Sample preparation on solid support

Abstract

To provide a method and a composition which rapidly and user-friendlily generate a library of tagged DNA fragments from target DNA, and are easily applicable to a nucleic acid analysis method such as next-generation sequencing and an amplification method.SOLUTION: Provided is a method and a composition for using transposase and transposon end parts which are immobilized in order to generate an immobilized library of double-stranded target DNA which is 5'-tagged on a surface. The method is useful for generating 5'- and 3'- tagged DNA fragments to be used in various processes including large-scale parallel DNA sequencing.SELECTED DRAWING: Figure 1a

Description

[Technical Field]

[0001] RELATED APPLICATIONS This application claims priority from U.S. Provisional Application No. 61 / 750,682, filed January 9, 2013. No. 6,119,793, which is incorporated herein by reference in its entirety. [Background technology]

[0002] There are various methods and applications, which are described in detail below. DNA) target molecules and generate a library of tagged DNA molecules from them. In many cases, the goal is to use the DNA as a template in a DNA sequencing reaction. For use, smaller DNA molecules (e.g., DNA fragments) can be synthesized into larger The key is to generate it from large dsDNA molecules. Summary of the Invention [Problem to be solved by the invention]

[0003] Currently, there are several methods for fragmenting and tagging double-stranded DNA for use in next-generation sequencing. Many of the methods used are wasteful of DNA and require expensive equipment for fragmentation. , and the procedures for fragmentation, tagging, and recovery of tagged DNA fragments are difficult. It is tedious, laborious, time-consuming, inefficient, expensive, and requires extensive testing to compare sample nucleic acids. Furthermore, many of these methods require relatively large amounts of sample nucleic acid from which they were generated. Generate tagged DNA fragments that are not entirely representative of the sequences contained within. Thus, what is required in the technique is to extract tagged DNA fragments from the target DNA. It offers speed and ease of use when generating libraries for next-generation sequencing. This method can be easily applied to nucleic acid analysis methods such as detection and amplification methods. [Means for solving the problem]

[0004] overview Here, methods and compositions are presented for nucleic acid sample preparation on a solid support. The methods and compositions are particularly directed to the transposon synthesis of DNA using transposon compositions immobilized on a solid support. The present invention relates to methods and compositions for fragmenting and tagging The methods and compositions provided herein can be used, for example, to generate tagged DNA fragments. Examples include next generation sequencing, and the like, to generate libraries of In certain preferred embodiments, the present invention is useful for use in target DNA. , including any dsDNA of interest (including double-stranded cDNA prepared from RNA) genomic, subgenomic, transcriptomic, or metagenomic analysis from any source for the preparation of linear ssDNA fragments on a solid support for analysis of RNA expression or RNA sequencing. do.

[0005] Thus, an immobilized library of tagged DNA fragments is prepared herein. A method is presented, which comprises: (a) providing a solid support with transposome complexes immobilized thereon; The first transposome complex comprises a first a transposase that binds to a polynucleotide, the first polynucleotide comprising: i) a 3' portion containing the transposon end (also called transposon terminus) sequence, and (ii) (b) the target DNA is transposable and contains a first tag that includes a first tag domain; fragmented by merging, and the 3' transposon end of the first polynucleotide under conditions whereby the sequence is transferred to the 5' end of at least one strand of the fragment, applying the target DNA to a solid support, thereby forming at least one strand An immobilized library of double-stranded fragments is generated in which each fragment is 5'-tagged with a first tag. In some embodiments, the transposome complex comprises the transposome. A second polynucleotide comprising a region complementary to the sposon end sequence is included. (c) providing a transposome complex in solution, and determining whether the target DNA is in solution. under conditions whereby the transposome complex fragments the transposome. The first step is to contact the liposome complex with the immobilized fragment, thereby Immobilized nucleic acid fragments having the desired moiety are obtained in solution. In one embodiment, the transposome complex in solution comprises a second tag. The immobilized nucleic acid fragments may be provided with a second tag, such that the second tag is generated in solution. The first and second tags can be different or the same.

[0006] Also provided herein are immobilized tagged DNA prepared according to the above method or other methods. A solid support having a library of fragments is presented. A support is presented having a transposome complex immobilized thereon; There, the transposome complex contains a transposer that binds to a first polynucleotide. The polynucleotide comprises (i) a 3' portion containing a transposon end sequence, and and (ii) a first tag comprising a first tag domain.

[0007] Also presented herein is a method for generating a flow cell, which involves attaching multiple transporters to a solid support. and immobilizing a transposome complex, the transposome complex comprising a first polymerase chain reaction product. a transposase that binds to a polynucleotide, and the first polynucleotide comprises (i ) a 3' portion including a transposon end sequence, and (ii) a first tag including a first tag domain. Includes:

[0008] The method further comprises providing a solid support having a plurality of first polynucleotides immobilized thereon. and providing a solid support for the transposase holoenzyme and the second polynucleotide. the second polynucleotide is contacted with a transposon end In some embodiments of the method, the immobilization step comprises immobilizing a region of complementary sequence to the target sequence. In particular, (a) a solid support having amplification primers coupled thereto. (b) hybridizing a second polynucleotide to one of the amplification primers; The second oligonucleotide is complementary to the transposon end sequence. (c) a region complementary to the first tag and the second polynucleotide; a first polynucleotide that is hybridized to a first polynucleotide that is directly immobilized on a solid support; Amplification primers are used with polymerase to generate a duplex containing the and (d) contacting the solid support with a transposase holoenzyme. whereby the transposome complex is assembled on the solid support. This includes being (or being)

[0009] Here we also present a population of microparticles with transposomes immobilized on them. a transposome complex comprising a first polynucleotide and a transposase that binds to a second polynucleotide, where the first polynucleotide The polynucleotide is immobilized at its 5' end on the surface of the microparticle, and the second polynucleotide is immobilized at its 5' end on the surface of the microparticle. hybridized to the 3' end of the first polynucleotide, The fragment contains (i) a 3' portion containing a transposon end sequence, and (ii) a first tag domain. The first tag contains the immobilized tagged DNA fragment. contacting the target DNA with said population of microparticles to form tagged DNA fragments. A method for producing an immobilized library is presented.

[0010] Also, here, indexing is directed to longer sequence reads. Libraries of tagged DNA fragments for index-directed assembly We present a method for generating a ly that involves a population of microparticles with immobilized traps. and preparing a transposome complex, the transposome complex comprising: A first polynucleotide and a second polynucleotide comprising an index domain associated with a microparticle. The target DNA is applied to a population of microparticles containing a transposase that binds to the microparticles. The goal is to generate immobilized DNA fragments tagged with index domains. In certain embodiments of the above method, the first polynucleotide is generated. The nucleotide is immobilized at its 5' end on the surface of the microparticle, and the second polynucleotide is immobilized at the first the first polynucleotide is hybridized to the 3' end of the polynucleotide, and the first polynucleotide is (ii) a 3' portion including a transposon end sequence; and (iii) an index domain; and wherein the population of microparticles includes at least a plurality of index domains; and wherein the first polynucleotide on each microparticle has the same index domain. share.

[0011] Also presented herein is a method for sequencing multiple target DNA molecules, which includes: A number of target DNAs are immobilized on a solid support with transposome complexes immobilized thereon. However, under conditions where the target DNA is fragmented by the transposome complex, whereby an immobilized library of double-stranded fragments is a first portion of each target DNA is generated and the first portion of each target DNA is pre-assembled at a first location on the solid support. and each second portion of target DNA is attached to the solid support. and linked by each target DNA; The immobilized library of double-stranded fragments is then used to generate a set of positions that are in contact with each other. mapping the library; determining the sequence of said first and second portions of target DNA; and to define that the first and second portions are linked by said target DNA, and correlating said set of positions to define the sequence of the target DNA molecule. .

[0012] In some embodiments of the methods and compositions provided herein, the transposome complex mm 2 At least 10 per 3 , 10 4 , 10 5 , 10 6 The complex is present on the solid support at a density of 0.1 μg / ml. In some embodiments, the transposome complex includes, for example, a Tn5 transposer. These include hyperactive transposases such as

[0013] In some embodiments of the methods and compositions provided herein, the tag domain may include, for example, For example, a region for cluster amplification is included. In some embodiments, a tag domain contains a region for priming the sequencing reaction.

[0014] In some embodiments of the methods and compositions provided herein, the solid support may include, for example, Examples include microparticles, patterned surfaces, wells, and the like. In this manner, the transposome complexes are randomly distributed on the solid support. In an embodiment, the transposome complexes are dispensed onto a patterned surface.

[0015] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will become apparent from the description and drawings, and from the claims. It becomes clear. [Brief explanation of the drawings]

[0016] [Figure 1a] 1 illustrates a general concept according to one embodiment. [Figure 1b] 1 illustrates a general concept according to one embodiment. [Figure 2a]FIG. 1 is a schematic diagram illustrating a tagmentation reaction. [Figure 2b] FIG. 10 is a schematic diagram with the resulting bridge redrawn to clarify the nature of the resulting bridge. [Figure 3a] Figure 3a illustrates an embodiment in which two forms of transposomes assemble on the surface of a flow cell. Addition of DNA to the flow cell results in tagmentation and coupling (pairing) of the DNA to the transposome. Figure 3a also shows the different types of resulting clusters: P7:P7, P5:P5, and P5:P7 clusters. [Figure 3b] 1 illustrates an embodiment in which two forms of transposomes are assembled into a flow cell. [Figure 4a] Another embodiment is illustrated in Figure 4a, where only one form of surface-bound transposome (e.g., P5 transposome) is present, resulting in a bridge with the same tag sequence at each end. Additional transposomes are added to further fragment (hereafter referred to as fragmentation) the bridge structure and incorporate an additional tag sequence (P7). [Figure 4b] This generates pairs of clusters obtained from each transpososome stump (transpososome stump) that demonstrate amplification and represent two adjacent fragments in the original intact DNA sample (Figure 4b). [Figure 5a] Figures 5a, 5b, 5c, and 5d illustrate different methods for assembling surface-bound transposome complexes. Figure 5a shows one embodiment of this method for assembling surface-bound transposome complexes. [Figure 5b] Figures 5a, 5b, 5c, and 5d illustrate different methods for assembling surface-bound transposome complexes. Figure 5b shows that the transposome complexes are assembled in solution, and immobilization includes the additional step of ligating the first polynucleotide to a splint oligonucleotide coupled to a solid support. [Figure 5c]Figures 5a, 5b, 5c, and 5d illustrate different methods for assembling surface-bound transposome complexes: Figure 5c shows that transposome dimers are assembled by hybridizing a looped oligonucleotide to an immobilized first polynucleotide. [Figure 5d] We show that transposome complexes can be assembled in a standard paired-end flow cell with amplification primers immobilized thereon. [Figure 6a] The design for the experiment described in Example 1 is described. [Figure 6b] The design for the experiment described in Example 1 is described. [Figure 7] Representative data from experiments conducted according to the design described in Example 1 are described. [Figure 8] Representative data from experiments conducted according to the design described in Example 1 are described. [Figure 9] 1 is an illustration of the assembly and subsequent tagmentation of bead-bound transposomes according to one embodiment. [Figure 10] 1 is an illustration of the assembly and subsequent tagmentation of bead-bound transposomes according to one embodiment. [Figure 11] 1 is an illustration of the assembly and subsequent tagmentation of bead-bound transposomes according to one embodiment. [Figure 12] 1 is an illustration of the assembly and subsequent tagmentation of bead-bound transposomes according to one embodiment. [Figure 13] 1 is an illustration of a surface-bound transposome according to one embodiment. [Figure 14] 1 is an illustration of tagmentation performed on bead-bound transposomes according to one embodiment. [Figure 15] 1 is an illustration of subfragment tagging and barcoding (hereafter referred to as barcoding) performed on bead-bound transposomes according to one embodiment. [Figure 16]1 is an illustration of subfragment tagging and barcoding performed on bead-bound transposomes according to one embodiment. [Figure 17] Illustrative example of transposome assembly as described in Example 3. [Figure 18a] Illustrative example of transposome assembly as described in Example 3. [Figure 18b] Results using transposome assembly as described in Example 3 are described. DETAILED DESCRIPTION OF THE INVENTION

[0017] A detailed description follows below. The current protocol for sequencing a nucleic acid sample is: Routinely employ sample preparation processes that convert DNA or RNA templates into libraries. These methods can result in loss of DNA sample and often However, fragmentation requires expensive equipment. Additionally, sample preparation methods are often difficult, tedious, and inefficient.

[0018] In the manufacturing method of the standard sample, each template (each mold) has an insert (insert) It contains adapters at either end, and the number of steps modifies both the DNA and RNA. It is often necessary to perform a purification step to obtain the desired product of the modification reaction. These steps are performed to determine the amount of ATP in solution prior to the addition of the fragments to the flow cell. They are carried out by adding a hy- drolyzed cellulose to the end of a primer that is covalently attached to the surface. The hybridized fragments are coupled to the surface by a primer extension reaction that copies the fragments. These "seed" templates then undergo several cycles of amplification. This results in monoclonal clusters of templates that are copied through

[0019] Adapter DNA in solution ready for cluster generation and sequencing The number of steps required to convert the template to be modified with α-transposase This can be minimized by using mediated fragmentation and tagging. The process is referred to herein as "tagging" and often involves the use of transposon end sequences. Transpososome complexes containing the transposase enzyme complexed with the adaptor containing the sequence. Tagging involves the simultaneous fragmentation of DNA and the separation of double-stranded fragments. This results in the ligation of adapters to the 5' ends of both strands. Following a purification step to remove the transposase enzyme, additional sequences are extracted by PCR. Append to the end of the adaptation fragment.

[0020] Solution-based tagmentation has drawbacks and requires several labor-intensive steps. In addition, bias can be introduced during the PCR amplification step. The methods and compositions presented overcome these drawbacks and facilitate sample manipulation or transfer. Unbiased sample preparation to occur on a single solid support with minimal requirements for , allowing clustering and sequencing.

[0021] The present disclosure provides transposomes precoupled to the surface of a flow cell. The complex effectively fragments, tags, and immobilizes intact DNA within the flow cell. In certain embodiments, the present invention relates to the surprising discovery that transposons can be used to One or more of the strands containing the adapters have their 5' ends The intact DNA is attached to the surface of the flow cell via a ion exchange membrane. When the nucleotide sequence is 0.01, the tagmentation reaction occurs similarly to how it occurs in solution-based tagmentation reactions. However, the resulting product fragments are physically attached by their ends to the surface of the flow cell. The transposome adapter sequences allow for subsequent cluster generation and sequencing. It can contain an array that

[0022] The methods and compositions presented herein have several advantages over solution-based tagmentation methods. For example, purified, partially purified, or unpurified intact DNA templates can be used. The sample is loaded directly onto the flow cell to generate clusters without prior sample preparation. It is also possible to read the contiguity of the sequence information in the original intact DNA. Connectivity is physically preserved by juxtaposing tagged fragments on the surface of the flow cell. As an added benefit, DNA can be physically linked to the surface of the flow cell, allowing for Such purification of the reagents after further manipulation of the NA is achieved by flow-through in the channels of the flow cell. - This can be achieved by buffer exchange.

[0023] Tagging on solid supports

[0024] In accordance with the above, there is provided herein a method for preparing an immobilized library of tagged DNA fragments. In some embodiments, the method includes: (a) a solid support having immobilized thereon a target molecule; By preparing a transposome complex, The body includes a transposase bound to a first polynucleotide, The domain contains (i) a 3' portion containing the transposon end sequence, and (ii) a first tag domain. (b) the target DNA is fragmented by the transposome complex; and and the 3' transposon end sequence of the first polynucleotide is located in at least one of the fragments. The target DNA is transferred to the 5' end of the other strand under conditions that The method may include applying the at least one strand to a support, whereby the at least one strand is An immobilized library of double-stranded fragments 5'-tagged with a single tag is generated. .

[0025] As used herein, the term "transposome complex" generally refers to a complex that is non-transposable to double-stranded nucleic acid. Refers to a covalently bound transposase enzyme. For example, the complex may be non-covalently bound. Under conditions that support the formation of synaptic complexes, preinducible nucleotides are synthesized together with double-stranded transposon DNA. The double-stranded transposon DNA can be a bated transposase enzyme. , including, but not limited to, Tn5 DNA, portions of Tn5 DNA, transposon end compositions, transposon A mixture of poson end components or a transposase such as a hyperactive Tn5 transposase It may contain other double-stranded DNAs capable of interacting with the transposase.

[0026] "Transposase" refers to a transposon end-containing composition (e.g., a transposon, transposon transposon end, transposon end composition) to form a functional complex, and Insertion or transposition of transposon end-containing compositions into heavy-strand target DNA ), which can be incubated, for example, in an in vitro rearrangement reaction. Transposases as presented herein also refer to enzymes that can The present invention relates to a method for the preparation of integrase-containing medicaments for the preparation of ... As exemplified by the disclosure of published application 2010 / 0120098, transposases, trans Transposomes and transposome complexes are generally well known to those skilled in the art. No. 6,299,499, the contents of which are incorporated herein by reference in their entirety. Many embodiments involve the use of Tn5 transposase and / or hyperactive Tn5 transposase. Although the present invention refers to a method for tagging transposon ends with 5' tags, it is not sufficient for its intended purpose. Any transposition system capable of rapid insertion and fragmentation of target DNA. It will be understood that a fusion system may be used in the present invention. In such a case, a preferred transposition system would be to randomly or nearly randomly transpose the 5' tag. It is possible to insert the transposon ends and fragment the target DNA.

[0027] The term "transposon end" refers to a transposon that is functional in an in vitro transposition reaction. Nucleotide sequences required to form a complex with the sase or integrase enzyme ("transposon end sequences"). In this study, transposon ends form functional complexes with transposase in the transposition reaction. As a non-limiting example, the transposon ends can be 19 bp (19 base pairs). outer end ("OE") transposon ends, inner end ("IE") transposon ends, or "mosaic" sequences recognized by wild-type or variant (mutant) Tn5 transposases. Ends" ("ME") transposon ends, or the disclosure of U.S. Patent Application Publication No. 2010 / 0120098 The R1 and R2 transposon ends described in The transposon ends are inserted into the in vitro transposition reaction. Suitable for forming a functional complex with the transposase or integrase enzyme present in the For example, the transposon ends can be It can contain DNA, RNA, modified bases, unnatural bases, modified backbones, and either or both The term "DNA" refers to the DNA strands at the ends of the transposon. As used throughout this disclosure in reference to compositions, any suitable nucleic acid or nucleic acid analogue may be used. It should be understood that the transposon end may be utilized.

[0028] The term "transferred strand" refers to the transferred portion of both transposon ends. Similarly, the term "non-transferred strand" refers to the non-transferred portion of both "transposon ends." The 3' end of the transferred strand is attached to the target DNA in an in vitro transposition reaction. The non-transferred strand is joined or transferred to the transposon by a sequence complementary to the end of the transferred transposon. The transposon terminal sequences are shown, and the transposon binds to or transposes to the target DNA in an in vitro transposition reaction. It will not be done.

[0029] In some embodiments, the transferred and non-transferred strands are covalently linked. For example, in some embodiments, the transferred and non-transferred strand sequences are contained within a single oligonucleotide. On nucleotides, an example is provided in a hairpin configuration. The free ends of the strands are not directly attached to the target DNA by the transposition reaction, but are The transferred strand is linked to the transferred strand by the loop of the hairpin structure. The non-transferred strand is indirectly attached to the DNA fragment. Additional examples of methods for preparing and using inspososomes are described in U.S. Patent Application Publication No. 2010 / 0120098. No. 6,299,523, the disclosure of which is incorporated herein by reference in its entirety. nothing.

[0030] The terms "tag" and "tag domain," as used herein, refer to the desired and intended Refers to a portion or domain of a polynucleotide that exhibits a sequence for a purpose or application. Some embodiments presented herein have a 3' portion that includes a transposon end sequence. a transposome complex comprising a polynucleotide encoding a target gene, and a tag comprising a tag domain; A tag domain may include any sequence that serves any desired purpose. For example, in some embodiments, the tag domain can be linked to one or more restriction endonucleases. In some embodiments, the tag domain comprises a cleavage enzyme recognition site. It contains one or more regions suitable for hybridization with a primer of interest. In an embodiment, the tag domain is used for hybridization with a primer for a sequencing reaction. Any other suitable features may be incorporated into the tag domain. It will be understood that in some embodiments, the tag domain may be 5 or 6. In some embodiments, the tag domain comprises a sequence having a length between 100 bp and 200 bp. comprises a sequence having a length between 10 and 100 bp. In some embodiments, the tag The domain comprises a sequence having a length between 20 and 50 bp. In some embodiments, the tag The domains are 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 and 200 It includes sequences having a length of between 100 and 150 bp.

[0031] In the methods and compositions provided herein, the transposome complex is attached to a solid support. In some embodiments, the transposome complex is immobilized, e.g., via one or more polynucleotides, such as a polynucleotide containing a sposon end sequence. In some embodiments, the transposome complex is immobilized on a solid support. The transposase enzyme can be immobilized via a linker molecule that couples it to the substrate. In some embodiments, the transposase enzyme and the polynucleotide Both the molecules and the nucleic acid are immobilized on a solid support. The terms "immobilized" and "attached" are used interchangeably herein when referring to Both terms are used interchangeably, directly and indirectly, unless otherwise expressly or by context. is intended to encompass indirect, covalent or non-covalent attachment. In some embodiments, the covalent attachment of the present invention may be preferred, but is often not required. All molecules (e.g., nucleic acids) are attached to the support, which allows the support to be used for, e.g., nucleic acid amplification and / or or under conditions intended for use in applications requiring sequencing. It is either stabilized or left attached.

[0032] Certain embodiments of the present invention involve the use of biomolecules, such as polynucleotides. a layer or coating of an intermediate material containing reactive groups that allow covalent attachment to consisting of an inert substrate or matrix that is functionalized, for example, by applying A solid support (e.g., glass slide, polymer beads, etc.) can be used. Examples of such supports include, but are not limited to, inert substrates such as glass. Polyacrylamide hydrogels supported on the substrate, in particular, those described in WO (International Publication) 2005 / 065814 and and polyacrylamide hydrochlorides such as those described in US (U.S. Patent Application Publication) 2008 / 0280773. and 4,000,000 hydroxybenzoates, the contents of which are incorporated herein by reference in their entirety. In such embodiments, the biomolecule (e.g., polynucleotide) is transferred to an intermediate material (e.g., a hydrogel). Although the intermediate material itself can be directly attached to the polymer (e.g., a polymer gel) by covalent bonding, It can be attached to a substrate or matrix (e.g., a glass substrate) by covalent bonding. "Covalent attachment to a solid support" refers accordingly to this type of arrangement (arrangement). should be interpreted as including the

[0033] The terms "solid surface," "solid support," and other grammatical equivalents are used herein to refer to transposon. Suitable for attachment of the phosphosome complex or can be modified to be suitable for it As will be appreciated by those skilled in the art, the number of possible substrates is very large. Suitable substrates include, but are not limited to, glass and modified or functionalized glass, plastics ( Acrylic, polystyrene and styrene copolymers and other materials, polypropylene, Polyethylene, polybutylene, polyurethane, TeflonTM , etc.), polysaccharides (polysaccharides lyride), nylon or nitrocellulose, ceramic, resin, silica or silica-based Materials containing silicon and modified silicon, carbon, metals, inorganic glasses, plastics These include polymers such as acrylic, plastics, plastics, optical fiber bundles, and a wide variety of other polymers. Solid supports and solid surfaces useful for some embodiments include flow cell apparatus (flow cell apparatus). An exemplary flow cell is described in more detail below. .

[0034] In some embodiments, the solid support is configured to carry transposons in an ordered pattern. "Patterned Surface" includes a patterned surface suitable for immobilizing the endothelial cell complex. refers to the placement of different regions in or on the exposed layer of a solid support. For example, one or more of the regions may be characterized by the presence of one or more transposome complexes. The feature is that it can be separated by gap regions where the transposome complex is not present. In some embodiments, the pattern may be an xy format of features in rows and columns. In some embodiments, the pattern may include a feature and / or interstitial area. In some embodiments, the pattern may be a repeating arrangement of features and / or or a random arrangement of gap regions. The sposome complexes are randomly distributed on the solid support. The transposome complexes are distributed on a patterned surface. Exemplary patterned surfaces that can be used in the article are described in U.S. Patent Application No. No. 13 / 661524 or U.S. Patent Application Publication No. 2012 / 0316086A1, each of which is incorporated herein by reference. Incorporated by reference.

[0035] In some embodiments, the solid support has wells or depressions in its surface. This generally includes an array of photonic crystals (e.g., a holographic image) using a variety of techniques, including but not limited to photolithography. including lithography, stamping, molding and micro-etching techniques. and can be prepared as known in the art. As will be appreciated, the technique used will depend on the composition and shape of the array substrate.

[0036] The composition and geometry of the solid support can vary depending on its use. In some embodiments, the solid support may be, for example, a slide, a chip, a microchip, or The surface of the substrate is thus flat (planar) and / or has a planar structure such as an array. In some embodiments, the solid support may be in the form of a flow layer. As used herein, the term "flow cell" refers to a solid a chamber including a surface across which one or more fluid reagents can flow; The flow cell and associated fluidic systems and methods of the present disclosure are readily Examples of detection platforms that can be used include, for example, the Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497; US 7057026; WO 91 / 0667 8;WO 07 / 123744;US 7329492;US 7211414;US 7315019;US 7405281, and US 2008 / 0 108082, each of which is incorporated herein by reference.

[0037] In some embodiments, the solid support or surface thereof may be, for example, a tube or container. In some embodiments, the solid support may be non-planar, such as the interior or exterior surface of a vessel. The carriers include microspheres or beads. By "cross-spheres" or "beads" or "particles" or grammatical equivalents, small By discrete particles, it is meant. Suitable bead compositions include, but are not limited to, plastic, ceramic, Polymers, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, Reasol, carbon graphite, titanium dioxide, latex or cross-linked dye Xtran, for example, Sepharose, cellulose, nylon, cross-linked Solid supports include micelles and Teflon, as well as any other solid supports outlined herein. All materials may be used. Bangs Laboratories, Fishers Ind. Microsphere Detection Group from Bioreactors, Inc., Fishers, Indiana The Microsphere Detection Guide is a helpful guide. The microspheres are magnetic microspheres or beads.

[0038] The beads need not be spherical; irregular particles can be used. Additionally, the beads may be porous. The size of the beads may be in the nanometer range, i.e. They range from 100 nm to millimeters, i.e., 1 mm, and are about 0.2 microns (μm) to about 200 Preferably, the beads are up to about 0.5 to about 5 microns in size, and particularly preferred are beads up to about 0.5 to about 5 microns in size. However, in some embodiments, smaller or larger beads may be used. It can be used.

[0039] Figures 1a and 1b generally illustrate a method according to one embodiment. Solid supports coated with oligonucleotides are shown, some of which contain ME sequences. The active transposons contain a sequence that, in the presence of Tn5, physically couples to a solid support. The density of these surfaces bound to the transposome is higher than that of the ME sequence. By varying the concentration of grafted oligonucleotides, including This can be adjusted by the amount of transposase added to the support. For example, in some embodiments, the transposome complex comprises mm 2 At least 10 per 3 , 10 4 , 10 5 , or at least 10 6 The complex is present on the solid support at a density of 0.1 μg / ml.

[0040] When double-stranded DNA is added to the solid support, the transposome complex binds to the added DNA. The NA is tagmented and thus coupled to the surface at both ends. In some embodiments, the length of the bridged fragment is determined by the surface area of ​​the fragment. This can be varied by changing the density of the transposome complex. In embodiments, the resulting bridged fragments are 100 base pairs, 200 base pairs, 30 0 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, 700 base pairs, 800 base pairs, 900 base pairs, 1000 base pairs, 1100 base pairs, 1200 base pairs, 1300 base pairs, 1400 base pairs, 1500 base pairs, 1600 base pairs, 1700 base pairs, 1800 base pairs, 1900 base pairs, 2000 base pairs, 2100 base pairs, 2200 base pairs, 2300 bases pairs, 2400 base pairs, 2500 base pairs, 2600 base pairs, 2700 base pairs, 2800 base pairs, 2900 base pairs, 3000 base pairs, 3100 base pairs, 3200 base pairs, 3300 base pairs, 3400 base pairs, 3500 base pairs, 3600 base pairs, 3700 base pairs, 3800 base pairs, 3900 base pairs, 4000 base pairs, 4100 base pairs, 4200 base pairs, 4300 bases pairs, 4400 base pairs, 4500 base pairs, 4600 base pairs, 4700 base pairs, 4800 base pairs, 4900 base pairs, 5000 base pairs, 10,000 base pairs, fewer than 30,000 base pairs, or fewer than 100,000 base pairs. In such embodiments, as exemplified by the disclosures of US Patent Nos. 7,985,565 and 7,115,400, The bridge fragments are then amplified into clusters using standard cluster chemistry. , the contents of each of which are incorporated herein by reference in their entirety. .

[0041] In some embodiments, the length of the template is preferably determined by the standard clustering method. For example, in some embodiments, The template lengths are 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, and 500 base pairs. , 600 base pairs, 700 base pairs, 800 base pairs, 900 base pairs, 1000 base pairs, 1100 base pairs, 1200 base pairs , 1300 bp, 1400 bp, 1500 bp, 1600 bp, 1700 bp, 1800 bp, 1900 bp base pairs, 2000 base pairs, 2100 base pairs, 2200 base pairs, 2300 base pairs, 2400 base pairs, 2500 base pairs, 26 00 base pairs, 2700 base pairs, 2800 base pairs, 2900 base pairs, 3000 base pairs, 3100 base pairs, 3200 base pairs , 3300 base pairs, 3400 base pairs, 3500 base pairs, 3600 base pairs, 3700 base pairs, 3800 base pairs, 3900 base pairs base pairs, 4000 base pairs, 4100 base pairs, 4200 base pairs, 4300 base pairs, 4400 base pairs, 4500 base pairs, 46 00 base pairs, 4700 base pairs, 4800 base pairs, 4900 base pairs, 5000 base pairs, 10000 base pairs, 30000 base pairs In such an embodiment, the first The second tagmentation reaction involves further fragmenting the bridge, as shown, for example, in Figure 4a. The second tagmentation reaction can be performed by adding transposomes from In this way, the internal span of the bridge can be eliminated and further sequencing steps can be performed. Anchor anchors to the surface that can be transformed into clusters ready for grafting. In certain embodiments, the length of the template is is within a range defined by upper and lower limits selected from those exemplified above. It is possible.

[0042] In certain embodiments, DNA is immobilized by surface tagmentation prior to cluster generation. A can be imaged. For example, immobilized DNA can be intercalated. The DNA molecules on the surface were stained with an interchelating dye and then Clusters can be imaged to keep a record of the positions of the clusters. After generation and sequencing, the coordinates of the clusters are determined by their positions on the original backbone. and thus along the molecular and genome assembly. Alignment of reads is supported.

[0043] In some embodiments, the step of applying target DNA includes applying a biological sample to the solid support. The biological sample may be any type of sample containing DNA. and can be deposited on a solid surface for tagmentation. For example, samples may contain DNA in various states of purification, including purified DNA. However, the sample does not need to be completely purified and can be, for example, DNA mixed with proteins, other nucleic acid species, other cellular components and / or any other contaminants In some embodiments, as shown in Example 2 below, biological The sample may contain DNA, proteins, other nucleic acid species, other cellular components and / or other components found in vivo. This includes mixtures of other contaminants present in approximately the same proportions as those produced. In some embodiments, the components are found in the same ratio as they are in intact cells. In some embodiments, the biological sample contains 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, Have a 260 / 280 ratio less than 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, or less than 0.60 In some embodiments, the biological sample has a pH of at least 2.0, 1.9, 1.8, 1.7, 1. 260 / 280 ratio of 6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, or at least 0.60 The methods provided herein allow DNA to be bound to a solid support. After surface-bound tagmentation has occurred, other contaminants can be removed by washing the solid support. The biological sample can be, for example, a crude cell lysate or whole cells. For example, crude cell lysate applied to a solid support by the methods described herein can be used. Lysates are prepared by one or more of the separation steps traditionally used to isolate nucleic acids from other cellular components. Exemplary separation steps are described in Maniatis et al. Molecular Cloning: A Laboratory Manual, 2nd Edition, 1989, and Short Protocols in Molecular Biology and the associated IEEE Protocols for the Promotion of Radioisotopes (PAPR), edited by Ausubel et al., which are incorporated herein by reference. Sink in.

[0044] Thus, in some embodiments, the biological sample may include, for example, blood, Sputum, serum, lymph, mucus, sputum, urine, semen, cerebrospinal fluid, bronchial aspirate, excrement (feces), and macerated tissue, or their lysates, or any DNA-containing Other biological specimens may be included. The biological sample is then passed through the flow cell and thereafter of the methods and compositions presented herein, which can be added to the lysis and purification steps of One advantage is that all of this can be achieved by simply flowing the necessary reagents through the flow cell. This occurs in the flow cell without any transfer or processing steps. Examples 1 and 2 below demonstrate this. The methods and compositions provided herein demonstrate successful application of crude cell lysates.

[0045] Figures 2a and 2b further illustrate a tagmentation reaction according to one embodiment. Thus, the transposome contains a dimer of Tn5, where each monomer binds a double-stranded molecule. That is, it contains an ME adaptor. One strand of the ME adaptor is covalently bonded to the The transposome binds the target DNA and then transfers it to the DNA backbone. In this case, two nicks are generated 9 bases apart on either strand. Although the figure shows a 9 base pair gap between the transposon and the nucleotide sequence, in other embodiments, The cleavage membrane can generate gaps of 7, 8, 9, 10, 11, or 12 base pairs between the nicks. Only one of the two strands of each ME adapter is the 5' strand at each nick position. This strand, the "transfer strand," is ligated via its 5' end to the The resulting bridges are grafted onto the surface. To clarify the nature of the geometry, it is redrawn in Figure 2b.

[0046] Figures 3a and 3b show an example of the invention in practice. Two forms of transposomes are shown in Figure 3a. The first form contains the P7 transposome, which assembles on the surface of the cell. In this study, the “transfer strand” of the ME adapter contains an amplification domain (P7) and a sequencing platform. The extended oligonucleotides link the transposome to the surface containing the nucleotides (S2). The second form includes the P5 transposome, in which The "transfer strand" of the ME adapter is the amplification domain (P5) and the sequencing primer. The transposome contains an extended oligonucleotide sequence that links the transposome to a surface containing β-transposome (S1). Addition of DNA to the flow cell results in tagmentation and binding of the DNA to the transposome. Three types of bridges result: P5-P5, P7-P7 and P5-P7. After cluster formation and linearization (Fig. 3b), either the P5-P5 or P7-P7 clusters were eliminated. As shown in Figure 3b, if linearization occurs through P5, then Only the P5-P7 and P7-P7 bridges remain. The P7-P7 cluster is linearized by this reaction. The P7-P7 cluster was then prepared for sequencing. Lead 2 is removed during linearization.

[0047] The methods described herein further include providing a transposome complex in solution; and target DNA is fragmented by the transposome complex solution in the liquid phase (solution the additional step of contacting the (liquid-phase) transposome complex with the immobilized fragment. whereby immobilized nucleic acid fragments having one end can be obtained in solution. In some embodiments, the transposome complexes in solution can be obtained by: The method may further comprise generating immobilized nucleic acid fragments having second tags, the second tags in solution. The first and second tags may be different or the same. It is possible.

[0048] In some embodiments, one form of surface-bound transposome is primarily a solid For example, in some embodiments, at least 50%, 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% % of the tags present on the solid support comprise the same tag domain. After the initial tagmentation reaction with surface-bound transposomes, at least 50%, 55%, and 60% , 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% % of the bridge structures contain the same tag domain at each end of the bridge. Perform further fragmentation of the bridge by adding transposomes from the solution. In some embodiments, most or all of the fluid-phase transposomes All of these are tag domains that are different from the tag domains present on the bridge structure generated in the first tagmentation reaction. For example, in some embodiments, at least 50%, 55%, 60%, %, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least The tag present in 99% of the liquid-phase transposomes is the tag generated in the first tagging reaction. It contains a tag domain that is different from the tag domain present on the ridge structure.

[0049] Figures 4a and 4b illustrate this embodiment. The solid support shown in Figure 4a contains a tag sequence (e.g., The transposomes contained only a single type of surface-bound transposome (P5 transposome). In this example, all tagmentation bridges are P5-P5 bridges. The P7 transposome is A bridge is added from solution and creates a template for all surface-bound molecules P5-P7. The surface-bound fragments can then be converted into clusters (e.g., (See Figure 4b for example.) Also, pairs of clusters are adjacent in the original intact DNA sample. Two fragments representing the respective transpososome ends would result (Fig. 4b).

[0050] Also presented herein are solid supports with tagged DNA molecules immobilized thereon. A fragment library, prepared according to the method described above.

[0051] Physical maps of immobilized polynucleotide molecules

[0052] Also provided herein are methods for generating physical maps of immobilized polynucleotides. The method advantageously comprises the step of: to identify clusters that may contain the first and second parts from Any two classes resulting from the immobilized polynucleotides can be The relative proximity of the targets thus determines the alignment of the sequence information obtained from the two clusters. Specifically, the ion exchange rate between any two given surfaces on a solid surface provides useful information for the purpose of The distance between the clusters is the distance between the two clusters from the same target polynucleotide molecule. A positive correlation was found between the probability of the object being a target and the probability of the object being a target, which is explained in more detail in WO 2012 / 025250. No. 6,119,793, which is incorporated herein by reference in its entirety.

[0053] As an example, in some embodiments, a long ds stretched over the surface of a flow cell. DNA molecules are tagged in situ and attached to the surface of the flow cell. A line of sDNA bridges is then generated, and a physical map of the immobilized DNA is then generated. After the immobilized DNA is amplified, a physical map can be generated in this way. Specifically, the physical map is based on the method described in WO 2012 / 02 As explained in the incorporated materials of No. 5250, the The sequence data is used to calculate the probability of linkage.

[0054] In some embodiments, the physical map is a map of DNA molecules immobilized across a solid surface. This is produced by imaging DNA to establish the location of the In an embodiment, the immobilized DNA is attached to a solid support by adding an imaging agent (contrast agent), and The signal is then detected by the imaging agent. In an embodiment, the imaging agent is a detectable label. Labels include, but are not limited to, protons, haptens, radionuclides, enzymes, fluorescent labels, chemical These include electroluminescent labels, and / or chromogenic agents (color-forming agents). In some embodiments, the imaging agent is an intercalating dye. ) or non-intercalating DNA binding agents. Intercalating dyes or non-intercalating DNA binding agents such as those known in the art are limiting. Although not disclosed herein, any of the above may be used, including those described in US 2012 / 0282617, the entirety of which is incorporated herein by reference. is incorporated herein by reference.

[0055] In some embodiments, the immobilized double-stranded fragments are cleaved to release free ends (see Figure 4a). To achieve this, the bridge structure is further fragmented prior to cluster formation (Fig. 4b). This technique is well known, as exemplified by the incorporated material in WO 2012 / 025250. Cleavage can be carried out using any suitable method known in the art. , modified nucleotides, such as uracil, as described in WO 2012 / 025250 by incorporating a restriction endonuclease site, or Bridge the solution-phase transposome complex to a DNA structure as described elsewhere herein. can be generated by applying

[0056] In certain embodiments, the plurality of target DNA molecules is placed on a flow cell comprising a plurality of nanochannels. The nanochannels have transposome complexes immobilized in them. As used in, the term nanochannel refers to a narrow channel through which long linear DNA molecules are flowed. In some embodiments, only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60 70, 80, 90, 100, 200, 300, No more than 400, 500, 600, 700, 800, 900, or 1000 targets (at most 1000) Individual long strands of DNA are flowed into each nanochannel. Individual nanochannels are separated by physical barriers, which are the individual channels of the target DNA. This prevents individual long strands from interacting with multiple nanochannels. In such cases, the solid support may be at least 10, 50, 100, 200, 500, 1000, 3000, 5000, 10000, 3 In some embodiments, the nanochannels may comprise 0,000, 50,000, 80,000, or at least 100,000 nanochannels. In this manner, transposomes bound to the surface of the nanochannel tag DNA. Contiguity mapping then determines, for example, the length of one of these channels. This can be done by following the clusters along The long strand of target DNA may be at least 0.1 kilobyte (kb), 1 kilobyte, 2 kilobytes, or byte, 3 kilobyte, 4 kilobyte, 5 kilobyte, 6 kilobyte, 7 kilobyte, 8 kilobyte 9 KB, 10 KB, 15 KB, 20 KB, 25 KB, 30 KB bytes, 35kB, 40kB, 45kB, 50kB, 55kB, 60 KB, 65 KB, 70 KB, 75 KB, 80 KB, 85 KB , 90KB, 95KB, 100KB, 150KB, 200KB, 250KB 100KB, 300KB, 350KB, 400KB, 450KB, 500KB byte, 550 KB, 600 KB, 650 KB, 700 KB, 750 KB , 800KB, 850KB, 900KB, 950KB, 1000KB, 50 00 KB, 10000 KB, 20000 KB, 30000 KB, or at least In some embodiments, the target DNA may be as long as 50,000 kilobytes. Strands are 0.1 kb, 1 kb, 2 kb, 3 kb, and 4 kb. , 5KB, 6KB, 7KB, 8KB, 9KB, 10KB , 15KB, 20KB, 25KB, 30KB, 35KB, 40KB byte, 45 KB, 50 KB, 55 KB, 60 KB, 65 KB, 70 KB KB, 75 KB, 80 KB, 85 KB, 90 KB, 95 KB, 100 KB, 150 KB, 200 KB, 250 KB, 300 KB, 350 KB 100KB, 400KB, 450KB, 500KB, 550KB, 600KB byte, 650 KB, 700 KB, 750 KB, 800 KB, 850 KB , 900 KB, 950 KB, or 1000 KB in length As an example, a 1000-well plate with immobilized tagged products mapped in a nanochannel Flow cells with 10 or more nanochannels require short "positioning" leads. In some embodiments, the method can be used to sequence the genome of an organism. The immobilized tagged products mapped in the nanochannel were analyzed using the solution haptic device used. In some implementations, the problem of fading (phase synchronization) can be addressed. To solve this problem, we used immobilized tagged products mapped in nanochannels. It can be used.

[0057] Amplification and sequencing of immobilized DNA fragments

[0058] amplification.

[0059] The present disclosure further provides methods for increasing the number of immobilized DNA fragments produced according to the methods provided herein. Immobilized DNA generated by surface-bound transposome-mediated tagmentation Fragment A can be amplified according to any suitable amplification method known in the art. In some embodiments, the immobilized DNA fragments are amplified on the solid support. In this embodiment, the solid support is the same solid support on which surface-bound tagmentation occurs. In such embodiments, the methods and compositions provided herein involve sample preparation. from the initial sample introduction step, through amplification, and optionally through the sequencing step. This allows for both to proceed on the same solid support.

[0060] For example, in some embodiments, the immobilized DNA fragments are subjected to cluster amplification techniques. The amplification is performed using a method exemplified by the disclosures of U.S. Pat. Nos. 7,985,565 and 7,115,400. The contents of each are incorporated herein by reference in their entirety. The incorporated material of U.S. Patent Nos. 7,985,565 and 7,115,400 is to form arrays consisting of clusters or "colonies" of immobilized nucleic acid molecules. In this paper, we describe a method for solid-phase nucleic acid amplification that allows the nucleic acid to be immobilized on a solid support. Each cluster or colony on such an array is composed of multiple identical immobilized polynucleotides. from a single strand and a plurality of identical immobilized complementary polynucleotide strands. Such formed arrays are generally referred to herein as "clustered arrays." For example, solid phases such as those described in U.S. Pat. Nos. 7,985,565 and 7,115,400 The products of the amplification reaction are immobilized polynucleotide strands and immobilized complementary strands. The DNA is formed by annealing a pair of DNA fragments, with both strands at their 5' ends on a solid support, preferably or immobilized via covalent attachment, so-called "bridged" structures. Cluster amplification methodology involves the use of immobilized nucleic acid templates to generate immobilized amplicons. Other suitable methodologies may also be used to produce the compounds according to the methods provided herein. This can be used to generate immobilized amplicons from immobilized DNA fragments. For example, one or more clusters or colonies may be Formed via solid-phase PCR whether one or both primers of each pair are immobilized It is possible.

[0061] In other embodiments, the immobilized DNA fragments are amplified in solution. In some embodiments, the immobilized DNA fragments are cleaved or otherwise removed from the solid support. The molecules are released from the carrier, and the amplification primers are then hybridized in solution with the released molecules. In other embodiments, amplification primers are hybridized to one or more initial amplification steps. The fragments are hybridized to immobilized DNA fragments for amplification, and then in solution for subsequent amplification. Thus, in some embodiments, the immobilized nucleic acid template is subjected to a liquid phase amplifying step. It can be used to generate precons.

[0062] Any amplification methodology described herein or commonly known in the art may be used to amplify immobilized DNA. It is understood that the method can be used with universal or target-specific primers to amplify fragments. Suitable methods for amplification include, but are not limited to, polymerase chain reaction (PCR). (PCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA) and nucleic acid sequence-based amplification width (NASBA), which are as described in U.S. Pat. No. 8,003,354, The above amplification methods may be used to amplify one or more For example, multiplex PCR can be used to amplify nucleic acids. PCR, including SDA, TMA, and NASBA, is used to amplify immobilized DNA fragments. In some embodiments, a primer specifically directed to a nucleic acid of interest can be , which is included in the amplification reaction.

[0063] Other suitable methods for amplification of nucleic acids include oligonucleotide extension and ligation. Ligation, rolling circle amplification (RCA) [Lizardi et al., Nat. G enet. (Nature Genetics) 19: 225-232 (1998)), which is incorporated herein by reference. Incorporation and oligonucleotide ligation assay (OLA) and U.S. Patent Nos. 7,582,420, 5,185,243, 5,679,524, and 5,573,907; EP (European Patent) EP 0 320 308 B1, EP 0 336 731 B1, EP 0 439 182 B1; WO 90 / 01069; WO 89 / 12696; and WO89 / 09835, all of which are incorporated by reference. These amplification methodologies may be designed to amplify immobilized DNA fragments. For example, in some embodiments, the amplification method may include Ligation probe amplification or oligation with primers specifically directed to the desired nucleic acid. An oligonucleotide ligation assay (OLA) reaction may be included. In some embodiments, the amplification method involves using primers specifically directed to the nucleic acid of interest. The amplification reaction may include a primer extension ligation reaction, which amplifies the nucleic acid of interest. Primer extension and ligation primers can be specifically designed to By way of non-limiting example, amplification is exemplified by U.S. Pat. Nos. 7,582,420 and 7,611,869. As described above, GoldenGate assay (Illumina, Inc.) Primers used for [San Diego, CA] each of which is incorporated herein by reference in its entirety. .

[0064] Exemplary isothermal amplification methods that can be used in the methods of the present disclosure include, but are not limited to: See, for example, Dean et al., Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United As exemplified by the study by the Journal of the American College of Cardiology (2002) 99:5261-66, Multiple Displacement Amplification (MDA), or, for example, US The present invention includes isothermal strand displacement nucleic acid amplification exemplified by Japanese Patent No. 6214587. Each of these is incorporated herein by reference in its entirety. Other non-PCR-based methods that can be used include, for example, strand displacement amplification (SDA), e.g., Wa Walker et al., Molecular Methods for Virus Detection Molecular Methods for the Analysis of Chromosomes, Academic Press, Inc., 1995; U.S. Pat. No. 5, 455,166, and 5,130,238, and Walker et al., Nucl. Acids Res. Acids Research 20:1691-1696 (1992), or, for example, ultrastructural Hyperbranched strand displacement amplification (HSTA) (Lage et al., Genome Research 13:294-307 (2003) each of which is incorporated herein by reference in its entirety. The thermal amplification method uses strand-displacing Phi29 polymerase or Bst DNA polymerase large. fragment, used with 5'->3' exon for random primer amplification of genomic DNA The use of these polymerases is due to their high processivity and strand length. High processivity allows the polymerase to generate fragments that are 10-20 kb in length. As mentioned above, smaller fragments can be used to generate Polymerases with low processivity and strand displacement activity, such as Further description of the amplification reaction, conditions and components This is described in detail in the disclosure of U.S. Pat. No. 7,670,810, which is incorporated herein by reference in its entirety. and incorporated by.

[0065] Another nucleic acid amplification method useful in the present disclosure is tagged PCR, which A population of two domain primers was used, each with a random 3' region followed by a constant 5' region. For example, Grothues et al., Nucleic Acids Res. 21(5):1321-2 (1993) and is incorporated herein by reference in its entirety. One round is based on individual hybridization from a randomly synthesized 3' region. This is done to allow multiple initiations on heat-denatured DNA. Therefore, the initiation site is thought to be random throughout the genome. The primers complementary to the constant 5' region can be removed, and further replication can proceed with the addition of primers complementary to the constant 5' region. This can be done using a marker.

[0066] Sequencing.

[0067] The disclosure further provides sequences of immobilized DNA fragments produced according to the methods provided herein. Immobilized proteins produced by surface-bound transposome-mediated tagmentation The DNA fragments can be sequenced using any suitable sequencing methodology, such as direct sequencing. Sequencing by synthesis, sequencing by ligation, sequencing by hybridization Sequencing according to the following criteria, including sequence determination, nanopore sequencing, and the like. In some embodiments, the immobilized DNA fragments can be attached to a solid support. In some embodiments, the solid support for sequencing comprises a surface The same solid support on which binding tagmentation occurs. In some embodiments, sequencing The solid support for is the same solid support on which amplification occurs.

[0068] One preferred sequencing methodology is sequencing-by-synthesis (sequencing by synthesis). In SBS, a nucleic acid template (e.g., a target nucleic acid or its amplicon) is subjected to Extension of the nucleic acid primer along the template is monitored to determine the sequence of nucleotides in the template. The underlying chemical process can be polymerization (e.g., polymerase enzymes). In particular, polymerase-based SBS embodiments utilize fluorescence to Labeled nucleotides allow for the detection of the order and type of nucleotides added to the primer. in a template-dependent manner such that the output can be used to determine the sequence of the template. It is added to the primer (thereby extending the primer).

[0069] The flow cell is used for housing the amplified DNA fragments produced by the methods of the present disclosure. In such a format, one or more amplified DNA fragments are provided as a convenient solid support. The fragments are subjected to SBS or other detection techniques involving repeated application of reagents in cycles. For example, to initiate a first SBS cycle, one or more labeled nucleotides can be The enzyme, DNA polymerase, etc., is placed in a flow cell containing one or more amplified nucleic acid molecules. The primer extension incorporates labeled nucleotides. The sites where this occurs can be detected. Once a nucleotide is added to the primer, it forms a reversible termination site that terminates further primer extension. For example, nucleic acids having reversible terminator moieties can be used. The oxidase analogue is delivered to remove the moiety via a deblocking agent. A primer can be added so that further extension does not occur until the primer is For embodiments using reversible termination, a deblocking agent is delivered to the flow cell. Washing can be performed between the various delivery steps (before or after detection occurs). The cycle then extends the primer by n nucleotides. The sequence can be repeated n times to detect sequences of length n. The procedures, fluidic systems and detection platforms are also suitable for use with an amplicons produced by the methods of the present disclosure. It can be readily adapted for use with precons, see, for example, Bentley et al., Nature e, 456:53-59(2008), WO 04 / 018497;US 7057026;WO 91 / 06678;WO 07 / 123744;US 7 329492; US 7211414; US 7315019; US 7405281, and US 2008 / 0108082. and US Pat. No. 6,299,099, each of which is incorporated herein by reference.

[0070] Other sequencing procedures using cyclic reactions, such as pyrosequencing Pyrosequencing can be used to identify specific nucleic acids. When a nucleotide is incorporated into a nascent nucleic acid strand, it releases inorganic pyrophosphate (PPi). Detect [Ronaghi et al., Analytical Biochemistry Mistry) 242(1), 84-9(1996);Ronaghi, Genome Res. 11(1), 3-11(2001); Ronaghi et al., Science 281(5375), 363(1998); US 6210891; US ​​6258568 and US 6,274,320, each of which is incorporated herein by reference. In the case of ATP sulfurylase, the released PPi is converted to adenosine triphosphate (ATP). The level of ATP produced can be detected by immediately converting it to The sequencing reaction can be detected via luciferase-produced photons. It can be monitored via a luminescence (cold light) detection system. The excitation radiation source used for this is not necessary for pyrosequencing procedures. The amplicons produced according to the method can be adapted for pyrosequencing applications. Useful fluidic systems, detectors, and procedures are described, for example, in WIPO Patent Application No. PCT / US11 / 571 No. 11, US 2005 / 0191698 A1, US 7595883, and US 7244559, each of which is 1006 / 000262, incorporated herein by reference.

[0071] Some embodiments involve real-time monitoring of DNA polymerase activity. For example, the incorporation of nucleotides can be achieved by incorporating fluorophore-bearing polymers. Fluorescence resonance energy transfer (FRET) interaction between γ-phosphate-labeled nucleotides and γ-phosphate-labeled nucleotides can be detected via the action or with zeromode waveguides (ZMWs) Techniques and reagents for FRET-based sequencing are described, for example, in Levene et al., Science e 299, 682-686 (2003); Lundquist et al., Opt. Lett. Letters 33, 1026-1028 (2008); Korlach et al., Proc. Natl. Acad. Sci. USA, 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference. To be incorporated.

[0072] Some SBS embodiments include a nucleotide that is released upon incorporation of a nucleotide into an extension product. For example, sequencing based on the detection of released protons. Ion Torrent (Guilford, Connecticut) ), commercially available from a Life Technologies subsidiary Handheld Electrical Detectors and Related Technology, or US 2009 / 0026082 A1; US ​​2009 / 012758 9 A1; US ​​2010 / 0137143 A1; or US 2010 / 0282617 A1, each of which is incorporated herein by reference. The sequencing methods and systems described in the incorporated herein may be used. The present invention relates to a method for amplifying a target nucleic acid using kinetic exclusion. The methods described can be readily applied to substrates used to detect protons. More specifically, the methods described herein can be used to detect protons. This can be used to produce a clonal population of amplicons that can be analyzed.

[0073] Another useful sequencing technique is nanopore sequencing (see, e.g., Deamer et al. , Trends Biotechnol. 18, 147-151 (2000); D Eagler et al., Acc. Chem. Res. (Accounts of Chemical Research) 35:817-825 (200 2); see Li et al., Nat. Mater. 2:611-615 (2003). [See, e.g., [D.], the disclosures of which are incorporated herein by reference.] Some nanopores In embodiments, the target nucleic acid or individual nucleotides to be removed from the target nucleic acid are ligated through the nanopore. When nucleic acids or nucleotides pass through the nanopore, each nucleotide type can be identified by measuring the variation in the electrical conductivity of the pores. U.S. Patent No. 7,001,792; Soni et al., Clin. Chem. 53, 1996-2001 (2007); Healy, Nanomed. 2, 459-481 (2007 Cockroft et al., J. Am. Chem. Soc. (Journal of the American Can Chemical Society) 130, 818-820 (2008), the disclosures of which are incorporated herein by reference. [Incorporated by]

[0074] For array-based expression and genotyping analyses that can be applied to detection according to the present disclosure Exemplary methods of this invention are disclosed in U.S. Patent Nos. 7,582,420; 6,890,741; 6,913,884; or 6,355,431. or U.S. Patent Application Publication Nos. 2005 / 0053980 A1, 2009 / 0186349 A1 or US 2005 / 0181440 A1, each of which is incorporated herein by reference.

[0075] The advantage of the methods described herein is that they allow rapid and efficient analysis of multiple target nucleic acids in parallel. Thus, the present disclosure provides for the detection of Nucleic acids can be prepared and detected using techniques known in the art. Thus, the integrated system of the present disclosure provides one or more Delivering amplification and / or sequencing reagents to immobilized DNA fragments The system may include, for example, pumps, valves, relays, and other fluid components that can be manipulated. Components such as reservoirs, fluid lines, and other similar items are included. Constructing and / or using in an integrated system for the detection of target nucleic acids Exemplary flow cells are described, for example, in US 2010 / 0111768 A1 and US patent application Ser. No. 13 / 273666, each of which is incorporated herein by reference. As exemplified for the flow cell, one or more of the fluidic components of the integrated system The above can be used for amplification methods and for detection methods. In embodiments involving sequencing, one or more of the fluidic components of the integrated system may be any of the fluidic components described herein. For amplification methods and for sequencing in sequencing methods such as those exemplified above. It can be used to deliver sequence-determining reagents. The system may be connected to another fluidic system to perform an amplification method and to perform a detection method. The method can include generating amplified nucleic acids and determining the sequence of the nucleic acids. Examples of possible integrated sequencing systems include, but are not limited to, MiSeq TM Platform (I Illumina, Inc., San Diego, CA), and the device described in U.S. Patent Application No. 13 / 273,666. which are incorporated herein by reference.

[0076] Solid supports with immobilized transposomes and methods of preparation

[0077] Other embodiments provided herein include solid supports, such as flow cells. , and having transposome complexes immobilized thereon. In such a case, the transposome complex comprises a transposase bound to the first polynucleotide. the polynucleotide comprises (i) a 3' portion comprising a transposon end sequence; and and (ii) a first tag comprising a first tag domain. The density of the transposons can vary. For example, in some embodiments, The mm complex 2 At least 10 per square millimeter 3 , 10 4 , 10 5 , or at least Also 10 6 The complex is present on the solid support at a density of 0.1 μg / ml.

[0078] Also presented herein is a method for generating a flow cell for tagmentation. The method includes, for example, immobilizing a plurality of transposome complexes on a solid support. The transposome complex comprises a transposase bound to the first polynucleotide. The first polynucleotide comprises (i) a 3' portion containing a transposon end sequence, and (i i) It may include a first tag that includes a first tag domain.

[0079] The transposome complex can be immobilized to a solid support in a variety of ways. In one embodiment, The method includes providing a solid support having a plurality of first polynucleotides immobilized thereon. and providing a solid support comprising a transposase holoenzyme and a second polynucleotide. the second polynucleotide comprises contacting the second polynucleotide with a transposon end; In some embodiments, the second polynucleotide comprises a region complementary to the target sequence. The transposase holoenzyme is added to the immobilized first polynucleotide. In some embodiments, the second polynucleotide and the transposon are hybridized. Figure 5a shows the assembly of the surface-bound transposome complex. One embodiment of this method for enabling the transposase holoenzyme is shown. From the 5' surface grafted end, a cluster amplification primer (A.pr) and a sequencing primer Flow cell containing primer (S.pr) and oligonucleotide containing ME sequence (Figure 5a) Instead, the non-transferred ME strand is added to the The strands can first hybridize to the surface-grafted ME, and then Transposase was added to the flow cell.

[0080] In some embodiments, the transposome complexes assemble in solution. The immobilization involves splint oligonucleotides coupled to a solid support. This embodiment further comprises ligating the first polynucleotide to the Illustrated in Figure 5b. In some embodiments, the splint oligonucleotide is It can be extended with a polymerase before the next step occurs.

[0081] In some embodiments, the transposome dimer comprises a looped oligonucleotide. Assembled by hybridizing to an immobilized first polynucleotide. For example, a looped oligonucleotide may include a first end and a second end. and a first end complementary to the transposon end sequence of the first polynucleotide. The second end of the looped oligonucleotide corresponds to the second transposon end sequence. The second transposon end sequence can be complementary to the first polynucleotide in the liquid phase, for example. In some embodiments, the immobilized nucleic acid may be a portion of a nucleotide. The first polynucleotide and the second polynucleotide in the liquid phase are The sposon may contain end sequences, or their complements. In such embodiments, the looped oligonucleotide has non-cognate first and second ends. The transposon end sequences are complementary to each of the transposon end sequences. An example of this embodiment is shown in Figure 5c. As shown in Figure 5c, adjacent adapter pairs represent two oligonucleotides. This is produced by hybridizing to a surface-bound oligonucleotide. This is achieved by employing two non-cognate transposon end sequences (ME1 and ME2). Addition of the transposase holoenzyme results in the formation of an active "looped" transposase. The transposome complex was reconstituted, where one of the two adapters in each transposome was Only one side is coupled to the surface 5c.

[0082] In another embodiment, the transposome complexes are separated in a standard paired-end flow cell. , with amplification primers immobilized thereon (e.g. , HiSeq flow cell or MiSeq flow cell sold by Illumina Inc, San Diego, CA This is evident, for example, in the hybridization of the "Sprint" oligonucleotides. This can be achieved by surface-grafting one of the amplification primers. The splint then anneals to one or both species. Sequence determination primers and oligonucleotides containing the transposon end sequences of the transposase Surface plates grafted with polymerase and dNTPs to form oligonucleotide duplexes Addition of transposase acts as a template to extend the surface transposons. Assemble the sposome. This embodiment is illustrated in Figure 5d.

[0083] In any of the embodiments provided herein, the transposome complex is a homodimer. For example, as illustrated in Figure 11, The transposome can then use two P5-ME adapters at both sites, or alternatively two Similarly, as shown in Figure 11, the heterodimeric trans The liposome complex will contain both the P5-ME and P7-ME adaptors.

[0084] Tagging using transpososome beads

[0085] One embodiment presented herein is a population of microparticles with transposons immobilized thereon. The use of solid supports, such as beads, can offer several advantages over solution-based tagmentation. For example, In solution-based tagmentation, it is difficult to control the final fragment size of the tagmentation reaction. Fragment size is a function of the transposome pair, the amount and size of DNA, and the duration of the reaction. Even if these parameters are controlled, the size selection fraction (si ze selection fractionation) is usually shorter than the combined paired read length , an additional step is required to remove excess small fragments. The proposed method avoids these drawbacks. Specifically, bead-immobilized transposomes Regardless of the amount of transposome beads added to the tagmentation reaction, Allows selection of final fragment size as a function of spatial separation of the polypeptides. A further limitation of tagmentation is the amount of purification required for the products of the tagmentation reaction both before and after PCR amplification. This typically requires some form of In contrast, bead-based transposons require some transfer of the reaction from beads to tubes. The tagged products in the system are washed and subsequently resolved for amplification or other downstream processing. The sample can be released, thus avoiding the need for sample transfer. In embodiments where the liposomes are assembled with paramagnetic beads, purification of the tagmentation reaction products can be accomplished by This can be easily achieved by immobilizing the beads with a magnet and washing. Thus, in some embodiments, tagging and other techniques such as PCR amplification are used. All downstream processing can be performed in a single tube, vessel, droplet, or other container. The application, filed on November 6, 2013, entitled "INTEGRATED SEQUENCING APPARATUSES AND METHODS OF USE" No. 13 / 670,318, entitled "Sequencing Apparatus and Methods of Use," which is incorporated herein by reference. As exemplified in the disclosure of the present application, which is incorporated in its entirety by reference herein, certain embodiments may be In this study, sample tagging and downstream processing was performed on a microfluidic droplet-based device. For example, in a microfluidic droplet-based device, droplets containing target nucleic acids, washing Buffers or other reagents are applied to the surface containing the immobilized transposome complexes. Similarly, beads with transposomes immobilized on them can be used to pass through the membrane. The droplets containing the target nucleic acid, wash buffer, and the like are then transferred to a microfluidic droplet-based device. or other reagents.

[0086] In some embodiments, the immobilized transposome complex comprises a first polynucleoside. a transposase that binds to a first polynucleotide and a second polynucleotide; The oligonucleotide is anchored at its 5' end to the surface of the microparticle, and the second polynucleotide is hybridized to the 3' end of the first polynucleotide, The tagged region includes: (i) a 3' portion containing the transposon end sequence; and (ii) a first tagged region. The first tag containing the main.

[0087] Figures 9-12 provide illustrations of transposomes assembled on the surface of paramagnetic beads. FIG. 9 shows a bead surface with two different first polynucleotides immobilized thereon. The first polynucleotide shown contains a transposon end sequence (ME). One of the first polynucleotides to be analyzed is an amplification primer sequence (P5) and a sequencing primer sequence (S1). The other polynucleotides shown in Figure 9 contain a tag domain containing a mer sequence (Read 1). The reads contained different amplification primer sequences (P7) and sequencing primer sequences (Read 2). The first polynucleotide may also include an index tag. The tag may be unique to the bead or may be unique to one or more other bead species in the population. A single bead can have a single index immobilized on it. It may have a tag or it may have multiple index tags fixed to it. It's fine.

[0088] FIG. 10 shows the second polynucleotides that hybridize to each of the first polynucleotides. The second polynucleotide has a region complementary to the transposon end sequence of the first polynucleotide. In some embodiments, the second polynucleotide is 15 base pairs in length. In some embodiments, the second polynucleotide is approximately 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150 It can be 15, 16, 17, 18, 19, 20 base pairs or longer than 20 base pairs. In embodiments, the second polynucleotide is phosphorylated at its 5' end.

[0089] FIG. 11 shows the assembly of transposomes on the surface of beads, where the transposomes The posase enzyme is contacted with the immobilized oligonucleotide. When transposase is added to the beads, three types of transposome complexes are formed. The following complexes were identified: P5:P5, P7:P7, and P5:P7 complexes (Figure 11).

[0090] When transposome beads are added to a solution of target DNA in tagmentation buffer, When the beads are immersed in water, tagging occurs, linking the DNA to the surface of the beads. A library is generated.

[0091] Figure 12 shows that sequential tagging into DNA provides bridging molecules between transposomes. An embodiment is shown in which three types of transposomes are present (as in FIG. 11 ): ), three types of bridge complexes result: P5:P5, P7:P7, and P5:P7 complexes. The ratio of fusion is 25:25:50 respectively.

[0092] In some embodiments, the length of the crosslinked fragments is determined by the length of the transposons on the surface of the beads. This density can be determined by the density of the oligonucleotides on the surface. amount, amount of double-stranded transposon end complexes on the surface and transposome assembly The amount of transposase enzyme added during the tagmentation step is adjustable. Upon cleavage, the P5:P7 tagmentation product is released from the surface of the beads using any suitable method. In some embodiments, the tagged products can be generated by, for example, suppressive PCR. PCR (suppression PCR), step-out PCR, etc. In some embodiments, the tagmentation products are released from the beads by cleavage. Cleavage can be, for example, chemical, enzymatic, photochemical, or a combination thereof. Any suitable method for releasing one or more tagmentation products from a solid support may be used herein. It will be understood that the present invention can be utilized in the methods provided herein.

[0093] The DNA may be attached to the surface using any suitable method to increase the probability of contact. For example, in some embodiments, Precipitation of DNA onto a solid surface allows for the formation of a complex between the target DNA and the transposome complex on the solid surface. This can be used to increase contact. contacting the DNA with a solid support, as incorporated by reference in its entirety Any one of many methods known in the art can be used for this purpose. As will be understood by those skilled in the art, for example, the bars used in solid phase reversible immobilization (SPRI) technology Precipitating DNA onto surfaces, including any of a number of buffers, PEG, ethanol, or DNA can be precipitated onto the solid support by adding any of a variety of other agents known to inhibit the synthesis of DNA. It can be made to be a palace.

[0094] In some embodiments, the population of beads with immobilized transposome complexes is Mix the beads with an excess of beads that do not carry transposomes or oligonucleotides. This reduces the possibility of tagmentation across two or more different beads. Another method to reduce the possibility of tagmentation across two or more different beads is to This involves immobilizing the beads so that contact between the beads is minimized. As exemplified by the incorporated material of WO 2010 / 115122, Immobilizing beads via magnetism on the side of a solid surface such as a small centrifuge tube, or any This can be accomplished by any of a number of techniques known in the art, including other immobilization techniques. This can be done.

[0095] In some embodiments, the transpososome beads are, for example, prokaryotic or eukaryotic. It can be used to isolate and identify nucleic acids from single cells, such as biological cells. For example, in some embodiments, particles such as beads can be Coated with indexed transposomes that share an index (specific beads All transposomes present on the beads are different from the indexes present on other beads. The beads are then separated using various methodologies known in the art. For example, to deliver beads within a cell, Methods for this include, but are not limited to, gene guns, photothermal nanoblades (Wu ) et al., Anal Chem. (Analytical Chemistry) (2011) 4:1321-7], and cells Peptides used in conjunction with permeabilizers (Nitin et al., Ann Biomed Eng. (2009) 37: 2018-2027) and others DNA from single cells is transferred to indexed transposomes. Any suitable method for associating particles with It will be understood that this may be possible.

[0096] In some embodiments, the transposons are used as described in detail above. The beads can be covalently attached to the beads. The insposomes can be released from the beads upon application of a chemical or physical stimulus. Some examples of stimuli that can trigger the release of transposomes from the body include: In some embodiments, the transposons are The method involves the use of enzymes such as restriction endonucleases to bind to solid supports. In certain embodiments, the transposome is released from the bead. The beads (or alternatively, the released transmembrane) are then free to move within the cell. Once the chromatin or DNA is in contact with the ribosomal membrane, tagmentation can occur. In mammalian and prokaryotic systems, all genomic DNA is always accessible and available for tagging. It will be understood that certain embodiments may not necessarily be implemented in accordance with the present invention. So, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% ,15%,20%,25%,30%,35%,40%,45%,50%,55%,60%,65%,70%,75%, up to 80%, 85%, 90%, 95%, or 99% or more of the total DNA in a cell % are tagged by transposomes. The use of only tagged beads By grouping together reads that share the same index, we identify reads from the same cell. These reads originate from the same bead (and therefore Additionally or alternatively, in some embodiments, In this case, a cross-linking step can be performed to cross-link cellular DNA, while it is still possible to inside the nucleus of a cell. Such steps link together the DNA from a single cell. After DNA extraction from the cells, it is then combined with indexed beads. The mixture can be mixed and subjected to tagmentation as described in detail above. Additionally or alternatively, variations of this method include the typing of flow-sorted chromosomes. Flow-sorted chromosomes can be mixed with the indexed beads described above. A specific chromosome can be attached to a bead and the fragments generated through tagging can be analyzed. This means that the entire chromosome (haplotyping) ) could enable phasing of SNPs.

[0097] In some such embodiments, the total number of index marks successfully tagged is In some embodiments, the dual index approach involves: For example, as a way to increase the number of possible combinations of indexes, As an example, the use of two 8-base indexes can be 8 ×4 8 =4.3×10 9 Combination of This gives the theoretical number of

[0098] In some embodiments, the approach involves individual cells being tagged with multiple beads. For example, one approach is to The solution is to use beads of a size similar to that of the cells. In this way, it is ensured that the cells cannot accommodate more than one bead. Alternatively, another approach is to take advantage of cell-to-bead ratios that favor single-cell targeting. For example, if there are many more cells than beads, then the beads within the cells The Poisson distribution of beads is such that cells that have taken up a single bead are more likely to take up two or more beads. This means that it far exceeds the cell.

[0099] In some embodiments, the single cell approach described above involves the identification of two SNPs (or structural repeats). sequence) are present in the same cell. For example, in a heterogeneous population of cancer cells, two SNPs may be present in the same cell or in different cells. Knowing whether or not you have cancer can help you deliver the right cancer treatment.

[0100] In some embodiments, the single cell approach described above is used to study RNA. For example, a suitable enzyme for the reverse transcription step (i.e., reverse transcriptase) and Coating beads with oligonucleotides allows for single-cell detection. Gene expression can be analyzed. In one embodiment, reverse transcriptase, oligonucleotides and transposome-coated beads were introduced, and cytoplasmic RNA was converted to cDNA. It can be converted, tagged, and prepared inside the cell.

[0101] A method for long-read assembly using barcodes on immobilized transposomes

[0102] Barcode-assisted assembly of DNA fragments is a method for identifying individual long DNA segments within a population of DNA molecules. Isolation of the fragments and conversion of each molecule into a uniquely barcoded subfragment library When the entire population of subfragmented DNA molecules is sequenced, the subfragments are By referencing the barcodes contained in the It can be done.

[0103] Various methods are known for barcoding individual DNA molecules. For example, the "dilution method" Extreme dilution and separate wells so that each well contains only one or a few molecules of DNA Individual long molecules are separated by aliquoting them into compartments (eg, wells of a plate). Each well is physically separated, so library preparation requires a unique barcode along with The contents of the wells are then pooled and distributed. Another method is to determine the sequence of each droplet by combining a long DNA molecule, library preparation reagents, and each droplet. Another approach is to use emulsions containing unique barcodes to detect the intactness of the molecules ( to insert multiple twin barcodes along the length of DNA while maintaining its integrity. Using a large library of indexed looped transposome complexes to Subsequent cleavage between the barcode "twin" allows for the matching of the barcodes. This results in fragments that can be sequenced and reassembled. Each of these methods has drawbacks that are overcome by the barcoding method presented here. Accompanying that.

[0104] Presented here is the above method of isolating and barcoding individual long molecules. The method presented here involves the use of emulsions. It achieves the same physical separation benefits as emulsification without the need for dilution. This provides a much greater degree of complexity than that provided by the large number of "wells" available. The unique barcode of the method is important because the number of beads in a bead-based method is often rare. In a sense, the "wells" of the dilution method can be much larger than the number of wells of the dilution method. An additional benefit over emulsion methods is that bead-based methods do not require a definitive barcode. The distribution of the barcodes is uniform (i.e., one barcode per bead) and non-random (i.e., multiple barcodes per bead). The method presented here also allows for molecular integrity and proximity ( contiguity) is a looped transpososome complex as used in some other methods. In addition, the method presented here achieves the same initial preservation without the need for coalescence. does not require as much code space as some other methods do.

[0105] Therefore, in some embodiments presented herein, the barcoding method involves Providing a population of particles having transposome complexes immobilized thereon the transposome complex comprises a first polynucleotide and a second polynucleotide; In some embodiments, the first polynucleotide comprises a transposase that binds to the first polynucleotide. The peptide comprises an index domain that is associated with a microparticle. In some embodiments, the index domain can be unique to the microparticle. In some cases, the population of particles includes at least a plurality of index domains. In an embodiment, the index domain is located on more than one microparticle in the population of microparticles. In some embodiments, in the population of microparticles, the microparticles have multiple individual Includes the x domain.

[0106] The barcoding method presented here further applies target DNA to a population of microparticles. thereby providing an immobilized DNA fragment tagged with an index domain. As exemplified by the incorporated material of WO 2010 / 115122, Any suitable method for increasing the likelihood of such contact may be used to efficiently separate the DNA. It can be contacted with a surface-bound transposome.

[0107] The method may be performed using any of a variety of known formats, for example, tagging reagents and labeling. The combination of bead arrays for library preparation is followed by indexed sequencing. Measurement runs and bespoke data analysis can be performed subsequently. Any other suitable method for maintaining in static separation from the surface tagmentation and It can be used for indexing purposes. For example, microspheres ) can remain in the well, e.g., by attaching a group that can hold beads. Physical construction such as wells or small depressions in the material, or the use of other forces ( magnetic or compressed), or chemically modified, or chemically functionalized at the active site, e.g. functionalized moieties, electrostatically modified moieties, hydrophobic and / or hydrophilic functionalized moieties, or It's like an adhesive spot.

[0108] In some embodiments, the microspheres are non-covalently associated with the wells. However, typically the wells may be additionally chemically functionalized as described below. Alternatively, a cross-linking agent may be used, or a physical barrier may be used, for example, a beam It is a film or membrane on the glass.

[0109] In certain embodiments, the surface of the substrate is either covalently or non-covalently bonded to the surface of the substrate. for attachment of the microspheres of the present invention to discrete sites or locations on a substrate. In this context, The "chemically modified site" includes, but is not limited to, an amino group, a carboxyl group, an oxidase group, and the like. The addition of patterns of chemical functional groups, including hydroxyl and thiol groups, allows them to be They can also be used to covalently attach molecules to other molecules. containing reactive functional groups that can be used to bind to microspheres; Addition of adhesive patterns (previous chemical functionalization for adhesive application or direct application of adhesive) by either addition of a pattern of charged groups for electrostatic attachment of microspheres; Addition (similar to chemical functionality), for example, microspheres with charged groups on the opposite side of the site. when containing similarly hydrophobic or hydrophilic microspheres under appropriate experimental conditions The addition of hydroxybenzoates to the microsphere sites based on hydroaffinity. sites differentially hydrophobic to result in association This includes the addition of patterns of chemical functional groups that confer hydrophilicity or hydrophobicity. The use of hydrophobic moieties on beads allows for preferential bead association to the moieties in aqueous systems. As outlined above, a "pattern" in this sense includes a set of discrete The surface is uniform to allow for bead attachment and treatment of the surface to provide distinct sites. As will be appreciated by those skilled in the art, this can be achieved in a variety of ways. It can be achieved.

[0110] In certain embodiments, a multiplicity of beads containing surface-bound transposomes are generated. , where each bead contains many transposomes, but for any given bead All transposomes on the beads contain the same barcode. A collection of monoclonal barcoded transpososome oligonucleotides The production of is exemplified by the disclosure of U.S. Pat. No. 5,604,097, which is incorporated by reference in its entirety. For better integration, the method may be performed according to any one of a number of techniques known in the art. It can be executed.

[0111] FIG. 13 illustrates one embodiment, in which surface-bound transposomes have their 5' As shown in Figure 13, the oligonucleotides contain long oligonucleotides attached to a surface via their ends. The 3' end of the oligonucleotide is the Mosaic End (M) of the Tn5 transposase enzyme. Upstream of this sequence (close to the 5' end) is a barcode sequence, typically 6-8 bases in length. The upstream is a primer sequence. Additionally, a cleavage site may be included to allow the oligonucleotide to be cleaved from the surface. Its presence in the oligonucleotide is optional to the overall method. The second short oligonucleotide (non-transcribed strand, MENTS) is attached to the long surface graft. ed) hybridizes to the METS sequence at the 3' end of the oligonucleotide. The sposase dimers must be fused to form functional transposomes. It is assembled at the 3' end of the oligonucleotide.

[0112] Figure 14 shows barcoding on beads in an array of wells. As shown in Figure 14, When long molecules of dsDNA are added to an array of bead-immobilized transposomes, A given molecule encounters a bead and is transported multiple times by the transposome on the bead. Each fragment gets tagmented. It is immobilized on a bead, and the bead In a particular embodiment shown in FIG. The physical separation of the beads in the loop prevents DNA molecules from reaching the gap between the two beads. In other embodiments, the beads are in close contact and one or more DNA molecules are attached to two or more beads. In some embodiments, more than one DNA molecule may extend between beads. The probability of two alleles being tagged on the same bead is low, and is a function of the concentration of DNA added, the number of beads, and the number of barcodes. For example, , 0.1 × haplom to avoid two alleles occurring in the same well 1000 genome equivalents (50,000 genome equivalents) with unique barcodes on 1 million beads each You need to load it into

[0113] Figure 15 shows the transfer of barcoded and tagged molecules to a sequencing reaction. As shown in Figure 15, once tagmentation is complete, the DNA is split into individual fragments, pooled, and They are then transferred from the bead surface into a solution so that they can be prepared for sequencing. Sequencing and assembly by referencing barcodes is performed on the original long tagged This allows the sequence of the DNA molecule to be recreated, thus eliminating long or pseudo-SNPs. This allows for similarly long leads and fading.

[0114] The release of barcoded surface tagged fragments into solution is known in this technology. This can be achieved using any suitable methodology, such as those described in In some embodiments, the tagged molecule is attached at the 5' end of a surface-bound oligonucleotide. can be cleaved from the surface of the beads via the presence of a cleavage moiety (See Figure 13.) The cleavage moiety can be any suitable moiety for cleaving a nucleic acid strand from a solid support. Examples of methods utilizing cleavage moieties include, but are not limited to, and WO 2006 / 064199, which are cleavage methods and cleavage components thereof, restriction endonuclease cleavage, chemical cleavage, including those incorporated by reference in These include cleavage, RNase cleavage, photochemical cleavage, and others like it.

[0115] Cleavage using a cleavage component gives a molecule with the following format: 5'-primer-barcode-ME-insert-ME-barcode-primer-3'

[0116] The "primer" region refers to additional sequences that may be added, such as amplification and sequencing primers. Columns allow for PCR step-out primers to hybridize, and hybridization For example, amplification primers P5 and P6 can be used as a primer for amplification of P7 can be added. Once added, suppressive PCR can be performed by adding, for example, a P5 adapter on one end. can be used to enrich for molecules with P7, and others with P7.

[0117] In some embodiments, amplification is by suppression PCR to add P5 and P7 adapter sequences. In another embodiment, the step-out primer can be directly from the bead. Each bead can have two types of surface-grafted oligonucleotides, Here, the primer sequence is (as in Figure 13) P5-Read 1 sequencing primer or P7-Read 2 sequencing primer. This means that the mixed P5-P7 As mentioned above, these are either detached from the beads or and then followed by suppression PCR to enrich for P5 / P7 molecules, or It can be amplified directly from the signal.

[0118] In some embodiments, a single transposome type (e.g., P5-Read1-Barcode) Once surface tagging is complete, different amplification and and / or a second transposome carrying a sequencing primer to cleave the bridge molecule. This allows for either cleavage or amplification from the bead surface. All molecules are provided with the same adapter format that allows for either Additional sample-specific barcodes allow multiple samples to be pooled by this method. Figure 16 shows an example of this embodiment. As shown in Figure 16, the liquid phase transposome carrying the P7 amplification primer sequence The immobilized fragment is added to the beads along with the immobilized bridge-tagged product. A polymerase chain containing a wide primer sequence and a barcode index tag (i9) that is unique to the bead. As shown in Figure 16, after the second tagging reaction, each The fragments were then amplified with fixed ends bearing the P7 and P5 primer sequences. It has an origin. [Example]

[0119] Example 1 Surface tagmentation in a flow cell

[0120] This example describes experiments confirming the embodiment shown in Figures 4a and 4b (surface tagging). (followed by solution tagmentation).

[0121] Experiments on an 8-lane flow cell were performed under multiple conditions and conditions, as shown in Figures 6a and 6b. Lane 6 shows a complete end-to-end description of the method. Indicates: unfragmented E. coli genomic DNA is added to the flow cell, which results in the surface-bound transposons The naked ME sequence was then selectively dehybridized. To do this, heat is applied to the flow cell, so that the transposomes are then transported into solution. When added from the first, they were eliminated as targets for the second tagmentation reaction. After the second tagmentation reaction, clusters are generated and sequenced, forming "paired-end" (2 Sequencing metrics are given in slide 7. In lane 6, 73.14% of the clusters passed through the filter, and This indicates that 74.69 of the inserts are aligned. Provide sufficient data to obtain plots and estimates of library diversity (Figure 8 reference).

[0122] Other lanes contained the following controls: Lane 1 shows everything was pumped correctly, cluster generation and sequencing PhiX DNA was used as a positive control to ensure that the include. Lane 2 is another control lane, showing surface-bound transfectants in the absence of target DNA. The flow cell (FC) contains a standard paired-end FC. This involves the use of an oligonucleotide containing a tagged primer sequence and an ME' sequence (non-transcribed ME' strand). The oligonucleotide was hybridized to the P7 surface oligonucleotide. The first extension resulted in a double-stranded transposon with ME ends. Transposomes were assembled in solution and run on a FC (6.25 nM / lane). The P5 transposome binds the transposon to the surface of the double stranded strand. The tagmentation products are then converted into clusters. Lane 3 shows tagging of surface-bound transposons in the absence of target DNA; in this example To convert ds surface-bound transposons into single-stranded oligonucleotides only in This prevents tagmentation of the target DNA prior to addition of P5 transposomes from solution. Therefore, the FC was heated to 75°C to prevent tagmentation of these constructs. Lane 4 shows the addition of surface-bound transposomes to the conditions in lane 3. In the method, an oligonucleotide containing a tagmentation primer sequence and an ME' sequence is attached to the P7 surface. The first extension hybridized to the double-stranded transposon carrying the ME. Following this, the P7 transposome was added to the lane at a 50x concentration of Tn5 enzyme. The FCs were assembled on the surface by incubating at 30°C for 30 minutes. The FC was then heated to 75°C before adding the P5 transposomes from the solution. Lane 5 contains the same conditions as lane 4, only in this case, the P5 transposomes were removed from solution. Instead of adding E. coli, a 900 bp library (P5 / P7 ends) was added to the surface-bound transcript of P7. Sposomes were added to determine whether they remained active after the heating step. Lane 6 shows an example of the realization of the present invention. In this example, FC is converted from standard paired-end FC to and an oligonucleotide comprising a tagmentation primer sequence and an ME' sequence. The first extension was a double-stranded oligonucleotide with ME ends. P7 transpososomes are obtained by adding Tn5 enzyme to the lanes at a 50x concentration. and assemble onto the surface of the FC by incubating for 30 min at 30 °C. Target DNA (300 ng of unfragmented E. coli genomic DNA) was added to the FC lane and and an incubation step at 55°C for 15 minutes were performed to allow tagmentation to occur. P7 surface-bound transposomes were washed away using PBI (Qiagen), and The FC was then heated to 75°C before adding the P5 transposomes from the solution. After the addition of P5 transposomes and a 15 min incubation step at 55 °C, the Stand displacement extension reactions are performed by using 9-amino-3-methyl-2-propanol groups generated in the DNA backbone by a transposition reaction. The stand displacement extension reaction was carried out to fill the 0-bp gap. The P5 transposome is prepared by adding 1000 ribosomal RNA and incubating at 65°C for 5 minutes. The final step was washing away. At this point, all surface-bound molecules were attached to the P5-P7 templates. should be a cluster and can therefore be converted into a cluster. Lane 7 contains the same conditions as lane 6, only in this instance the heating step is The data was omitted to highlight the effect of heat on star count and % aligned. Lane 8 contains a negative control, in which P7 transposomes bind to ds surface-bound transposomes. Assembled on the surface at 50x concentration in the presence of a saturating concentration of transposon, but the target No DNA was added. This indicates that the surface-bound P7 transposomes were not directly connected to their neighboring surface-bound transposomes. This allows for evaluation of whether transposons are tagged.

[0123] Example 2 Surface-bound sample preparation from E. coli scrapes

[0124] E. coli samples (5mm x 2mm scrapes from the bacterial lawn on the agar plate) The cells were scraped off and resuspended in a tube containing water and glass beads. Mix using a vortex mixer to disrupt the cells, then remove the cell debris. The supernatant (containing the cell lysate and containing proteins and nucleic acids) was collected by centrifugation. (having) and immobilizing the transposomes according to the protocol described in Example 1. Genome Analyzer flow cell (Illumina, Inc.) Lumina, San Diego, CA).

[0125] Cluster generation is performed using the Cluster Station sample preparation device. Cluster generation was performed on a flow cell using a 3D printer (Illumina, Inc., San Diego, CA). Afterwards, a paired-end sequencing run was performed with 36 base reads in each direction.

[0126] For read 1, 58.99% of the clusters passed the filter, and 92.16 of these were analyzed. For read 2, 58.99% of the clusters passed the filter, and 55.0% of these These data were obtained by aligning crude cell lysates with transposome-immobilized The results are surprisingly robust. Check that:

[0127] Example 3

[0128] This example shows the effect of surface-bound oligonucleotides on the addition of liquid-phase transposomes to the flow cell. A method for avoiding tagmentation of the peptide duplex is described.

[0129] One method for assembling transposomes on the surface of a flow cell is to use standard A paired-end flow cell was used, and polymerase was used to create a double-stranded ME sequence. P5 and / or P7 surface groups form extendable overhangs that can be extended Hybridization of 'splint' oligonucleotides with rafted oligonucleotides At this stage, the transposase enzyme hybridizes functional surface-bound transposons. Only part of the duplex can be added to form a transpososome (Fig. 5d). When forming sposomes, the remaining 'naked' ME duplexes are then transported to the immediate surface Tagged transposomes either bound or added from solution. Furthermore, fully assembled surface transposons can be targets for transcription. The system also reacts with nearby surface-bound transposomes or transposomes added from solution. It contains a dsDNA segment upstream of the ME sequence that can be targeted for tagging by the nucleotide sequence. Unwanted tagmentation is filtered out using a purity filter that aligns to the target genome. This is manifested in a decrease in the proportion of clusters passing through rs.

[0130] An example of this effect is shown in Figure 18b by comparing lanes 4 and 5 versus lanes 6 and 7. In lanes 6 and 7, the transposomes were Assembled using a long splint oligonucleotide, it is extended and transfected. After sposome assembly, surface-bound duplexes with at least 50 double-stranded bases are generated. In use, only 22.59% of the surface tagmentation reaction (Figure 18a) and subsequent sequencing were performed. and 15.52% of the clusters aligned to the target E. coli, respectively (Fig. 18b).

[0131] Avoid generating a population of clusters containing sequences that do not align to the target genomic DNA To achieve this, transposome assembly was performed as shown in Figure 17. The oligonucleotide is extended with a polymerase to create a duplex containing a double-stranded ME sequence. Surface grafting of P5 and / or P7 to form extendible overhangs that could be The oligonucleotide was first hybridized to the splint oligonucleotide. After removing the leutide by de-annealing and washing it off, , followed by even shorter oligonucleotides, only about 12 bases long, but preferably 16 bases long. The oligonucleotide is placed by hybridizing it to the 3' end of the surface-attached ME oligonucleotide. The transposase creates a transposome that does not contain any exposed dsDNA. Any 'naked' DNA that does not contain a transposase was added to assemble the DNA. The duplexes are selectively deannealed by incubating the flow cell at 60°C. and removed by washing under flow conditions to remove short double-stranded DNA.

[0132] The beneficial effect of this approach on assembling transposomes is This can be seen in Figures 4 and 5, where surface-bound transposomes are used in this method. The resulting nucleotides were assembled and used in a surface tagmentation reaction (Figure 18a). The percentage of clusters aligned to was 22.59 and 15.0 for lanes 6 and 7, respectively. 52% to 96.47 and 96.41 for lanes 4 and 5, respectively (Fig. 18b). .

[0133] Throughout this application, various publications, patents and / or patent applications are referenced. The disclosures of these publications are incorporated herein by reference in their entireties into this application. be absorbed.

[0134] The term encompassed is used herein to include not only the recited elements but also additional elements. It is intended to be open-ended, including exhaustive.

[0135] A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. a solid support having a plurality of transposome complexes immobilized thereon; a plurality of sites on a surface of the solid support; a plurality of transposome complexes non-covalently attached to respective sites of the plurality of sites; Each of the transposome complexes is a dimer, and the dimer comprises: (a) a first monomer comprising a first transposase non-covalently bound to a first double-stranded nucleic acid comprising a first polynucleotide and a second polynucleotide; the first polynucleotide is immobilized on the solid support via its 5' end; and (i) a 3' portion containing a first transposon end sequence; and (ii) a first tag region; the second polynucleotide is a first monomer that includes a region complementary to the first transposon end sequence; and (b) a second monomer comprising a second transposase non-covalently bound to a second double-stranded nucleic acid comprising a third polynucleotide and a fourth polynucleotide; the third polynucleotide is immobilized on the solid support via its 5' end; and (i) a 3' portion containing a second transposon end sequence; and (ii) a second tag region; the fourth polynucleotide comprises a second monomer comprising a region complementary to the second transposon end sequence; A solid support, wherein the first tag region and the second tag region are identical in a first subset of the plurality of transposome complexes, and the first tag region and the second tag region are different in a second subset of the plurality of transposome complexes.

2. The solid support of claim 1 , wherein the plurality of sites are patterned on the surface.

3. The solid support of claim 1 , wherein the surface is the surface of a bead.

4. The solid support of claim 3 , wherein the beads are paramagnetic.

5. The solid support of claim 3 , wherein the beads are located within the wells of a flow cell.

6. The solid support of claim 1 , wherein the surface is a lane of a flow cell.

7. 2. The solid support of claim 1, wherein the first polynucleotide is longer than the second polynucleotide such that a 5' portion of the first polynucleotide, including the 5' end, is single-stranded.

8. 2. The solid support of claim 1, wherein the first tag region is within a 5' portion of the first polynucleotide that includes the 5' end.

9. The solid support of claim 1 , wherein the first tag region comprises an index sequence.

10. The solid support of claim 9, which is a bead and wherein the index sequence is unique to each individual bead.

11. 2. The solid support of claim 1, wherein the first tag region, the second tag region, or both the first tag and second tag regions comprise an amplification primer sequence.

12. The solid support of claim 1 , wherein the dimer is a homodimer.

13. 2. The solid support of claim 1, wherein the first tag region of the first monomer is a first amplification primer sequence and the second tag region of the second monomer is a second amplification primer sequence that is different from the first amplification primer sequence.

14. The solid support of claim 1 , wherein the plurality of transposome complexes comprises a hyperactive Tn5 transposase or a Tn5 transposase.

15. 1. A method of producing a flow cell, comprising: providing a flow cell comprising a solid support having a patterned surface comprising a plurality of sites; and immobilizing a plurality of transposome complexes on the solid support to non-covalently attach individual transposome complexes to individual sites; Each of the transposome complexes is a dimer, and the dimer comprises: (a) a first monomer comprising a first transposase non-covalently bound to a first double-stranded nucleic acid comprising a first polynucleotide and a second polynucleotide; the first polynucleotide is immobilized on the solid support via its 5' end; and (i) a 3' portion containing a first transposon end sequence; and (ii) a first tag region; the second polynucleotide is a first monomer that includes a region complementary to the first transposon end sequence; and (b) a second monomer comprising a second transposase non-covalently bound to a second double-stranded nucleic acid comprising a third polynucleotide and a fourth polynucleotide; the third polynucleotide is immobilized on the solid support via its 5' end; and (i) a 3' portion containing a second transposon end sequence; and (ii) a second tag region; the fourth polynucleotide comprises a second monomer comprising a region complementary to the second transposon end sequence; The method, wherein the first tag region and the second tag region are identical in a first subset of the plurality of transposome complexes, and the first tag region and the second tag region are different in a second subset of the plurality of transposome complexes.

16. 1. A method for generating a sequencing library, comprising: providing a flow cell surface having a solid support on which transposome complexes are immobilized within lanes of the flow cell surface, the transposome complexes comprising a dimer, the dimer comprising: (a) a first monomer comprising a first transposase non-covalently bound to a first double-stranded nucleic acid comprising a first polynucleotide and a second polynucleotide; the first polynucleotide is immobilized on the solid support via its 5' end; and (i) a 3' portion containing a first transposon end sequence; and (ii) a first tag region; the second polynucleotide is a first monomer that includes a region complementary to the first transposon end sequence; and (b) a second monomer comprising a second transposase non-covalently bound to a second double-stranded nucleic acid comprising a third polynucleotide and a fourth polynucleotide; the third polynucleotide is immobilized on the solid support via its 5' end; and (i) a 3' portion containing a second transposon end sequence; and (ii) a second tag region; the fourth polynucleotide comprises a second monomer comprising a region complementary to the second transposon end sequence; the first tag region and the second tag region are identical in a first subset of the plurality of transposome complexes, and the first tag region and the second tag region are different in a second subset of the plurality of transposome complexes; and applying target DNA to said transposome complex, thereby generating an immobilized DNA fragment.

17. 17. The method of claim 16, wherein the first tag region of the first monomer is a first amplification primer sequence and the second tag region of the second monomer is a second amplification primer sequence that is different from the first amplification primer sequence.

18. 17. The method of claim 16, further comprising generating clusters from the immobilized DNA fragments using one or more primers that hybridize to the first tag region.

19. 20. The method of claim 18, further comprising generating sequence data for the strands of the cluster.

20. 20. The method of claim 19, further comprising assembling the sequence of the target DNA using the sequence data.

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