herein as the attached fragments <NUM>.

As mentioned herein, the attached fragments <NUM> may be introduced to the flow cell, where additional processing may be performed to complete library preparation. This is shown schematically in <FIG>, which will be described further in reference to the methods disclosed herein.

The methods disclosed herein may utilize a flow cell <NUM>, an example of which is depicted in <FIG>. The flow cell <NUM> includes a substrate <NUM> that at least partially define a lane or flow channel <NUM>.

The substrate <NUM> may be a single layer/material. Examples of suitable single layer substrates include epoxy siloxane, glass, modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene (such as TEFLON® from Chemours), cyclic olefins/cyclo-olefin polymers (COP) (such as ZEONOR® from Zeon), polyimides, etc.), nylon (polyamides), ceramics/ceramic oxides, silica, fused silica, or silica-based materials, aluminum silicate, silicon and modified silicon (e.g., boron doped p+ silicon), silicon nitride (Si<NUM>N<NUM>), silicon oxide (SiO<NUM>), tantalum pentoxide (Ta<NUM>O<NUM>) or other tantalum oxide(s) (TaOx), hafnium oxide (HaO<NUM>), carbon, metals, inorganic glasses, or the like. The substrate <NUM> may also be a multi-layered structure. Some examples of the multi-layered structure include glass or silicon, with a coating layer of tantalum oxide or another ceramic oxide at the surface. Other examples of the multi-layered structure include an underlying support (e.g., glass or silicon) having a patterned resin thereon. Still other examples of the multi-layered substrate may include a silicon-on-insulator (SOI) substrate.

In an example, the substrate <NUM> may have a diameter ranging from about <NUM> to about <NUM>, or a rectangular sheet or panel having its largest dimension up to about <NUM> feet (~ <NUM> meters). In an example, the substrate <NUM> is a wafer having a diameter ranging from about <NUM> to about <NUM>. In another example, the substrate <NUM> is a die having a width ranging from about <NUM> to about <NUM>. While example dimensions have been provided, it is to be understood that a substrate <NUM> with any suitable dimensions may be used. For another example, a panel may be used that is a rectangular support, which has a greater surface area than a <NUM> round wafer.

In the example shown in <FIG>, the flow cell <NUM> includes flow channels <NUM>. While several flow channels <NUM> are shown, it is to be understood that any number of channels <NUM> may be included in the flow cell <NUM> (e.g., a single channel <NUM>, four channels <NUM> etc.). Each flow channel <NUM> is an area defined between two bonded components (e.g., the substrate <NUM> and a lid or two substrates <NUM>), which can have fluids (e.g., those describe herein) introduced thereto and removed therefrom. Each flow channel <NUM> may be isolated from each other flow channel <NUM> so that fluid introduced into any particular flow channel <NUM> does not flow into any adjacent flow channel <NUM>. Some examples of the fluids introduced into the flow channels <NUM> may introduce reaction components (e.g., contiguity preserved library fragments <NUM>, <NUM>' (e.g., on the solid support <NUM> or attached to one another), polymerases, sequencing primers, nucleotides, etc.), washing solutions, deblocking agents, etc..

The flow channel <NUM> may be defined in the substrate <NUM> using any suitable technique that depends, in part, upon the material(s) of the substrate <NUM>. In one example, the flow channel <NUM> is etched into a glass substrate <NUM>. In another example, the flow channel <NUM> may be patterned into a resin of a multi-layered substrate <NUM> using photolithography, nanoimprint lithography, etc. In still another example, a separate material (not shown) may be applied to the substrate <NUM> so that the separate material defines the walls of the flow channel <NUM> and the substrate <NUM> defines the bottom of the flow channel <NUM>.

In an example, the flow channel <NUM> has a rectilinear configuration. The length and width of the flow channel <NUM> may be smaller, respectively, than the length and width of the substrate <NUM> so that portion of the substrate surface surrounding the flow channel <NUM> is available for attachment to a lid (not shown) or another substrate <NUM>. In some instances, the width of each flow channel <NUM> can be at least about <NUM>, at least about <NUM>, at least about <NUM>, at least about <NUM>, at least about <NUM>, or more. In some instances, the length of each lane/flow channel <NUM> can be at least about <NUM>, at least about <NUM>, at least about <NUM>, at least about <NUM>, or more. The width and/or length of each flow channel <NUM> can be greater than, less than or between the values specified above. In another example, the flow channel <NUM> is square (e.g., <NUM> x <NUM>).

The depth of each flow channel <NUM> can be as small as a monolayer thick when microcontact, aerosol, or inkjet printing is used to deposit a separate material that defines the flow channel walls. The depth may be larger when a separate material (not shown) is used to bond the lid to the substrate <NUM> or to bond two substrates <NUM> together. For other examples, the depth of each flow channel <NUM> can be about <NUM>, about <NUM>, about <NUM>, about <NUM>, or more. In an example, the depth may range from about <NUM> to about <NUM>. In another example, the depth may range from about <NUM> to about <NUM>. In still another example, the depth is about <NUM> or less. It is to be understood that the depth of each flow channel <NUM> be greater than, less than or between the values specified above.

Different examples of the architecture within the flow channels <NUM> of the flow cell <NUM> are shown <FIG>.

In the example shown in <FIG>, the flow cell <NUM> includes a single layer substrate 38A and the flow channel <NUM> defined at least partially in the single layer substrate 38A.

A polymeric hydrogel <NUM> is present in the flow channel <NUM>. An example of the polymeric hydrogel <NUM> includes an acrylamide copolymer, such as poly(N-(<NUM>-azidoacetamidylpentyl)acrylamide-co-acrylamide, PAZAM. PAZAM and some other forms of the acrylamide copolymer are represented by the following structure (I):
<CHM>
wherein:.

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

The molecular weight of PAZAM and other forms of the acrylamide copolymer may range from about <NUM> kDa to about <NUM> kDa or from about <NUM> kDa to about <NUM> kDa, or may be, in a specific example, about <NUM> kDa.

In some examples, PAZAM and other forms of the acrylamide copolymer are linear polymers. In some other examples, PAZAM and other forms of the acrylamide copolymer are a lightly cross-linked polymers.

In other examples, the polymeric hydrogel <NUM> may be a variation of the structure (I). In one example, the acrylamide unit may be replaced with N,N-dimethylacrylamide
<CHM>
In this example, the acrylamide unit in structure (I) may be replaced with
<CHM>
where RD, RE, and RF are each H or a C1-C6 alkyl, and RG and RH are each a C1-C6 alkyl (instead of H as is the case with the acrylamide). In this example, q may be an integer in the range of <NUM> to <NUM>,<NUM>. In another example, the N,N-dimethylacrylamide may be used in addition to the acrylamide unit. In this example, structure (I) may include
<CHM>
in addition to the recurring "n" and "m" features, where RD, RE, and RF are each H or a C1-C6 alkyl, and RG and RH are each a C1-C6 alkyl. In this example, q may be an integer in the range of <NUM> to <NUM>,<NUM>.

As another example of the initial polymeric hydrogel, the recurring "n" feature in structure (I) may be replaced with a monomer including a heterocyclic azido group having structure (II):
<CHM>
wherein R<NUM> is H or a C1-C6 alkyl; R<NUM> is H or a C1-C6 alkyl; L is a linker including a linear chain with <NUM> to <NUM> atoms selected from the group consisting of carbon, oxygen, and nitrogen and <NUM> optional substituents on the carbon and any nitrogen atoms in the chain; E is a linear chain including <NUM> to <NUM> atoms selected from the group consisting of carbon, oxygen and nitrogen, and optional substituents on the carbon and any nitrogen atoms in the chain; A is an N substituted amide with an H or a C1-C4 alkyl attached to the N; and Z is a nitrogen containing heterocycle. Examples of Z include <NUM> to <NUM> ring members present as a single cyclic structure or a fused structure.

As still another example, the polymeric hydrogel <NUM> may include a recurring unit of each of structure (III) and (IV):
<CHM>
wherein each of R1a, R2a, R1b and R2b is independently selected from hydrogen, an optionally substituted alkyl or optionally substituted phenyl; each of R3a and R3b is independently selected from hydrogen, an optionally substituted alkyl, an optionally substituted phenyl, or an optionally substituted C7-C14 aralkyl; and each L<NUM> and L<NUM> is independently selected from an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker.

It is to be understood that other molecules may be used to form the polymeric hydrogel <NUM>, as long as they are functionalized to graft oligonucleotide primers <NUM>, <NUM> thereto. Other examples of suitable polymer layers include those having a colloidal structure, such as agarose; or a polymer mesh structure, such as gelatin; or a cross-linked polymer structure, such as polyacrylamide polymers and copolymers, silane free acrylamide (SFA), or an azidolyzed version of SFA. Examples of suitable polyacrylamide polymers may be synthesized from acrylamide and an acrylic acid or an acrylic acid containing a vinyl group, or from monomers that form [<NUM>+<NUM>] photo-cycloaddition reactions. Still other examples of suitable polymeric hydrogels <NUM> include mixed copolymers of acrylamides and acrylates. A variety of polymer architectures containing acrylic monomers (e.g., acrylamides, acrylates etc.) may be utilized in the examples disclosed herein, such as branched polymers, including star polymers, star-shaped or star-block polymers, dendrimers, and the like. For example, the monomers (e.g., acrylamide, acrylamide containing the catalyst, etc.) may be incorporated, either randomly or in block, into the branches (arms) of a star-shaped polymer.

To introduce the polymeric hydrogel <NUM> into the flow channel <NUM>, a mixture of the polymeric hydrogel <NUM> may be generated and then applied to the substrate 38A (having the flow channel <NUM> defined at least partially therein). In one example, the polymeric hydrogel <NUM> may be present in a mixture (e.g., with water or with ethanol and water). The mixture may then be applied to the substrate surfaces (including in the flow channel(s) <NUM>) using spin coating, or dipping or dip coating, spray coating, or flow of the material under positive or negative pressure, or another suitable technique. These types of techniques blanketly deposit the polymeric hydrogel <NUM> on the substrate 38A (e.g., in the flow channel <NUM> and on interstitial regions <NUM> surrounding the flow channel <NUM>). Other selective deposition techniques (e.g. involving a mask, controlled printing techniques, etc.) may be used to specifically deposit the polymeric hydrogel <NUM> in the flow channel <NUM> and not on the interstitial regions <NUM>.

In some examples, the substrate surface (including the portion that is exposed in the flow channel <NUM>) may be activated, and then the mixture (including the polymeric hydrogel <NUM>) may be applied thereto. In one example, a silane or silane derivative (e.g., norbornene silane) may 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 generate surface-activating agent(s) (e.g., -OH groups) that can adhere to the polymeric hydrogel <NUM>.

Depending upon the polymeric hydrogel <NUM>, the applied mixture may be exposed to a curing process. In an example, curing may take place at a temperature ranging from room temperature (e.g., about <NUM>) to about <NUM> for a time ranging from about <NUM> millisecond to about several days.

In some examples, polishing may then be performed in order to remove the polymeric hydrogel <NUM> from the interstitial regions <NUM> at the perimeter of the flow channel(s) <NUM>, while leaving the polymeric hydrogel <NUM> on the surface in the flow channel(s) <NUM> at least substantially intact.

The flow cell <NUM> also includes amplification primers <NUM>, <NUM>.

A grafting process may be performed to graft the amplification primers <NUM>, <NUM> to the polymeric hydrogel <NUM> in the flow channel <NUM>. In an example, the amplification primers <NUM>, <NUM> can be immobilized to the polymeric hydrogel <NUM> by single point covalent attachment at or near the <NUM>' end of the primers <NUM>, <NUM>. This attachment leaves i) an adapter-specific portion of the primers <NUM>, <NUM> free to anneal to its cognate nucleic acid fragment (e.g., the P5' portion attached to the fragment <NUM>, <NUM>') and ii) the <NUM>' hydroxyl group free for primer extension. Any suitable covalent attachment may be used for this purpose. Examples of terminated primers that may be used include alkyne terminated primers, which can attach to an azide moiety of the polymeric hydrogel <NUM>. Specific examples of suitable primers <NUM>, <NUM> include P5 and P7 primers used on the surface of commercial flow cells sold by Illumina Inc. for sequencing on HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NEXTSEQ™, NEXTSEQDX™, NOVASEQ™, GENOME ANALYZER™, ISEQ™, and other instrument platforms.

In an example, grafting may involve flow through deposition (e.g., using a temporarily bound or permanently bonded lid), dunk coating, spray coating, puddle dispensing, or by another suitable method that will attach the primer(s) <NUM>, <NUM> to the polymeric hydrogel <NUM> in the flow channel <NUM>. Each of these example techniques may utilize a primer solution or mixture, which may include the primer(s) <NUM>, <NUM>, water, a buffer, and a catalyst. With any of the grafting methods, the primers <NUM>, <NUM> react with reactive groups of polymeric hydrogel <NUM> in the flow channel <NUM> and have no affinity for the surrounding substrate 38A. As such, the primers <NUM>, <NUM> selectively graft to the polymeric hydrogel <NUM> in the flow channel <NUM>.

In the example shown in <FIG>, the flow cell <NUM> includes a multilayer substrate 38B, which includes a support <NUM> and a patterned material <NUM> positioned on the support <NUM>. The patterned material <NUM> defines depressions <NUM> separated by interstitial regions <NUM>. The depressions <NUM> are located within each of the flow channel(s) <NUM>.

In the example shown in <FIG>, the patterned material <NUM> is positioned on the support <NUM>. It is to be understood that any material that can be selectively deposited, or deposited and patterned to form the depressions <NUM> and the interstitial regions <NUM> may be used for the patterned material <NUM>.

As one example, an inorganic oxide may be selectively applied to the support <NUM> via vapor deposition, aerosol printing, or inkjet printing. Examples of suitable inorganic oxides include tantalum oxide (e.g., Ta<NUM>O<NUM>), aluminum oxide (e.g., Al<NUM>O<NUM>), silicon oxide (e.g., SiO<NUM>), hafnium oxide (e.g., HfO<NUM>), etc..

As another example, a resin may be applied to the support <NUM> and then patterned. Suitable deposition techniques include chemical vapor deposition, dip coating, dunk coating, spin coating, spray coating, puddle dispensing, ultrasonic spray coating, doctor blade coating, aerosol printing, screen printing, microcontact printing, etc. Suitable patterning techniques include photolithography, nanoimprint lithography (NIL), stamping techniques, embossing techniques, molding techniques, microetching techniques, printing techniques, etc. Some examples of suitable resins include a polyhedral oligomeric silsesquioxane resin (POSS)-based resin, a non-POSS epoxy resin, a poly(ethylene glycol) resin, a polyether resin (e.g., ring opened epoxies), an acrylic resin, an acrylate resin, a methacrylate resin, an amorphous fluoropolymer resin (e.g., CYTOP® from Bellex), and combinations thereof.

As used herein, the term "polyhedral oligomeric silsesquioxane" (POSS) refers to a chemical composition that is a hybrid intermediate (e.g., RSiO<NUM>) between that of silica (SiO<NUM>) and silicone (R<NUM>SiO). An example of POSS can be that described in <NPL>. In an example, the composition is an organosilicon compound with the chemical formula [RSiO<NUM>/<NUM>]n, where the R groups can be the same or different. Example R groups for POSS include epoxy, azide/azido, a thiol, a poly(ethylene glycol), a norbornene, a tetrazine, acrylates, and/or methacrylates, or further, for example, alkyl, aryl, alkoxy, and/or haloalkyl groups. The resin composition disclosed herein may comprise one or more different cage or core structures as monomeric units. The polyhedral structure may be a T<NUM> structure, such as:
<CHM>
and represented by:
<CHM>. This monomeric unit typically has eight arms of functional groups R<NUM> through R<NUM>.

The monomeric unit may have a cage structure with <NUM> silicon atoms and <NUM> R groups, referred to as T<NUM>, such as:
<CHM>
or may have a cage structure with <NUM> silicon atoms and <NUM> R groups, referred to as T<NUM>, such as:
<CHM>
The POSS-based material may alternatively include T<NUM>, T<NUM>, or T<NUM> cage structures. The average cage content can be adjusted during the synthesis, and/or controlled by purification methods, and a distribution of cage sizes of the monomeric unit(s) may be used in the examples disclosed herein.

As shown in <FIG>, the patterned material <NUM> includes the depressions <NUM> defined therein, and interstitial regions <NUM> separating adjacent depressions <NUM>. Many different layouts of the depressions <NUM> may be envisaged, including regular, repeating, and non-regular patterns. In an example, the depressions <NUM> are disposed in a hexagonal grid for close packing and improved density. Other layouts may include, for example, rectilinear (rectangular) layouts, triangular layouts, and so forth. In some examples, the layout or pattern can be an x-y format of depressions <NUM> that are in rows and columns. In some other examples, the layout or pattern can be a repeating arrangement of depressions <NUM> and/or interstitial regions <NUM>. In still other examples, the layout or pattern can be a random arrangement of depressions <NUM> and/or interstitial regions <NUM>. The pattern may include spots, pads, stripes, swirls, lines, triangles, rectangles, circles, arcs, checks, plaids, diagonals, arrows, squares, and/or cross-hatches.

The layout or pattern of the depressions <NUM> may be characterized with respect to the density of the depressions <NUM> (number of depressions <NUM>) in a defined area. For example, the depressions <NUM> may be present at a density of approximately <NUM> million per mm<NUM>. The density may be tuned to different densities including, for example, a density of about <NUM> per mm<NUM>, about <NUM>,<NUM> per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, or more, or less. It is to be further understood that the density of depressions <NUM> in the patterned material <NUM> can be between one of the lower values and one of the upper values selected from the ranges above. As examples, a high density array may be characterized as having depressions <NUM> separated by less than about <NUM>, a medium density array may be characterized as having depressions <NUM> separated by about <NUM> to about <NUM>, and a low density array may be characterized as having depressions <NUM> separated by greater than about <NUM>. While example densities have been provided, it is to be understood that any suitable densities may be used. The density of the depressions <NUM> may depend, in part, on the depth of the depressions <NUM>. In some instances, it may be desirable for the spacing between depressions <NUM> to be even greater than the examples listed herein.

The layout or pattern of the depressions <NUM> may also or alternatively be characterized in terms of the average pitch, or the spacing from the center of the depression <NUM> to the center of an adjacent depression <NUM> (center-to-center spacing) or from the edge of one depression <NUM> to the edge of an adjacent depression <NUM> (edge-to-edge spacing). The pattern can be regular, such that the coefficient of variation around the average pitch is small, or the pattern can be non-regular in which case the coefficient of variation can be relatively large. In either case, the average pitch can be, for example, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or more or less. The average pitch for a particular pattern of depressions <NUM> can be between one of the lower values and one of the upper values selected from the ranges above. In an example, the depressions <NUM> have a pitch (center-to-center spacing) of about <NUM>. While example average pitch values have been provided, it is to be understood that other average pitch values may be used.

The size of each depression <NUM> may be characterized by its volume, opening area, depth, and/or diameter.

Each depression <NUM> can have any volume that is capable of confining a fluid. The minimum or maximum volume can be selected, for example, to accommodate the throughput (e.g., multiplexity), resolution, nucleotides, or analyte reactivity expected for downstream uses of the flow cell <NUM>. For example, the volume can be at least about <NUM>×<NUM>-<NUM> µm<NUM>, at least about <NUM>×<NUM>-<NUM> µm<NUM>, at least about <NUM><NUM>, at least about <NUM><NUM>, at least about <NUM><NUM>, at least about <NUM><NUM>, or more. Alternatively or additionally, the volume can be at most about <NUM>×<NUM><NUM> µm<NUM>, at most about <NUM>×<NUM><NUM> µm<NUM>, at most about <NUM><NUM>, at most about <NUM><NUM>, at most about <NUM><NUM>, at most about <NUM><NUM>, or less.

The area occupied by each depression opening can be selected based upon similar criteria as those set forth above for the volume. For example, the area for each depression opening can be at least about <NUM>×<NUM>-<NUM> µm<NUM>, at least about <NUM>×<NUM>-<NUM> µm<NUM>, at least about <NUM><NUM>, at least about <NUM><NUM>, at least about <NUM><NUM>, at least about <NUM><NUM>, or more. Alternatively or additionally, the area can be at most about <NUM>×<NUM><NUM> µm<NUM>, at most about <NUM><NUM>, at most about <NUM><NUM>, at most about <NUM><NUM>, at most about <NUM><NUM>, at most about <NUM>×<NUM>-<NUM> µm<NUM>, or less. The area occupied by each depression opening can be greater than, less than or between the values specified above.

The depth of each depression <NUM> can large enough to house some of the polymeric hydrogel <NUM>. In an example, the depth may be at least about <NUM>, at least about <NUM>, at least about <NUM>, at least about <NUM>, at least about <NUM>, or more. Alternatively or additionally, the depth can be at most about <NUM>×<NUM><NUM> µm, at most about <NUM>, at most about <NUM>, or less. In some examples, the depth is about <NUM>. The depth of each depression <NUM> can be greater than, less than or between the values specified above.

In some instances, the diameter or length and width of each depression <NUM> can be at least about <NUM>, at least about <NUM>, at least about <NUM>, at least about <NUM>, at least about <NUM>, at least about <NUM>, or more. Alternatively or additionally, the diameter or length and width can be at most about <NUM>×<NUM><NUM> µm, at most about <NUM>, at most about <NUM>, at most about <NUM>, at most about <NUM>, at most about <NUM>, or less (e.g., about <NUM>). In some examples, the diameter or length and width is about <NUM>. The diameter or length and width of each depression <NUM> can be greater than, less than or between the values specified above.

In the example shown in <FIG>, the polymeric hydrogel <NUM> is positioned within each of the depressions <NUM>. The polymeric hydrogel <NUM> may be applied as described in reference to <FIG>, so that the polymeric hydrogel <NUM> is present in the depressions <NUM> and not present on the surrounding interstitial regions <NUM>.

While not shown in <FIG>, it is to be understood that the flow cell <NUM> may also include a lid attached to the substrate <NUM>. In an example, the lid may be bonded to at least a portion of the substrate <NUM>, e.g., at some of the interstitial regions <NUM>. The bond that is formed between the lid and the substrate <NUM> may be a chemical bond, or a mechanical bond (e.g., using a fastener, etc.).

The lid may be any material that is transparent to an excitation light that is directed toward the substrate <NUM>. As examples, the lid may be glass (e.g., borosilicate, fused silica, etc.), plastic, or the like. A commercially available example of a suitable borosilicate glass is D <NUM>®, available from Schott North America, Inc. Commercially available examples of suitable plastic materials, namely cyclo olefin polymers, are the ZEONOR® products available from Zeon Chemicals L.

The lid may be bonded to the substrate <NUM> using any suitable technique, such as laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma activation bonding, glass frit bonding, or others methods known in the art. In an example, a spacer layer may be used to bond the lid to the substrate <NUM>. The spacer layer may be any material that will seal at least some of the substrate <NUM> and the lid together. In some examples, the spacer layer can be a radiation-absorbing material that aids in bonding of the substrate <NUM> and the lid.

In other examples, the flow cell <NUM> may also include an additional patterned or non-patterned substrate <NUM> attached to the substrate <NUM>. The substrates <NUM> may be bonded as described herein.

The method generally includes generating a series of time-based clustering images for a plurality of contiguity preserved library fragments <NUM>, <NUM>' from a genome sample. Each time-based clustering image in the series is sequentially generated by introducing, to a flow cell <NUM>, a respective sample including some of the contiguity preserved library fragments <NUM>, <NUM>', wherein the some of the contiguity preserved library fragments <NUM>, <NUM>' are attached to a solid support <NUM> (<FIG>) or are attached to each other (e.g., attached fragments <NUM> shown in <FIG>); initiating release of the contiguity preserved library fragments <NUM>, <NUM>' from the solid support <NUM> or from each other <NUM>; amplifying the contiguity preserved library fragments <NUM>, <NUM>' to generate a plurality of respective template strands; staining the respective template strands; and imaging the respective template strands.

With the method(s) disclosed herein, the contiguity preserved library fragments <NUM>, <NUM>' are used in combination with sequential amplification and imaging in order to generate the series of time-based clustering images. Each time-based clustering image records the spatial location and orientation of the template strands generated using the particular sample. As such, these images can be used to identify cluster (template strand) locations, where the cluster is derived from a particular sample that was introduced to the flow cell at a particular time.

The methods may vary slightly depending upon whether the complexes <NUM> are introduced into the flow cell <NUM> or whether the attached fragments <NUM> are introduced into the flow cell. The various examples will now be described.

In this example, a genome sample (e.g., sample <NUM>) is fragmented to form a plurality of contiguity preserved fragments <NUM>, <NUM>', each of which is attached to a solid support <NUM>. It is to be understood that all of the contiguity preserved fragments <NUM>, <NUM>' from the genome sample <NUM> may not be attached to the same solid support <NUM>; but rather, contiguity preserved fragments <NUM>, <NUM>' associated with particular portions of the genome sample <NUM> may be attached to respective solids supports <NUM>. As such, the genome sample <NUM> is fragmented to form a plurality of the complexes <NUM> or <NUM>'. This may be accomplished as described in reference to <FIG>, <FIG>, <FIG>, or using any other contiguity preserving method that incorporates adapters <NUM>, <NUM> or <NUM>', <NUM> to each of the fragments <NUM>, <NUM>' so that they are sequencing ready fragments <NUM>, <NUM>'.

The complexes <NUM> or <NUM>' formed using the genome sample <NUM> may be incorporated into a mixture. As such, each of the plurality of contiguity preserved fragments <NUM>, <NUM>' is also incorporated into the mixture. The liquid carrier of the mixture may be a buffer, such as Tris-HCl buffer or <NUM>. 5x saline sodium citrate (SSC) buffer.

The liquid carrier may be added to the plurality of contiguity preserved fragments <NUM>, <NUM>' (e.g., present in complexes <NUM> or <NUM>') to initially form the mixture, and then the mixture may be diluted with additional liquid carrier to generate a predetermined number of dilution samples that are to be individually introduced to the flow cell <NUM> (or an individual lane <NUM> thereof).

The final volume of the mixture that is generated, and thus the dilution of the mixture, may be controlled in any desirable manner. In some instances, the dilution may depend upon the volume of the flow cell <NUM> (or, for example, each channel <NUM> of a multi-channel flow cell) and the desired number of samples to be introduced to the flow cell <NUM>. In one example, the volume of the flow cell <NUM> (or channel <NUM> thereof) may be used as the limiting dilution factor. As such, in some examples, the carrier liquid may be added to dilute the mixture to the predetermined volume, where the predetermined volume is based on i) the volume of the flow cell <NUM> as a limiting dilution and ii) the predetermined number of dilution samples to be introduced to the flow cell <NUM>. As an example, the flow cell <NUM> or one lane <NUM> of the flow cell <NUM> may have a volume of about <NUM>µL and the desired number of samples may be <NUM>. In this example, the mixture may be diluted to about <NUM>,<NUM>µL. As another example, the flow cell <NUM> or one lane <NUM> of the flow cell <NUM> may have a volume of about <NUM>µL and the desired number of samples may be <NUM>. In this example, the mixture may be diluted to about <NUM>,<NUM>µL.

The desired number of dilution samples may depend, for example, upon the volume of the flow cell <NUM> and the desired resolution of the individual template strands in the respective time-based clustering images. With smaller volume flow cells <NUM>, it may be desirable to have a more dilute mixture so that each individual dilution sample to be introduced to the flow cell <NUM> contains fewer contiguity preserved library fragments <NUM>, <NUM>' (than if a less dilute mixture were used). In this type of flow cell <NUM>, fewer contiguity preserved library fragments <NUM>, <NUM>' will lead to fewer template strands, which may improve the resolution of the template stands in the respective time-based clustering images. In an example, the desired number of samples may range from about <NUM> samples to about <NUM> samples. In other examples, the desired number of dilution samples to be prepared from the mixture may be over <NUM>. The upper limit on the number of dilution samples may depend, in part, upon the desired time frame in which the overall method is to take place.

The mixture may then be divided into the predetermined number of dilution samples. In one example, diluted mixture may be divided so that all of the dilution samples are generated at the same time. In another example, the predetermined volume of any one dilution sample may be separated from the bulk mixture when it is time for that sample to be introduced to the flow cell <NUM>.

<FIG> illustrate an example of a non-patterned flow cell <NUM> (e.g., as shown in <FIG>) from a top view during different stages of the generation of the time-based clustering images.

As shown schematically in <FIG>, the flow cell <NUM> may be introduced into a system which includes a flow cell receptacle <NUM>; a fluidic control system <NUM> including delivery fluidics <NUM> to respectively deliver a dilution sample 56A and a stain (not shown) to a flow cell <NUM> positioned in the flow cell receptacle <NUM>; an illumination system <NUM> positioned to illuminate the flow cell <NUM> positioned in the flow cell receptacle <NUM>; a detection system <NUM> positioned to capture an image of the flow cell <NUM> positioned in the flow cell receptacle <NUM>; and a controller <NUM> in operative communication with the fluidic control system <NUM>, the illumination system <NUM>, and the detection system <NUM>, the controller <NUM> to cause the delivery fluidics <NUM> to introduce the dilution sample 56A to the flow cell <NUM> positioned in the flow cell receptacle <NUM>; cause the delivery fluidics <NUM> to introduce the stain to the flow cell <NUM> positioned in the flow cell receptacle <NUM> after template strands 58A are generated in the flow cell <NUM> positioned in the flow cell receptacle <NUM> from contiguity preserved library fragments present in the dilution sample 56A; cause the illumination system <NUM> to illuminate the stained template strands in the flow cell <NUM> positioned in the flow cell receptacle <NUM>; and cause the detection system <NUM> to image the illuminated, stained template strands in the flow cell <NUM> positioned in the flow cell receptacle <NUM>.

When in position, the flow cell <NUM> is in fluid communication with the fluidic control system <NUM> (e.g., pumps, valves, and the like) and is in optical communication with an illumination system <NUM> and a detection system <NUM>.

In <FIG>, a first of the dilution samples 56A (including some of the complexes <NUM> or <NUM>', shown as 10A in <FIG>) is introduced into the flow cell <NUM>. The introduction of any of the respective dilution samples 56A (or e.g., 56B in <FIG>) involves fluidically directing one of the dilution samples 56A, 56B to the flow cell <NUM>. The dilution sample 56A may be introduced, e.g., in a cartridge <NUM>, and the fluidic control system <NUM> may fluidically transport dilution sample 56A from the cartridge <NUM> to the flow channel <NUM> of the flow cell <NUM> using the delivery fluidics <NUM> (e.g., pumps, valves, and the like).

Given the concentration of complexes 10A in the dilution sample 56A, most, if not all, of the complexes 10A will settle onto the polymeric hydrogel <NUM> and any primers <NUM>, <NUM> thereon (which are not shown in <FIG>). In some examples, the complexes 10A may settle and remain in the flow channel <NUM> or in the depressions <NUM> due to the depth of the flow channel <NUM> or the depressions <NUM>. In other examples, the flow channel <NUM> or the depressions <NUM> may include a capture site that the complexes 10A adhere to.

It is to be understood that some complexes 10A may not settle, and these complexes 10A will be removed from the flow cell <NUM> before further processes. As such, some examples of the method then include washing away non-trapped complexes 10A from the flow cell <NUM>. Washing may involve introducing a fluid into the flow cell <NUM>. The flow may push any complexes 10A that have not settled and/or adhered out through an exit port of the flow cell <NUM>.

This example of the method then includes initiating the release of the contiguity preserved library fragments <NUM>, <NUM>' from the respective solid supports <NUM> to which they are attached. In this example, the sequencing-ready nucleic acid fragments <NUM>, <NUM>' (including the contiguity preserved library fragments <NUM>, <NUM>' and the adapters <NUM>, <NUM> or <NUM>', <NUM> attached thereto) are released from the respective solid supports <NUM>. In <FIG>, the release of the sequencing-ready nucleic acid fragments <NUM>, <NUM>' from the solid supports <NUM> is represented by the arrows pointing outward from each solid support <NUM>.

The release of the sequencing-ready nucleic acid fragments <NUM>, <NUM>' may be initiated in several different ways. In one example, initiating release involves heating the flow cell <NUM>. In this example, the system may include a heater <NUM>. The controller <NUM> may cause the heater <NUM> to initiate release of some of the contiguity preserved library fragments <NUM>, <NUM>' from the solid support <NUM> or from each other. As an example, temperatures greater than <NUM> may be used to at least partially break the bonds, and thus initiate the release of the sequencing-ready nucleic acid fragments <NUM>, <NUM>'. In another example, initiating release involves introducing a cleaving agent to the flow cell <NUM>. The fluidic control system <NUM> may be used to deliver the cleaving agent. The cleaving agent may initiate chemical, enzymatic, or photochemical release of the sequencing-ready nucleic acid fragments <NUM>, <NUM>' from the solid support <NUM>. In these examples, another stimulus, such as heat or light, may trigger the cleaving agent to release the sequencing-ready nucleic acid fragments <NUM>, <NUM>' from the solid support <NUM>. As one example, free biotin may be introduced as the cleaving agent, and heating to about <NUM> may be used to induce biotin-oligo release from the solid support <NUM>.

The released sequencing-ready nucleic acid fragments <NUM>, <NUM>' transport from the solid support <NUM> and seed onto the polymeric hydrogel <NUM>. More specifically, the amplification primers <NUM>, <NUM> seed the released sequencing-ready nucleic acid fragments <NUM>, <NUM>' in a relatively confined manner. In an example, seeding is accomplished through hybridization between the first or second sequence of the fragment <NUM>, <NUM>' and a complementary one of the primers <NUM>, <NUM> on the polymeric hydrogel <NUM> in the flow cell <NUM>. Seeding may be performed at a suitable hybridization temperature for the fragment sequencing-ready nucleic acid fragments <NUM>, <NUM>' and the primer(s) <NUM>, <NUM>. The heater <NUM> may be controlled to bring the flow cell <NUM> to the seeding temperature.

A washing process may be performed to remove the beads.

The seeded sequencing-ready nucleic acid fragments <NUM>, <NUM>' can then be amplified using any suitable method, such as cluster generation. In one example of cluster generation, the released sequencing-ready nucleic acid fragments <NUM>, <NUM>' are copied from the hybridized primers <NUM>, <NUM> by <NUM>' extension using a high-fidelity DNA polymerase. The original sequencing-ready nucleic acid fragments <NUM>, <NUM>' are denatured, leaving the copies immobilized within the flow channel <NUM> or some of the depressions <NUM>. Any clonal amplification process may be used. In one example, isothermal bridge amplification may be used to amplify the immobilized copies. For example, the copied templates loop over to hybridize to an adjacent, complementary primer <NUM>, <NUM>, and a polymerase copies the copied templates to form double stranded bridges, which are denatured to form two single stranded strands. These two strands loop over and hybridize to adjacent, complementary primers <NUM>, <NUM> and are extended again to form two new double stranded loops. The process is repeated on each template copy by cycles of isothermal denaturation and amplification to create dense clonal clusters. Each cluster of double stranded bridges is denatured. In an example, the reverse strand is removed by specific base cleavage, leaving forward template polynucleotide strands. It is to be understood that clustering results in the formation of several template strands 58A in the flow channel <NUM> or some of the depressions <NUM>. In some examples, the controller <NUM> causes the heater <NUM> to run a thermal cycle to amplify the seeded sequencing-ready nucleic acid fragments <NUM>, <NUM>'.

<FIG> illustrates clusters 60A of template strands 58A generated from the complexes 10A of the first dilution sample 56A (compartment). The clusters 60A in <FIG> are outlined for clarity. While <FIG> illustrates four clusters 60A, it is to be understood that the number of clusters 60A will depend upon the number of complexes 10A introduced in the sample 56A, as well as the number of sequencing-ready nucleic acid fragments <NUM>, <NUM>' released from each solid support <NUM>. A cluster 60A is generated from each of the released sequencing-ready nucleic acid fragments <NUM>, <NUM>'. Moreover, the released sequencing-ready nucleic acid fragments <NUM>, <NUM>' may diffuse across the flow cell surface, and thus clusters 60A may be generated across the flow cell surface.

After generating the clusters 60A for the first dilution sample 56A, a stain is introduced into the flow cell <NUM>. Any fluorescent stain that is capable of staining the template strands 58A may be used. Examples of suitable fluorescent stains include the SYBR® family of dyes from Molecular Probes, Inc. (e.g., SYBR® Green, SYBR® Gold, SYBR® Safe, etc.), ethidium bromide, propidium iodide, crystal violet, EVAGREEN® dye (from Biotium), DAPI (<NUM>',<NUM>-diamidino-<NUM>-phenylindole), or the like. The stain is introduced into the flow cell <NUM>, e.g., from a second cartridge (not shown), allowed to incubate for a suitable time period to stain the template strands 58A, and then is flushed out of the flow cell <NUM>.

The illumination system <NUM> may then be used to illuminate the stained template strands 58A in the flow cell <NUM>. The illumination system may include a light source and a plurality of optical components Examples of light sources may include lasers, arc lamps, LEDs, or laser diodes. The optical components may be, for example, reflectors, dichroics, beam splitters, collimators, lenses, filters, wedges, prisms, mirrors, detectors, and the like. The illumination system may be operatively positioned to direct an excitation light to the flow cell surface that corresponds to the stain used.

The detection system <NUM> may be used to capture an image I<NUM> of the fluorescing template strands 58A. This image I<NUM> is the time-based clustering image for the dilution sample 56A because it depicts a spatial location and orientation of the template strands 58A associated with the dilution sample 56A. Any suitable camera may be used to capture an image I<NUM> of the clusters 60A on the flow cell <NUM>.

The image I<NUM> may be electronically stored for subsequent retrieval and use. As such, some examples of the system include an electronic storage component <NUM> to store the image I<NUM>. In the electronic record, the image I<NUM> may be linked to the dilution sample 56A. The image I<NUM> may also be assigned a temporal record. As such, some examples of the method include assigning each time-based clustering image I<NUM> in the series a temporal record of the introduction of the respective sample including the some of the contiguity preserved library fragments. The temporal record may include a time stamp indicating when the dilution sample 56A was introduced and/or imaged, a step number in an introduction and/or imaging sequence (e.g., sample <NUM> of <NUM>, sample <NUM> of <NUM>,. sample X of <NUM>), or combinations thereof.

A wash may take place after the clusters 60A on the flow cell <NUM> are imaged. Water, a buffer, or another mild wash solution may be used.

The processes shown and described in reference to <FIG> are then repeated with a second dilution sample 56B (shown in <FIG>).

In <FIG>, the second dilution sample 56B is introduced into the flow cell <NUM>. The complexes 10B (which may be complexes <NUM> or <NUM>') settle and/or adhere to the flow cell surface.

As shown in <FIG>, the method then includes initiating the release of the contiguity preserved library fragments <NUM>, <NUM>' from the respective solid supports <NUM> to which they are attached. In this example, the sequencing-ready nucleic acid fragments <NUM>, <NUM>' (including the contiguity preserved library fragments <NUM>, <NUM>' and the adapters <NUM>, <NUM> or <NUM>', <NUM> attached thereto) are released from the respective solid supports <NUM>.

The released sequencing-ready nucleic acid fragments <NUM>, <NUM>' transport from the solid support <NUM> and seed onto the polymeric hydrogel <NUM>. The seeded sequencing-ready nucleic acid fragments <NUM>, <NUM>' can then be amplified using any suitable method, such as cluster generation. It is to be understood that this round of clustering results in the formation of several more template strands 58B in the flow channel <NUM> or some of the depressions <NUM>.

<FIG> illustrates clusters 60B of template strands 58B generated from the respective complexes 10B of the second dilution sample 56B (compartment). The clusters 60A and 60B in <FIG> are outlined for clarity. While <FIG> illustrates three clusters 60B, it is to be understood that the number of clusters 60B will depend upon the number of complexes 10B introduced in the sample 56B, as well as the number of sequencing-ready nucleic acid fragments <NUM>, <NUM>' released from each solid support <NUM>.

After generating the clusters 60B for the second dilution sample 56B, the stain is again introduced into the flow cell <NUM>. The same stain used to stain the template strands 58A may be used to stain the template strands 58B and any subsequently generated template strands.

The illumination system <NUM> may then be used to illuminate the stained template strands 58A and 58B in the flow cell <NUM>. The detection system <NUM> may be used to capture an image I<NUM> of the fluorescing template strands 58A and 58B in the respective clusters 60A and 60B.

The image I<NUM> may also be electronically stored for subsequent retrieval and use. In the electronic record, the image I<NUM> may be linked to the dilution sample 56B. The image I<NUM> may also be assigned a temporal record. The temporal record may include a time stamp indicating when the dilution sample 56B was imaged, a step number in a sequence (e.g., sample <NUM> of <NUM>), or combinations thereof.

A wash may take place after the clusters 60A, 60B on the flow cell <NUM> are imaged. Water, a buffer, or another mild wash solution may be used.

The processes shown and described in reference to <FIG> may then be repeated, e.g., using the described system, for the number of dilution samples derived from the original mixture. Each additional image I<NUM>, I<NUM>,. Ix will depict new clusters 60C, 60D,. 60X of template stands 58C, 58D,. 58X generated with the introduction of a respective dilution sample 56C, 56D,. All of the images I<NUM>, I<NUM>,. Ix obtained for the respective dilution samples 56A, 56B,. 56X are associated with a particular mixture, and thus a particular longer nucleic acid molecule <NUM>.

Because each sequential image I<NUM>, I<NUM>, I<NUM>,. Ix depicts a newly formed cluster 60B, 60C, 60D,. 60X with respect to the immediately preceding image I<NUM>, I<NUM>, I<NUM>,. Ix, image subtraction may be used to generate a resolved cluster image for each sample introduced subsequent to the first sample 56A. Some example resolved cluster images RIx are shown in <FIG>.

<FIG> depicts images I<NUM>, I<NUM>, I<NUM>, I<NUM>, I<NUM>, I<NUM> taken for six different dilution samples 56A, 56B, 56C, 56D, 56E, 56F that are sequentially introduced, amplified, stained, and imaged as described in reference to <FIG>. As depicted, new clusters 60A, 60B, 60C, 60D, 60E, and 60F are respectively generated for each of the newly introduced and processed dilution samples 56A, 56B, 56C, 56D, 56E, 56F.

Because image I<NUM> for the first sample 56A includes clusters 60A from one sample, a resolved cluster image does not need to be generated, as the original image I<NUM> can be used for spatial identification of the clusters 60A. For each sample introduced subsequent to the first sample 56A, a resolved cluster image RI<NUM>, RI<NUM>, RI<NUM>, RI<NUM>, Rls is generated for each of the other images I<NUM>, I<NUM>, I<NUM>, I<NUM>, I<NUM>. As one example, image I<NUM> (depicting template strands 58A in clusters 60A) may be subtracted from image I<NUM> (depicting template strands 58A in clusters 60A and template strands 58B in clusters 60B) to generate a resolved cluster image RI<NUM> for the second sample 56B. This resolved cluster image RI<NUM> depicts a spatial location and orientation of the template strands 58B in clusters 60B associated with the second sample 56B. For another example, image I<NUM> (depicting template strands 58A in clusters 60A) and image I<NUM> (depicting template strands 58A in clusters 60A and template strands 58B in clusters 60B) may be subtracted image I<NUM> (which depict template strands 58A in clusters 60A, template strands 58B in clusters 60B, and template strands 58C in clusters 60C) to generate a resolved cluster image RI<NUM> for a third sample, e.g., 56C. This resolved cluster image RI<NUM> depicts a spatial location and orientation of the template strands 58C in clusters 60C associated with a third sample 56C. The resolved cluster image RI<NUM>, RI<NUM>, and RI<NUM> may be generated in a similar manner by subtracting any of the preceding images.

It is to be understood that image subtraction may be performed for any of the I<NUM>, I<NUM>, I<NUM>,. Ix in a series. The resulting resolved cluster image RIx for any given dilution sample 56X depicts the spatial location and orientation of the template strands 58X associated with that dilution sample 56X.

The resolved cluster images may be stored for subsequent analysis.

In this example, a genome sample is fragmented to form a plurality of contiguity preserved fragments <NUM>, <NUM>' that are attached to one another, e.g., as attached fragments <NUM>. It is to be understood that all of the contiguity preserved fragments <NUM>, <NUM>' from the genome sample may not be attached to one another; but rather, the process shown in <FIG> may result in the formation of several attached fragments <NUM>.

The attached fragments <NUM> formed using the genome sample may be incorporated into a mixture. As such, each of the plurality of contiguity preserved fragments <NUM>, <NUM>' is also incorporated into the mixture. The liquid carrier of the mixture may be a buffer, such as Tris-HCl buffer or <NUM>. 5x saline sodium citrate (SSC) buffer.

The liquid carrier may be added to a plurality of attached fragments <NUM> to initially form the mixture, and then the mixture may be diluted with additional liquid carrier to generate a predetermined number of dilution samples that are to be individually introduced to the flow cell <NUM> (or an individual lane <NUM> thereof).

The final volume of the mixture that is generated, and thus the dilution of the mixture, may be controlled in any desirable manner and as described herein.

In this example method, the attached fragments <NUM> shown in <FIG> are introduced to the flow channel as part of one of the dilution samples. An example of this dilution sample 66A is depicted in <FIG>.

As shown in <FIG>, within the flow cell <NUM>, the transposases <NUM> are removed from the attached fragments <NUM>. This may be accomplished, for example, using SDS or proteinase. The removal of the transposases <NUM> liberates respective contiguity preserved fragments <NUM>, <NUM>' (and any strands <NUM>', <NUM>' directly or indirectly attached thereto) from adjacent contiguity preserved fragments <NUM>, <NUM>'. In other words, sub-fragments <NUM> of the attached fragments <NUM> are released, and are capable of seeding onto the polymeric hydrogel <NUM> via the transferred strands <NUM>'. As shown schematically in <FIG>, the transferred strands <NUM>' hybridize to respective and complementary primers <NUM> on the surface of the flow cell <NUM>. In some instances, heat may be applied during hybridization. The application of heat may depend upon the melting temperature of the transferred strands <NUM>'. As one example, the P5' portion of the transferred strand <NUM>' hybridizes to the complementary P5 amplification primer <NUM> attached to the polymeric hydrogel <NUM>.

A washing solution may be flowed through the flow channel of the flow cell <NUM> to remove the transposases <NUM> from the flow cell <NUM>. An example of a suitable washing solution includes SDS, which may remove the transposase. A second wash solution, such as TRIS or a hybridization wash buffer, may be used to rinse out the flow cell <NUM>.

Prior to amplification, this example of the method further includes introducing a second sequence portion (e.g., non-transferred strands <NUM>') to each of the hybridized contiguity preserved library fragments <NUM>, <NUM>' at an end that opposed to the hybridized end. As such, in some examples of the method, each of the contiguity preserved library fragments <NUM>, <NUM>' includes a first sequence portion at a first end that hybridizes to a first primer sequence <NUM> on a surface of the flow cell <NUM>; and prior to amplification, the method further comprises attaching a second sequence portion to each of the hybridized contiguity preserved library fragments <NUM>, <NUM>' at a second end that is opposed to the first end, the second sequence portion being identical to a second primer sequence <NUM> on the surface of the flow cell <NUM>, so that the copy of the second sequence portion can hybridize to the second primer sequence <NUM>.

The introduction of the second sequence portion may be performed using extension ligation. In an example, extension ligation may be initiated (as represented by the arrows <NUM> in <FIG>) to join the non-transferred strands <NUM>' to the corresponding fragments <NUM>, <NUM>'. In an example, extension ligation may be initiated by introducing an extension ligation mix to the flow cell <NUM>, and heating to a suitable temperature for enzyme activity (e.g., ranging from about <NUM> to about <NUM>).

The extension ligation mix may include a ligation enzyme (e.g., DNA ligase) that catalyzes the formation of a bond between a non-transferred strand <NUM>' and its corresponding fragment <NUM> or <NUM>'. As described in reference to <FIG>, the non-transferred strands <NUM>' include a second sequencing primer sequence (e.g., a read <NUM> sequencing primer sequence) and a second sequence (P7) that is identical to at least a portion of another of the amplification primers <NUM> (P7) on the flow cell surface. This second sequence enables a complementary copy, e.g., P7', to be generated during amplification that can hybridize to the amplification primer <NUM> (P7) on the flow cell surface during clustering. As such, ligation results in the formation of sequencing ready library fragments <NUM>, <NUM>' attached to the flow cell surface.

The extension ligation mix may also include a blocking group that attaches to the exposed ends of the primers <NUM> to prevent undesired extension at these primers <NUM>. Alternatively, primers <NUM> may be grafted to the surface with blocking groups (e.g., a <NUM>' phosphate) attached thereto. In still other example, blocking groups may not be used.

As the resulting fragments <NUM>, <NUM>' are attached to one another, heating may be used to dissociate the fragments <NUM>' from the fragments <NUM>. The fragments <NUM>' that are not hybridized to the primers <NUM> may be removed from the flow cell <NUM> with a wash.

When used, any blocked primers <NUM> may then be unblocked (e.g., using kinase or another suitable de-blocking agent) so that amplification can be performed. In this example, amplification may be performed using any suitable method, such as cluster generation. Cluster generation may be performed as described herein in reference to <FIG>. The system described in reference to <FIG> may be used.

<FIG> illustrates clusters 70A of template strands 68A generated from the attached fragments <NUM> of the dilution sample 66A (shown in <FIG>). The clusters 70A in <FIG> are outlined for clarity. While <FIG> illustrates four clusters 70A, it is to be understood that the number of clusters 70A will depend upon the number of attached fragments <NUM> introduced in the sample 66A, as well as the number of sequencing-ready nucleic acid fragments <NUM>, <NUM>' released from the attached fragments <NUM>.

After generating the clusters 70A for the first dilution sample 66A, a stain is introduced into the flow cell <NUM> as described in reference to <FIG>. The stain is introduced into the flow cell <NUM>, allowed to incubate for a suitable time period to stain the template strands 68A, and then is flushed out of the flow cell <NUM>.

The illumination system <NUM> may then be used to illuminate the stained template strands 68A in the flow cell <NUM>, and the detection system <NUM> may be used to capture an image I<NUM> of the fluorescing template strands 68A. This image I<NUM> is the time-based clustering image for the dilution sample 66A because it depicts a spatial location and orientation of the template strands 68A associated with the dilution sample 66A.

The image I<NUM> may be electronically stored for subsequent retrieval and use. In the electronic record, the image I<NUM> may be linked to the dilution sample 66A. The image I<NUM> may also be assigned a temporal record. The temporal record may include a time stamp indicating when the dilution sample 66A was introduced and/or imaged, a step number in an introduction and/or imaging sequence (e.g., sample <NUM> of <NUM>, sample <NUM> of <NUM>,. sample X of <NUM>), or combinations thereof.

A wash may take place after the clusters 70A on the flow cell <NUM> are imaged. Water, a buffer, or another mild wash solution may be used.

The processes shown and described in reference to <FIG> and <FIG> are then repeated with a second dilution sample 66B (shown in <FIG>).

In <FIG>, the second dilution sample 66B is introduced into the flow cell <NUM>. The attached fragments <NUM> may be broken up into the sub-fragments <NUM> as described in reference to <FIG>. One transferred strand <NUM> of at least some of the sub-fragments <NUM> will hybridize to complementary amplification primers <NUM> on the flow cell <NUM>. Extension ligation and the other processes described in reference to <FIG> may then be performed, which results in sequencing-ready nucleic acid fragments <NUM> attached to the flow cell surface.

Cluster generation may be performed as described herein in reference to <FIG>.

<FIG> illustrates clusters 70B of template strands 68B generated from the attached fragments <NUM> of the second dilution sample 66B. The clusters 70A and 70B in <FIG> are outlined for clarity. While <FIG> illustrates two clusters 70B, it is to be understood that the number of clusters 70B will depend upon the number of attached fragments <NUM> introduced in the sample 66B, as well as the number of sequencing-ready nucleic acid fragments <NUM>, <NUM>' released from the attached fragments <NUM>.

After generating the clusters 70B for the second dilution sample 66B, the stain is again introduced into the flow cell <NUM>. The same stain used to stain the template strands 68A may be used to stain the template strands 68B and any subsequently generated template strands.

The illumination system <NUM> may then be used to illuminate the stained template strands 68A and 68B in the flow cell <NUM>. The detection system <NUM> may be used to capture an image I<NUM> of the fluorescing template strands 68A and 68B in the respective clusters 70A and 70B.

The image I<NUM> may also be electronically stored for subsequent retrieval and use. In the electronic record, the image I<NUM> may be linked to the dilution sample 66B. The image I<NUM> may also be assigned a temporal record. The temporal record may include a time stamp indicating when the dilution sample 56B was imaged and/or introduced, a step number in a sequence (e.g., sample <NUM> of <NUM>), or combinations thereof.

A wash may take place after the clusters 70A, 70B on the flow cell <NUM> are imaged. Water, a buffer, or another mild wash solution may be used.

The processes shown and described in reference to <FIG> may then be repeated for the number of dilution samples derived from the original mixture. Each additional image I<NUM>, I<NUM>,. Ix will depict new clusters 70C, 70D,. 70X of template stands 68C, 68D,. 68X generated with the introduction of a respective dilution sample 66C, 66D,. All of the images I<NUM>, I<NUM>,. Ix obtained for the respective dilution samples 66A, 66B,. 66X are associated with a particular mixture, and thus a particular longer nucleic acid molecule <NUM>.

Because each sequential image I<NUM>, I<NUM>, I<NUM>,. Ix depicts a newly formed cluster 70B, 70C, 70D,. 70X with respect to the immediately preceding image I<NUM>, I<NUM>, I<NUM>,. Ix, image subtraction may be used to generate a resolved cluster image for each sample introduced subsequent to the first sample 66A. The resolved cluster images RIx may be generated as described in reference to <FIG>.

It is to be understood that image subtraction may be performed for any of the I<NUM>, I<NUM>, I<NUM>,. Ix in a series. The resulting resolved cluster image RIx for any given dilution sample 66X depicts the spatial location and orientation of the template strands 68X associated with that dilution sample 66X.

Rather than introducing individual dilution samples 56A, 66A, the diluted mixture may be diffused into the flow cell in predetermined volumes, and the processing on the flow cell <NUM> may take place as described herein to amplify, stain, and record images of the generated template strands 58X, 68X. The diffusion may be controlled so that one predetermined volume is introduced at a time.

As such, some examples of the method include generating a time-based clustering image for each of the limiting dilution samples introduced to the flow cell <NUM> by: controlling diffusion of the mixture into the flow cell so that one of the limiting dilution samples is introduced to the flow cell <NUM> at a time; initiating release of contiguity preserved library fragments <NUM>, <NUM>' from a solid support <NUM> or from each other (e.g., from attached fragments <NUM>) in the one of the limiting dilution samples in the flow cell <NUM>; amplifying the contiguity preserved library fragments <NUM>, <NUM>' to generate a plurality of respective template strands 58A, 68A; staining the respective template strands 58A, 68A; and imaging the respective template strands 58A, 68A.

When all of the dilution samples from a mixture are amplified and imaged as described herein, the flow cell <NUM> is ready for a sequencing operation. A variety of sequencing approaches or technologies may be used, including techniques often referred to as sequencing-by-synthesis (SBS), cyclic-array sequencing, sequencing-by-ligation, pyrosequencing, and so forth.

As one example, a sequencing by synthesis (SBS) reaction may be run on a system such as the HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NOVASEQ™, NEXTSEQDX™, ISEQ™, NEXTSEQ™, or other sequencer systems from Illumina (San Diego, CA). In SBS, extension of sequencing primers along the template strands 58A, 58B,. 58X is monitored to determine the sequence of nucleotides in the templates. The <NUM>'-ends of the template strands 58A, 58B,. 58X and any flow cell-bound primers <NUM>, <NUM> (not attached to the template strands 58A, 58B,. 58X) may be blocked to prevent interference with the sequencing reaction, and in particular, to prevent undesirable priming.

A sequencing primer may be introduced that hybridizes to a complementary sequence on the template strands 58A, 58B,. This sequencing primer renders the template strands 58A, 58B,. 58X ready for sequencing.

The underlying chemical process can be polymerization (e.g., catalyzed by a polymerase enzyme) or ligation (e.g., catalyzed by a ligase enzyme). In a particular polymerase-based SBS process, fluorescently labeled nucleotides are added to the sequencing primer in a template dependent fashion 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 a first SBS cycle, one or more labeled nucleotides, DNA polymerase, etc., may be delivered into/through the flow cell <NUM>, etc., where sequencing primer extension causes a labeled nucleotide to be incorporated. This incorporation can be detected through an imaging event. During an imaging event, the illumination system <NUM> may provide an excitation light to the flow cell <NUM>.

In some examples, the fluorescently labeled nucleotides can further include a reversible termination property that terminates further primer extension once a nucleotide has been added to the template. For example, a nucleotide analog having a reversible terminator moiety can be added to the template such that subsequent extension cannot occur until a deblocking agent is delivered to remove the moiety. Thus, for examples that use reversible termination, a deblocking reagent can be delivered to the flow cell <NUM>, etc. (after detection occurs).

Wash(es) may take place between the various fluid delivery steps. The SBS cycle can then be repeated n times to extend the template by n nucleotides, thereby detecting a sequence of length n.

While SBS has been described in detail, it is to be understood that the flow cell <NUM> described herein may be utilized with other sequencing protocol, for genotyping, or in other chemical and/or biological applications.

The sequencing reads that are obtained during the sequencing operation can be grouped together based on the resolved cluster images RI<NUM>, RI<NUM>,. The grouped sequencing reads can then be linked to a respective one of the dilution samples based on the resolved cluster images RI<NUM>, RI<NUM>,. An inference can be made that the grouped and linked sequencing reads originated from the same longer nucleic acid sample <NUM>. As such, some examples of the method include performing a sequencing operation on the flow cell <NUM> including the respective template strands for each of the plurality of library fragments; and grouping sequencing reads together in different groups based on the resolved clustering images RI, RI<NUM>, RI<NUM>,.

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

Barcode oligonucleotides (e.g., adapter <NUM>) were attached via a biotin linker to M280 streptavidin beads (Thermofisher) to form a beadpool. A universal transposome was hybridized to the complementary sequence at the end of the oligos to form a bead-linked transposome (BLT). The BLTs were then washed in a wash buffer, resuspended in a working buffer, and accessory proteins (single strand binding protein (Thermofisher) and double stranded binding proteins (Illumina) were added.

High molecular weight NA12878 DNA (extracted from cultured cells using the Qiagen MAGATTRACT® HMW DNA extraction protocol) was added to the BLT mix and the tubes were gently inverted to mix. The tubes were then incubated at room temperature for about <NUM> minutes to allow the DNA to wrap around the beads. A tagmentation buffer (containing magnesium chloride and tris actetate) was then added to each tube and the samples were incubated for about <NUM> minutes at about <NUM>. During this step, the transposome tagmented the DNA and the tagged DNA became attached to the BLTs. Following the tagmentation reaction, sodium dodecyl sulfate (SDS) was added and the samples were incubated at room temperature for about <NUM> minutes to denature the transposase. The tubes were then placed on a magnet and the supernatant was removed. The beads were washed with the wash buffer. Following the final wash, the beads were resuspended in a ligase mix (containing T7 ligase and its associated buffer, T7 ligase buffer from NEB). The samples were then mixed and left to incubate for about <NUM> minutes at room temperature. During this time, the gap in the transfer stand (where the transposome was initially hybridized) was ligated, thus physically attaching the tagged DNA to the bead. The samples were then placed on a magnet, the supernatant was removed, and the beads were again washed in the wash buffer.

The tagged beads were then split into two groups for removal of the non-transferred strands and introduction of a sample index (e.g., adapter <NUM>). One group (comp. group) was exposed to a comparative workflow where heat was used to remove the non-transferred strands. The other group (ex. group) was exposed to an example workflow where an exonuclease was used to remove the non-transferred strands.

group was resuspended in the wash buffer were heated to about <NUM> for about <NUM> minutes to denature off the non-transfer strands. The tubes containing the comp. group were then placed on a magnet, the supernatant was removed, and the beads were washed. The sample index, diluted in the wash buffer, was then added to the beads of the comp. group, and this mixture was incubated at about <NUM> for about <NUM> minute, followed by a slow temperature ramp down.

group was suspended in a T7 exonuclease mix (containing T7 exonuclease and NEB Buffer <NUM>), and was allowed to incubate at room temperature for about <NUM> minutes. The <NUM>' to <NUM>' exonuclease activity of the T7 exonuclease digested the non-transferred strands. The tubes containing ex. group were then placed on a magnet, the supernatant was removed, and the beads were washed. The sample index, diluted in the wash buffer, was then added to the beads of the ex. group, and this mixture was incubated at about <NUM> for about <NUM> minutes to allow the sample index to anneal.

The tubes respectively containing comp. group and the ex. group were placed on a magnet and the supernatant was removed. An extension ligation mix was added, and the samples were incubated for about <NUM> minutes at about <NUM>. The tubes were again placed on a magnet, the supernatant was removed, and the beads of the comp. group and the ex. group were washed in the wash buffer. Following the final wash, the beads were resuspended in the wash buffer.

Some beads of the comp. group and some beads of the ex. group were subsampled and used in a PCR reaction. In addition to the respective beads, each PCR mix consisted of Illumina PCR mix EPM, and P5 and P7 oligos. Each sample was amplified using PCR.

Following PCR, a subsample of the PCR supernatant of each of the comp. group and the ex. group was transferred to a fresh tube and a <NUM>. 50x - <NUM>. 62x size selection (solid phase reversible immobilization, SPRI) was performed using sample purification beads. The resulting libraries were eluted in a resuspension buffer. These libraries were sequenced on individual HISEQ™ <NUM> Rapid flow cells (using a standard 2x101 cycle read length). Following Fastq generation, the samples were aligned to the human genome (hg38) and the data imported into IGV.

<FIG> shows coverage obtained, using the library from each of the comp. group (labeled heat denaturation) and the ex. group (labeled T7 exonuclease), for an AT rich region of the human genome that is known to be negatively affected by high temperature steps in the library preparation protocol. The results for the comp. group library fragments whose non-transferred strands were removed via heat denaturation are shown at the top of <FIG>, and the results for the ex. group library fragments whose non-transferred strands were removed via exonuclease digestion are shown at the bottom of <FIG>. As illustrated, there was full coverage in the AT rich region for the ex. group library fragments, while there was only partial coverage in the same region for the comp. group library fragments. These results indicate that the use of the enzymatic digestion method disclosed herein increases library coverage and improves sequencing over AT rich regions of the genome when compared to heat denaturation.

Complexes were prepared as described in the method of <FIG> (multi-step ligation and digestion) and in the method of <FIG> (one pot ligation and digestion).

BLT generation, DNA binding, tagmentation, and exposure to SDS were performed as described in Example <NUM>. Following the removal of the transposase and the wash associated therewith, the tagged beads were then split into two groups for removal of the non-transferred strands via multi-step ligation and digestion (referred to as ex. group <NUM>) or via one pot ligation and digestion (referred to as ex. group <NUM>).

group <NUM> was resuspended in an E. Coli DNA ligase mix including E. Coli DNA ligase and its associated buffer, both from NEB. group <NUM> samples were then mixed and incubated for about <NUM> minutes at about <NUM>. The tubes containing the ex. group <NUM> samples were then placed on a magnet, the supernatant was removed, and the ex. group <NUM> beads were washed in the wash buffer. After the wash, the ex. group <NUM> beads were resuspended in a mix containing T7 Exonuclease and NEB Buffer <NUM> and were incubated at about <NUM> for about <NUM> minutes.

group <NUM> was resuspended in a combined ligase and exonuclease mix including E. Coli DNA ligase, T7 exonuclease, NAD+, and a CUTSMART™ buffer (from NEB) at about <NUM> for about <NUM> minutes.

The tubes containing ex. group <NUM> and ex. group <NUM> were then placed on a magnet, the supernatant was removed, and the respective beads were washed. The sample index, diluted in the wash buffer, was then added to the beads of each ex. group, and these mixtures were incubated at about <NUM> for about <NUM> minutes to allow the sample index to anneal.

The tubes respectively containing ex. group <NUM> and the ex. group <NUM> were placed on a magnet and the supernatant was removed. An extension ligation mix was added, and the samples were incubated for about <NUM> minutes at about <NUM>. The tubes were again placed on a magnet, the supernatant was removed, and the beads of the comp. group and the ex. group were washed in the wash buffer. Following the final wash, the beads were resuspended in the wash buffer.

Some beads of the ex. group <NUM> and some beads of the ex. group <NUM> were subsampled and used in a PCR reaction. In addition to the respective beads, each PCR mix consisted of Illumina PCR mix EPM, and P5 and P7 oligos. Each sample was amplified using PCR.

Following PCR, a subsample of the PCR supernatant of each of the ex. group <NUM> and the ex. group <NUM> was transferred to a fresh tube. Sample purification beads were added and a <NUM>. 5x SPRI was performed with the resulting library being eluted in a resuspension buffer. The cleaned-up libraries were then run on a Bioanalyzer <NUM> High Sensitivity chip and the trace in <FIG> was obtained. The results show that size profiles and yields of the libraries were comparable for the multi-step ligation and digestion and for the one pot ligation and digestion. These results indicate that the combined reagent formulation did not deleteriously affect ligation and did not result in digestion of the fragment or transferred strand.

The example bead bound library fragments (complexes) obtained using the library preparation methods described in Examples <NUM> and <NUM> may be divided into subsamples and diluted to form a plurality of dilution samples as described herein. The method described in reference to <FIG> (including clustering, staining, and imaging) may then be performed with each of the dilution samples to generate a series of time-based clustering images. When all of the dilution samples are amplified and imaged, the flow cell is ready for a sequencing operation. The library preparation techniques and time-based imaging techniques disclosed herein may be used together to efficiently and reliably reconstitute a long DNA fragment.

Furthermore, it is to be understood that the ranges provided herein include the stated range and any value or sub-range within the stated range, as if they were explicitly recited. For example, a range represented by from about <NUM> to about <NUM>, should be interpreted to include not only the explicitly recited limits of from about <NUM> to about <NUM>, but also to include individual values, such as about <NUM>, <NUM>, <NUM>, etc., and sub-ranges, such as from about <NUM> to about <NUM>, etc..

Claim 1:
A method, comprising:
generating a series of time-based clustering images for a plurality of contiguity preserved library fragments from a genome sample, wherein each time-based clustering image in the series is sequentially generated by:
introducing, to a flow cell, a respective sample including attached fragments, each attached fragment including some of the contiguity preserved library fragments attached to each other via transposases;
removing the transposases, thereby releasing sub-fragments of the attached fragments, wherein each of the sub-fragments includes a first sequence portion at a first end, whereby the released sub-fragments seed onto a surface of the flow cell by hybridization of the first sequence portion to a first primer sequence attached to a polymeric hydrogel on the surface of the flow cell;
attaching a second sequence portion to each of the hybridized sub-fragments at a second end that is opposed to the first end, the second sequence portion being identical to a second primer sequence attached to the polymeric hydrogel on the surface of the flow cell;
amplifying the seeded sub-fragments to generate a plurality of respective template strands;
staining the respective template strands; and
imaging the respective template strands.