Sample preparation on a solid support

Abstract

A method and composition are provided for using immobilized transposase and transposon ends to generate an immobilized library of double-stranded target DNA 5'-tagged on the surface. The method is useful for generating 5'- and 3'-tagged DNA fragments used in various processes including large-scale parallel DNA sequencing.

Description

Technology Field

[0001] Related applications

[0002] This application claims priority to U.S. provisional application No. 61 / 750,682, filed January 9, 2013, the entirety of which is incorporated herein by reference. Background Technology

[0003] There are various methods and applications where generating a library of fragmented and tagged DNA molecules from double-stranded DNA (dsDNA) target molecules is desired. Often, the purpose is to generate smaller DNA molecules (e.g., DNA fragments) from larger dsDNA molecules to be used as templates in DNA sequencing reactions.

[0004] Many methods currently used for the fragmentation and tagging of double-stranded DNA for next-generation sequencing are wasteful of DNA, require expensive equipment for fragmentation, and involve procedures for fragmentation, tagging, and recovery of tagged DNA fragments that are difficult, verbose, laborious, time-consuming, inefficient, costly, and require relatively large amounts of sample nucleic acids. Furthermore, many of these methods generate tagged DNA fragments that do not adequately represent the sequences contained in the sample nucleic acids from which they are produced. Therefore, what is needed in this field is a method that provides speed and ease of use when generating libraries of tagged DNA fragments from target DNA and can be easily applied to nucleic acid analysis methods such as next-generation sequencing and amplification.

[0005] Methods and compositions for preparing nucleic acid samples on a solid support are provided herein. The methods and compositions are particularly related to methods and compositions for fragmenting and tagging DNA using a transposon composition immobilized on a solid support. The methods and compositions provided herein are useful for generating a library of tagged DNA fragments for use, for example, in next-generation sequencing methods, etc. In some preferred embodiments, the invention relates to the preparation of linear ssDNA fragments on a solid support from target DNA (including double-stranded cDNA prepared from RNA) comprising any dsDNA of interest from any source for genomic, subgenomic, transcriptomic, or metagenomic analysis, or RNA expression analysis.

[0006] Accordingly, a method for preparing an immobilized library of tagged DNA fragments is provided herein, comprising: (a) providing a solid support having an immobilized transposome complex thereon, wherein the transposome complex comprises a transposase bound to a first polynucleotide, and the first polynucleotide comprises (i) a 3' portion comprising a transposon end sequence and (ii) a first tag comprising a first tag domain; and (b) applying the target DNA to the solid support under conditions in which the target DNA is fragmented by the transposome complex, thereby transferring the 3' transposon end sequence of the first polynucleotide to the 5' end of at least one strand of the fragment; and thereby producing an immobilized library of double-stranded fragments, wherein at least one strand is 5'-tagged with the first tag. In some embodiments, the transposome complex comprises a second polynucleotide comprising a region complementary to the transposon end sequence. The above method may further comprise the step of (c) providing a transposome complex in solution and contacting the transposome complex with a fixed fragment under conditions in which target DNA is fragmented by the transposome complex in solution; thereby obtaining a fixed nucleic acid fragment having one end in solution. In some embodiments, the transposome complex in solution may comprise a second tag such that the method produces a fixed nucleic acid fragment having a second tag, which is a second tag in solution. The first and second tags may be different or the same.

[0007] A solid support having a library of immobilized tagged DNA fragments prepared according to the above method or other methods is also provided herein. For example, a solid support having an immobilized transposome complex is provided herein, wherein the transposome complex comprises a transposase coupled to a first polynucleotide, and the polynucleotide comprises (i) a 3' portion comprising a transposon terminal sequence, and (ii) a first tag comprising a first tag domain.

[0008] Additionally, a method for generating a flowcell comprising immobilizing a plurality of transposome complexes on a solid support is provided herein, wherein the transposome complex comprises a transposase bound to a first polynucleotide, and the first polynucleotide comprises (i) a 3' portion comprising a transposon terminal sequence, and (ii) a first tag comprising a first tag domain.

[0009] The above method may further comprise providing a solid support having a plurality of first polynucleotides immobilized thereon, and contacting the solid support with a transposase holoenzyme and a second polynucleotide, wherein the second polynucleotide comprises a region complementary to the transposon end sequence. In some embodiments of the above method, immobilization comprises: (a) providing a solid support having a coupled amplification primer; (b) hybridizing the second polynucleotide to one of the amplification primers, wherein the second oligonucleotide comprises a region complementary to the transposon end sequence and a region complementary to the first tag; (c) extending the amplification primer using a polymerase to generate a duplex comprising the first polynucleotide hybridized to the second polynucleotide, wherein the first polynucleotide is directly immobilized on the solid support; and (d) contacting the solid support with a transposase holoenzyme to assemble a transposome complex on the solid support.

[0010] A population of microparticles having an immobilized transposome complex comprising a transposase bound to a first polynucleotide and a second polynucleotide is also provided herein; wherein the first polynucleotide is immobilized on the surface of the microparticle at its 5' end and the second polynucleotide is hybridized to the 3' end of the first polynucleotide; wherein the first polynucleotide comprises (i) a 3' portion comprising a transposon end sequence and (ii) a first tag comprising a first tag domain. A method for producing an immobilized library of tagged DNA fragments is also provided herein, comprising contacting target DNA with the population of microparticles to produce immobilized tagged DNA fragments.

[0011] Also provided herein is a method for generating a library of tagged DNA fragments for index-designated assembly for longer sequence reads, the method comprising the steps of providing a population of microparticles on which a transposome complex is immobilized, comprising a transposase and a second polynucleotide bound to a first polynucleotide comprising an index domain bound to the microparticles; and applying target DNA to the population of microparticles to generate an immobilized DNA fragment tagged with an index domain. In a specific embodiment of the method, the first polynucleotide is immobilized on the surface of the microparticle at its 5' end, and the second polynucleotide is hybridized to the 3' end of the first polynucleotide; wherein the first polynucleotide comprises (i) a 3' portion comprising a transposon end sequence, and (ii) an index domain; wherein the population of microparticles comprises at least a plurality of index domains, and the first polynucleotides on individual microparticles share the same index domain.

[0012] Additionally, a method for sequencing a plurality of target DNA molecules is provided in the present invention, comprising applying a plurality of target DNAs to a solid support having a fixed transposome complex thereon under conditions in which the target DNAs are fragmented by the transposome complex; generating a fixed library of double-stranded fragments (wherein a first portion of each target DNA is attached to the solid support at a first position of the solid support and a second portion of each target DNA is attached to the solid support at a second position of the solid support); mapping the fixed library of double-stranded fragments to generate a set of positions connected by each target DNA; determining the sequences of the first and second portions of the target DNA; and correlating the set of positions to determine whether the first and second portions are connected by the target DNA and determining the sequence of the target DNA molecule.

[0013] In some embodiments of the methods and compositions provided herein, the transsosome complex is mm 2 At least 10 per 3 , 10 4 , 10 5 , 10 6 The complex exists on a solid support at a density of the complex. In some embodiments, the transsosome complex includes an overactive transposase such as Tn5 transposase.

[0014] In some embodiments of the methods and compositions provided herein, the tag domain may include, for example, a region for cluster amplification. In some embodiments, the tag domain may include a region for priming a sequencing reaction.

[0015] In some embodiments of the method and composition provided herein, the solid support may comprise, for example, microparticles, patterned surfaces, wells, etc. In some embodiments, the transposome complex is optionally distributed on the solid support. In some embodiments, the transposome complex is distributed on the patterned surface.

[0016] Details of one or more embodiments are disclosed in the attached drawings and the following description. Other features, purposes, and advantages will be apparent from the description, drawings, and claims. Brief explanation of the drawing

[0017] FIG. 1a illustrates a general concept according to one embodiment. FIG. 1b illustrates a general concept according to one embodiment. Figure 2a is a schematic diagram illustrating a tagmentation reaction. Figure 2b is a schematic diagram of the generated bridge redrawed to clarify the characteristics of the generated bridge. FIG. 3a illustrates a specific example in which two types of transposoms are assembled on the surface of a flow cell. The addition of DNA to the flow cell results in DNA tagging and coupling to the transposoms. Also, different types of generated clusters: P7:P7, P5:P5, and P5:P7 clusters are shown in FIG. 3a. FIG. 3b illustrates a specific example in which two types of transposoms are assembled on the surface of a flow cell. FIG. 4a illustrates another specific example. In FIG. 4a, only one form of surface-bound transposome (e.g., P5 transposome) is present, forming a bridge with the same tag sequence at each end. Additional transposomes are added to further fragment the bridge structure and incorporate additional tag sequences (P7). Fig. 4b illustrates amplification generating pairs of clusters originating from each transsomatic stump representing two adjacent fragments from the original intact DNA sample (Fig. 4b). FIGS. 5a, 5b, 5c, and 5d illustrate different methods for assembling surface-bound transsosome complexes. FIG. 5a shows one embodiment of such a method for assembling surface-bound transsosome complexes. FIG. 5b illustrates the assembly of a transsosome complex in a soluble state, and immobilization includes an additional step of ligating a first polynucleotide to a splint oligonucleotide coupled to a solid support. FIG. 5c indicates that a transsosome dimer is assembled by hybridizing a loop oligonucleotide to the immobilized first polynucleotide. FIG. 5d indicates that the transsosome complex can be assembled to a standard pair of terminal folocells using an amplification primer immobilized thereon. FIG. 6a discloses the design for an experiment described in Example 1. FIG. 6b discloses the design for an experiment described in Example 1. FIG. 7 discloses representative data obtained from an experiment performed according to the design described in Example 1. FIG. 8 discloses representative data obtained from an experiment performed according to the design described in Example 1. FIG. 9 is an example of the assembly and subsequent tagging of a bead-coupled transsosome according to one embodiment. FIG. 10 is an example of the assembly and subsequent tagging of a bead-coupled transsosome according to one embodiment. FIG. 11 is an example of the assembly and subsequent tagging of a bead-coupled transsosome according to one embodiment. FIG. 12 is an example of the assembly and subsequent tagging of a bead-coupled transsosome according to one embodiment. FIG. 13 is an example of a surface-bonded transposome according to one embodiment. FIG. 14 is an example of tagging performed on a bead-linked transsosome according to one embodiment. FIG. 15 is an example of tagging and barcoding of partial fragments performed on a bead-linked transposome according to one embodiment. FIG. 16 is an example of tagging and barcoding of partial fragments performed on a bead-linked transposome according to one embodiment. FIG. 17 is an example of a transsosome assembly described in Example 3. FIG. 18a is an example of a transsosome assembly described in Example 3. FIG. 18b discloses the results using the transposome assembly described in Example 3. Specific details for implementing the invention

[0018] Current protocols for sequencing nucleic acid samples routinely employ sample preparation processes that convert DNA or RNA into a template library. This method results in DNA sample loss and often requires expensive machinery for fragmentation. Furthermore, sample preparation methods are often difficult, verbose, and inefficient.

[0019] In standard sample preparation methods, each template contains an adapter at one end of the insert, and many steps are often required for both modifying DNA or RNA and purifying the desired product of the modification reaction. These steps are performed in a lytic state, after which the fitting fragment is added to the flow cell, which is coupled to the surface by a primer extension reaction that copies the hybridized fragment on the end of a primer covalently attached to the surface. This 'seeding' template then generates monoclonal clusters of the copied template through several cycles of amplification.

[0020] The number of steps required to transform DNA into a lysed adapter-modified template prepared for cluster formation and sequencing can be minimized by using transposase-mediated fragmentation and tagging. This process, referred to herein as 'tagging,' often involves the modification of DNA by a transsosome complex comprising a transposase enzyme complexed with an adapter containing a transposon terminal sequence. Tagging results in simultaneous DNA fragmentation and ligation of the adapter to the 5' ends of both strands of the duplex fragment. After a purification step to remove the transposase enzyme, an additional sequence is added to the ends of the fitted fragment by PCR.

[0021] Dissolution-based tagging has disadvantages and requires multiple labor-intensive steps. Additionally, bias can be introduced during the PCR amplification step. The methods and compositions provided herein overcome these disadvantages and enable the preparation of non-biased samples, cluster formation, and sequencing, which occur on a single solid support with minimal requirements for sample manipulation or transfer.

[0022] This description relates to a surprising discovery that a transposome complex pre-coupled to the surface of a fluorocell can effectively fragment, tag, and immobilize intact DNA within the fluorocell. In a specific embodiment, one or more strands containing a transposome adapter are attached to the surface of the fluorocell via their 5' ends. When intact DNA is pumped into the fluorocell, the tagging reaction occurs in the same manner as in a lysis-based tagging reaction, but the resulting product fragments are physically attached to the surface of the fluorocell by their ends. The transposome adapter sequence may contain a sequence that enables subsequent cluster formation and sequencing.

[0023] The methods and compositions provided herein offer several advantages over lysis-based tagging methods. For example, purified, partially purified, or even unpurified intact DNA templates can be directly loaded onto a flow cell for cluster formation without prior sample preparation. Additionally, the continuity of sequence information in the original intact DNA can be physically preserved by the juxtaposition of the tagging fragment on the surface of the flow cell. As an additional advantage, since the DNA is physically linked to the surface of the flow cell, purification of the reagent after further manipulation of the DNA can be achieved by flow-through buffer exchange in the flow cell channels.

[0024] Tagging on a solid support

[0025] According to the above, a method for preparing an immobilized library of tagged DNA fragments is provided herein. In some embodiments, the method may comprise: (a) providing a solid support having an immobilized transposome complex thereon, wherein the transposome complex comprises a transposase bound to a first polynucleotide, and the first polynucleotide comprises (i) a 3' portion comprising a transposon end sequence and (ii) a first tag comprising a first tag domain; and (b) applying the target DNA to the solid support under conditions in which the target DNA is fragmented by the transposome complex, thereby transferring the 3' transposon end sequence of the first polynucleotide to the 5' end of at least one strand of the fragment; thereby producing an immobilized library of double-stranded fragments, wherein at least one strand is 5'-tagged with the first tag.

[0026] As used herein, the term “transfosome complex” generally refers to a transposase enzyme non-covalently bound to a double-stranded nucleic acid. For example, the complex may be a transposase enzyme pre-incubated with double-stranded transposon DNA under conditions supporting non-covalent complex formation. The double-stranded transposon DNA may, without limitation, comprise Tn5 DNA, a portion of Tn5 DNA, a transposon terminal composition, a mixture of transposon terminal compositions, or other double-stranded DNA capable of interacting with a transposase, such as a hyperactive Tn5 transposase.

[0027] "Transposase" means an enzyme capable of forming a functional complex with a transposon end-containing composition (e.g., transposon, transposon end, transposon end composition) and, for example, in an in vitro translocation reaction, catalyzing the insertion or translocation of the transposon end-containing composition into double-stranded target DNA that is co-incubated. The transposases provided herein may also include integrases from retrotransposons and retroviruses. Transposases, transpososomes, and transpososomal complexes are generally known to those skilled in the art, as exemplified by the description in US 2010 / 0120098, the full contents of which are incorporated herein by reference. Although many of the embodiments described herein refer to Tn5 transposases and / or hyperactive Tn5 transposases, it will be understood that any transposition system capable of inserting transposon ends into the 5'-tag and fragment of target DNA with sufficient efficiency for the intended purpose may be used in the present invention. In certain embodiments, a preferred transposition system may insert transposon ends into the 5'-tag and fragment of target DNA in any or nearly any manner.

[0028] The term “transposon end” means double-stranded nucleic acid DNA that represents only the nucleotide sequence (“transposon end sequence”) necessary to form a complex with a functional transposase or integrase enzyme in an in vitro translocation reaction. In some embodiments, the transposon end may form a functional complex with a transposase in a translocation reaction. As a non-limiting example, the transposon end may include a 19-bp outer end (“OE”) transposon end, an inner end (“IE”) transposon end, or a “mosaic end” (“ME”) transposon end recognized by wild-type or mutant Tn5 transposase, or the R1 and R2 transposon ends disclosed in the description of US 2010 / 0120098, the full contents of which are incorporated herein by reference. The transposon end may comprise any nucleic acid or nucleic acid analog suitable for forming a functional complex with a transposase or integrase enzyme in an in vitro translocation reaction. For example, the transposon end may comprise DNA, RNA, modified bases, non-natural bases, or modified backbones, and may include nicks on one or both strands. Although the term “DNA” is used throughout this description in relation to the composition of the transposon end, it should be understood that any suitable nucleic acid or nucleic acid analog may be used for the transposon end.

[0029] The term "transposed strand" refers to the transposed portion of the transposon end of both. Similarly, the term "non-transposed strand" refers to the non-transposed portion of the "transposon end" of both. The 3'-end of the transposed strand is connected to or transposed onto the target DNA in an in vitro transposition reaction. The non-transposed strand, which contains a transposon end sequence complementary to the transposed transposon end sequence, is not connected to or transposed onto the target DNA in an in vitro transposition reaction.

[0030] In some embodiments, the transposed strand and the non-transposed strand are covalently connected. For example, in some embodiments, the transposed strand and the non-transposed strand sequences are provided on a single oligonucleotide, for instance, in a hairpin arrangement. Thus, although the free end of the non-transposed strand is not directly connected to the target DNA by a transposition reaction, the non-transposed strand is connected to the transposed strand by loops of the hairpin structure, thereby indirectly attaching to the DNA fragment. Further examples of transposome structures and methods for manufacturing and using transposomes can be found in the description of US 2010 / 0120098, the full text of which is incorporated herein by reference.

[0031] As used herein, the terms “tag” and “tag domain” refer to a portion or domain of a polynucleotide representing a sequence for a desired intended purpose or application. Some embodiments provided herein include a polynucleotide having a 3’ portion containing a transposon terminal sequence, and a transposome complex containing a tag comprising a tag domain. The tag domain may include any sequence provided for any desired purpose. For example, in some embodiments, the tag domain includes one or more restriction endonuclease recognition sites. In some embodiments, the tag domain includes one or more regions suitable for hybridizing with a primer for a cluster amplification reaction. In some embodiments, the tag domain includes one or more regions suitable for hybridizing with a primer for a sequencing reaction. It will be understood that any other suitable features may be included in the tag domain. In some embodiments, the tag domain includes a sequence having a length of 5 to 200 bp. In some embodiments, the tag domain includes a sequence having a length of 10 to 100 bp. In some embodiments, the tag domain comprises a sequence having a length of 20 to 50 bp. In some embodiments, the tag domain comprises a sequence having a length between 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 to 200 bp.

[0032] In the methods and compositions provided herein, the transsosome complex is immobilized on a solid support. In some embodiments, the transsosome complex is immobilized on the support via one or more polynucleotides, such as a polynucleotide containing a transposon terminal sequence. In some embodiments, the transsosome complex may be immobilized via a linker molecule that couples a transposase enzyme to the solid support. In some embodiments, both the transposase enzyme and the polynucleotide are immobilized on the solid support. When referring to the immobilization of a molecule (e.g., nucleic acid) onto a solid support, the terms "immobilized" and "attached" are used interchangeably herein, and both terms are intended to include direct or indirect, covalent or non-covalent attachment unless explicitly or otherwise indicated by the context. In specific embodiments of the present invention, covalent attachment may be preferred, but all generally required is that the molecule (e.g., nucleic acid) remains fixed or attached to the support under the conditions in which the support is to be used, for example in an application requiring nucleic acid amplification and / or sequencing.

[0033] Certain embodiments of the present invention may utilize a solid support composed of a functionalized inert substrate or matrix (e.g., glass slide, polymer beads, etc.) by, for example, by the application of a layer or by coating an intermediate material containing reactive groups that allow for covalent attachment to biomolecules such as polynucleotides. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on inert lipids such as glass, particularly polyacrylamide hydrogels described in WO 2005 / 065814 and US 2008 / 0280773, the full contents of which are incorporated herein by reference. In such embodiments, the biomolecule (e.g., polynucleotide) may be directly covalently attached to the intermediate material (e.g., hydrogel), but the intermediate material may be non-covalently attached to the substrate or matrix (e.g., glass substrate). The term “covalent attachment to a solid support” should therefore be interpreted to include such types of arrangements.

[0034] The terms “solid surface,” “solid support,” and other grammatical equivalents herein refer to any material suitable for or suitablely modified for the attachment of the transposome complex. As understood by those skilled in the art, the number of possible substrates is very large. Possible substrates include, but are not limited to, glass and modified or functionalized glass; plastics (including acrylic, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon™, etc.); polysaccharides; nylon or nitrocellulose; ceramics; resins; silica or silica-based materials, e.g., silicon and modified silicon; carbon; metals; inorganic glass; plastics; optical fiber bundles; and various other polymers. Solid supports and solid surfaces that are particularly useful in some embodiments are located within a fluorocell device. Exemplary fluorocells are described in further detail below.

[0035] In some embodiments, the solid support comprises a patterned surface suitable for fixing the transposome complex in an aligned pattern. The “patterned surface” refers to an arrangement of different regions within or on the exposed layer of the solid support. For example, one or more of the regions may be feature regions where one or more transposome complexes are present. These feature regions may be separated by gap regions where the transposome complexes are not present. In some embodiments, the pattern may be a feature region in an xy format existing in rows and columns. In some embodiments, the pattern may be a repeating arrangement of feature regions and / or gap regions. In some embodiments, the pattern may be a random arrangement of feature regions and / or gap regions. In some embodiments, the transposome complexes are randomly distributed on the solid support. In some embodiments, the transposome complexes are distributed on the patterned surface. Exemplary patterned surfaces that can be used in the methods and compositions disclosed herein are described in U.S. Serial No. 13 / 661,524 or U.S. Patent Application Publication 2012 / 0316086 A1, each of which is incorporated herein by reference.

[0036] In some embodiments, the solid support comprises an array of wells or depressions on its surface. This can be manufactured as generally known in the art using various techniques, including, but not limited to, photolithography, stamping, forming, and microetching. As understood by those skilled in the art, the technique used will depend on the composition and shape of the array substrate.

[0037] The composition and geometry of the solid support may vary depending on the application. In some embodiments, the solid support is a planar structure such as a slide, chip, microchip, and / or array. As such, the surface of the substrate may be in the form of a planar layer. In some embodiments, the solid support comprises one or more surfaces of a flow cell. As used herein, the term "flow cell" refers to a chamber comprising a solid surface through which one or more fluid reagents can flow. Examples of flow cells, related fluid systems, and detection platforms that can be readily utilized in the methods described herein are, for example, the literature [Bentley et al., each incorporated herein by reference, Nature It is disclosed in 456:53-59 (2008), WO 04 / 018497; US 7,057,026; WO 91 / 06678; WO 07 / 123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281, and US 2008 / 0108082.

[0038] In some embodiments, the solid support or its surface is not flat, for example, the inner or outer surface of a tube or container. In some embodiments, the solid support comprises microspheres or beads. As used herein, “microsphere,” “bead,” “particle,” or grammatical equivalent means a distinct small particle. Suitable bead compositions include, but are not limited to, plastics, ceramics, glass polystyrene, methylstyrene, acrylic acid polymers, paramagnetic materials, thoria sol, carbon graphite, titanium dioxide, latex, or cross-linked dextran, such as Sepharose, cellulose, nylon, cross-linked micelles, and Teflon, as well as any other material outlined herein for the solid support. The “Microsphere Detection Guidelines” from Bangs Laboratories, Fishers Ind. are helpful guidelines. In certain embodiments, the microspheres are magnetic microspheres or beads.

[0039] The beads do not need to be spherical; irregular particles may be used. Alternatively or additionally, the beads may be porous. The bead size ranges from several nanometers, i.e., 100 nm, to several millimeters, i.e., 1 mm, and beads of about 0.2 microns to about 200 microns are preferred, and about 0.5 to about 5 microns are particularly preferred, but smaller or larger beads may also be used in some embodiments.

[0040] FIGS. 1a and 1b generally illustrate a method according to one embodiment. A solid support coated with grafted oligonucleotides is shown, some of which contain ME sequences, to form active transsosome complexes physically coupled to the solid support in the presence of Tn5. The density of these surface-coupled transsosomes can be controlled by varying the density of the grafted oligonucleotides containing ME sequences or by the amount of transposase added to the solid support. For example, in some embodiments, the transsosome complex is mm 2 At least 10 per 3 , 10 4 , 10 5 , or at least 10 6 It exists on a solid support at the density of the composite.

[0041] When double-stranded DNA is added to a solid support, the transposome complex will tag the added DNA to produce ds fragments coupled at both ends to the surface. In some embodiments, the length of the bridging fragments may be varied by changing the density of the transposome complex on the surface. In a specific embodiment, the length of the resulting bridged fragment is 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp, 1500 bp, 1600 bp, 1700 bp, 1800 bp, 1900 bp, 2000 bp, 2100 bp, 2200 bp, 2300 bp, 2400 bp, 2500 bp, 2600 bp, 2700 bp, 2800 bp, 2900 bp, 3000 bp, 3100 bp, 3200 bp, 3300 bp, 3400 bp, 3500 bp, 3600 bp, 3700 bp, 3800 bp, 3900 bp, 4000 bp, 4100 bp, 4200 bp, 4300 bp, 4400 bp, 4500 bp, 4600 bp, 4700 bp, 4800 bp, 4900 bp, 5000 bp, 10000 bp, less than 30000 bp or less than 100,000 bp. In such specific examples, the bridging fragment may subsequently be amplified into a cluster using standard cluster chemistry as exemplified by the descriptions in U.S. Patents 7,985,565 and 7,115,400, the contents of each of said patents are incorporated herein by reference in their entirety.

[0042] In some embodiments, the length of the mold is longer than can be suitably amplified using standard cluster chemistry. For example, in some embodiments, the length of the mold is 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp, 1500 bp, 1600 bp, 1700 bp, 1800 bp, 1900 bp, 2000 bp, 2100 bp, 2200 bp, 2300 bp, 2400 bp, 2500 bp, 2600 bp, 2700 bp, 2800 bp, 2900 bp, 3000 bp, 3100 bp, 3200 bp, 3300 bp, 3400 bp, 3500 bp, 3600 bp, 3700 bp, 3800 bp, 3900 bp, 4000 bp, 4100 bp, 4200 bp, 4300 bp, 4400 bp, 4500 bp, 4600 bp, 4700 bp, 4800 bp, 4900 bp, 5000 bp, 10000 bp, longer than 30000 bp or longer than 100,000 bp. In such a specific embodiment, the second tagging reaction can then be carried out by adding a transposome from the solution that further fragments the bridge, for example, as illustrated in FIG. 4a. Therefore, the second tagging reaction can remove the inner width of the bridge, leaving a short truncated end fixed to the surface that can be converted into a cluster prepared for an additional sequencing step. In a specific embodiment, the length of the mold may be within a range defined by upper and lower limits selected from those exemplified above.

[0043] In certain embodiments, DNA immobilized by surface tagging can be imaged prior to cluster generation. For example, the immobilized DNA can be stained with an interchelating dye and imaged to preserve a positional record of the DNA molecule's backbone on the surface. After cluster generation and sequencing, the coordinates of the clusters can be relative to their positions on the original backbone, thereby assisting in the alignment of reads according to molecular and genomic assemblies.

[0044] In some embodiments, the step of applying the target DNA includes adding a biological sample to the solid support. The biological sample may be any type that contains DNA and can be deposited on a solid surface for tagging. For example, the sample may contain DNA in various purified states, such as purified DNA. However, the sample does not need to be completely purified and may contain DNA mixed with, for example, proteins, other nucleic acid species, other cellular components, and / or any other contaminants. As demonstrated in Example 2 below, in some embodiments, the biological sample may contain a mixture of DNA, proteins, other nucleic acid species, other cellular components, and / or any other contaminants present in proportions roughly equal to those found in vivo. For example, in some embodiments, the components are found in proportions equal to those found in intact cells. In some embodiments, the biological sample has a 260 / 280 ratio of 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, less than 0.7, or less than 0.60. In some embodiments, the biological sample has a 260 / 280 ratio of at least 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, or at least 0.60. Since the method provided herein allows DNA to bind to a solid support, other contaminants can be removed by simply washing the solid support after surface-bound tagging has occurred. Biological samples may include, for example, unpurified cell lysates or whole cells. For example, in the method disclosed herein, the unpurified cell lysates applied to a solid support did not need to be processed into one or more separation steps traditionally used to separate nucleic acids from other cellular components. Exemplary separation steps are described in the literature incorporated herein by reference [Maniatis et al.It is disclosed in Molecular Cloning: A Laboratory Manual, 2nd Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al.

[0045] Accordingly, in some embodiments, the biological sample may include, for example, blood, plasma, serum, lymph fluid, mucus, sputum, urine, semen, cerebrospinal fluid, bronchial aspirate, feces, and macerated tissue, or lysates thereof, or any other biological sample including DNA. One advantage of the method and composition provided herein is that the biological sample can be added to the fluorocell, and subsequent lysis and purification steps can both occur in the fluorocell by simply flowing the necessary reagents into the fluorocell without additional transfer or processing steps. Examples 1 and 2 below demonstrate the successful application of unpurified cell lysates to the method and composition provided herein.

[0046] FIGS. 2a and 2b further illustrate a tagging reaction according to one embodiment. As illustrated in FIG. 2a, the transposome comprises a dimer of Tn5, namely an ME adapter, having each monomer that binds to a double-stranded molecule. One strand of the ME adapter is covalently attached to the surface of the fluorocell. The transposome binds to target DNA and creates two nicks on the DNA backbone separated by nine bases on either strand. Although FIG. 2a illustrates a 9 bp gap between the nicks, in other embodiments, the transposome may create a gap of 7, 8, 9, 10, 11, or 12 bp between the nicks. Only one of the two strands of each ME adapter is ligated to the 5' strand at each nick location. This strand, the 'transferred strand,' is grafted onto the surface of the fluorocell through its 5' end. To clarify the characteristics of the generated bridge, the generated bridge of the surface tagging is redrawed in Fig. 2b.

[0047] FIGS. 3a and 3b illustrate an example of the practical application of the invention. Two forms of transpososomes are assembled on the surface of a flurocell. The first form comprises a P7 transposome comprising an extended oligonucleotide sequence in which the 'transfer strand' of the ME adapter connects the transposome to a surface containing an amplification domain (P7) and a sequencing primer (S2). The second form comprises a P5 transposome comprising an extended oligonucleotide sequence in which the 'transfer strand' of the ME adapter connects the transposome to a surface containing an amplification domain (P5) and a sequencing primer (S1). The addition of DNA to the flurocell results in the tagging of DNA and coupling to the transposome. The results of the three types of bridging are P5-P5, P7-P7, and P5-P7. After cluster formation and linearization (Fig. 3b), the P5-P5 or P7-P7 clusters are removed (depending on the linearization). As illustrated in Fig. 3b, if linearization occurs through P5, only the P5-P7 and P7-P7 bridges remain. Since the P7-P7 cluster is not linearized by this reaction, it will not be sequenced. Subsequently, the P7-P7 cluster is removed during Read 2 linearization for sequenced sequences.

[0048] The method provided herein may further include the additional step of providing a transposome complex in a soluble state and contacting the solution-phase transposome complex with a fixed fragment under conditions in which target DNA is fragmented by the transposome complex solution; thereby obtaining a fixed nucleic acid fragment having one end in a soluble state. In some embodiments, the soluble transposome complex may comprise a second tag, so that the method produces a fixed nucleic acid fragment having the second tag, the second tag being in a soluble state. The first and second tags may be different or identical.

[0049] In some embodiments, a form of surface-bound transsosome is predominantly present on a solid support. 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 contain the same tag domain. In such embodiments, after an initial tagging reaction using surface-bound transsosomes, at least 50%, 55%, 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. The second tagging reaction may be performed by adding a transposome from the solution that further fragments the bridge. In some embodiments, most or all of the solution-phase transposome comprises a tag domain different from the tag domain present on the bridge structure generated in the first tagging reaction. 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 in the solution-phase transposome comprise a tag domain different from the tag domain present on the bridge structure generated in the first tagging reaction.

[0050] FIGS. 4a and 4b illustrate such specific embodiments. The solid support shown in FIG. 4a contains only surface-bound transpososomes (e.g., P5 transpososomes) containing a single type of tag sequence. In this example, all tagged bridges are P5-P5 bridges. P7 transpososomes are added from the solution and tagged as bridges, making all surface-bound molecules P5-P7 templates. The surface-bound fragments can subsequently be converted into clusters (see, for example, FIG. 4b). Furthermore, a pair of clusters will arise from each transposome truncated end representing two adjacent fragments from the original intact DNA sample (Fig. 4b).

[0051] A solid support having a library of tagged DNA fragments prepared according to the above method fixed thereon is also provided in the present invention.

[0052] Physical map of immobilized polynucleotide molecules

[0053] A method for generating a physical map of immobilized polynucleotides is also provided herein. The method can be advantageously used to identify clusters likely to contain linked sequences (i.e., first and second parts from the same target polynucleotide molecule). Accordingly, the relative proximity of any two clusters derived from the immobilized polynucleotides provides useful information for aligning sequence information obtained from the two clusters. Specifically, the spacing between any two clusters provided on a solid surface is certainly related to the likelihood that the two clusters originated from the same target polynucleotide molecule, which is described in more detail in WO 2012 / 025250, the full text of which is incorporated herein by reference.

[0054] As an example, in some embodiments, long dsDNA molecules extended over the surface of the flue cell are tagged in situ to generate lines of dsDNA bridges connecting across the surface of the flue cell. Additionally, a physical map of the fixed DNA can then be generated. Thus, the physical map can be associated with the physical relationships of the clusters after the fixed DNA is amplified. Specifically, the physical map is used to calculate the probability that sequence data obtained from any two clusters are linked, as described in the integrated data of WO 2012 / 025250.

[0055] In some embodiments, a physical map is generated by imaging DNA to establish the location of DNA molecules immobilized across a solid surface. In some embodiments, the immobilized DNA is imaged by adding an imaging agent to a solid support and detecting a signal from the imaging agent. In some embodiments, the imaging agent is a detectable label. Suitable detectable labels include, but are not limited to, protons, haptens, radionuclides, enzymes, fluorescent labels, chemiluminescent labels, and / or chromogenic agents. For example, in some embodiments, the imaging agent is an intercalating dye or a non-intercalating DNA binder. Any suitable intercalating dye or non-intercalating DNA binder known in the art may be used, including, but are not limited to, those disclosed in U.S. No. 2012 / 0282617, the full text of which is incorporated herein by reference.

[0056] In some embodiments, the fixed double-stranded fragment is further fragmented to release the free end prior to cluster formation (Fig. 4b) (see Fig. 4a). Cutting the bridged structure may be performed using any suitable method known in the art, as exemplified by the integrated material of WO 2012 / 025250. For example, cutting may occur by incorporating a modified nucleotide, such as uracil, as described in WO 2012 / 025250, by incorporating a restriction endonuclease site, or by applying a solution-phase transposome complex to the bridged DNA structure as otherwise described herein.

[0057] In certain embodiments, a plurality of target DNA molecules flow into a flow cell comprising a plurality of nano-channels, and the nano-channels have a plurality of transposome complexes immobilized thereon. As used herein, the term nano-channel refers to a narrow channel through which long linear DNA molecules flow. In some embodiments, individual long strands of target DNA not exceeding 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, 400, 500, 600, 700, 800, 900, or 1000 flow into each nano-channel. In some embodiments, individual nano-channels are separated by a physical barrier that prevents individual long strands of target DNA from interacting with multiple nano-channels. In some embodiments, the solid support comprises at least 10, 50, 100, 200, 500, 1000, 3000, 5000, 10000, 30000, 50000, 80000, or at least 100000 nano-channels. In some embodiments, transpososomes bound to the surface of the nano-channels tag the DNA. Continuity mapping can then be performed, for example, by following the cluster down the length of one of these channels. In some embodiments, the long strand of target DNA is at least 0.1kb, 1kb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 15kb, 20kb, 25kb, 30kb, 35kb, 40kb, 45kb, 50kb, 55kb, 60kb, 65kb, 70kb, 75kb, 80kb, 85kb, 90kb, 95kb, 100kb, 150kb, 200kb, 250kb, 300kb, 350kb, 400kb, 450kb, 500kb, 550kb, 600kb, 650kb, It may be 700kb, 750kb, 800kb, 850kb, 900kb, 950kb, 1000kb, 5000kb, 10000kb, 20000kb, 30000kb, or at least 50000kb in length. In some embodiments, the long strand of the target DNA is 0.1kb, 1kb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 15kb, 20kb, 25kb, 30kb, 35kb, 40kb, 45kb, 50kb, 55kb, 60kb, 65kb, 70kb, 75kb, 80kb, 85kb, 90kb, 95kb, 100kb, 150kb, 200kb, 250kb, 300kb, 350kb, 400kb, 450kb, 500kb, 550kb, The length is 600kb, 650kb, 700kb, 750kb, 800kb, 850kb, 900kb, 950kb, or 1000kb or less. As an example, the genome of an organism with short 'oriented' reads can be sequenced using a fluorocell having 1000 or more nano-channels with a fixed tagging product mapped to the nano-channel. In some embodiments, haplotypes can be analyzed using the mapped fixed tagging product in the nano-channel. In some embodiments, topology problems can be solved using the mapped fixed tagging product in the nano-channel.

[0058] Amplification and sequencing of fixed DNA fragments

[0059] AmplificationThis description further relates to the amplification of immobilized DNA fragments produced according to the method provided herein. Immobilized DNA fragments produced by surface-bound transposome-mediated tagging may be amplified according to any suitable amplification method known in the art. In some embodiments, the immobilized DNA fragments are amplified on a solid support. In some embodiments, the solid support is the same solid support on which surface-bound tagging occurs. In such embodiments, the method and composition provided herein allow sample preparation to proceed on the same support from the initial sample introduction step, during amplification, and optionally during the sequencing step.

[0060] For example, in some embodiments, the immobilized DNA fragment is amplified using a cluster amplification method as exemplified by the descriptions of U.S. Patents 7,985,565 and 7,115,400, the full contents of which are incorporated herein by reference. The combined material of U.S. Patents 7,985,565 and 7,115,400 describes a solid-state nucleic acid amplification method in which the amplification product is immobilized on a solid support to form an array consisting of clusters or “colonies” of immobilized nucleic acid molecules. Each cluster or colony on said array is formed from a plurality of identical immobilized polynucleotide strands and a plurality of identical immobilized complementary polynucleotide strands. The array thus formed is generally referred to as a “clustered array.” The product of a solid-phase amplification reaction, such as that described in U.S. Patents 7,985,565 and 7,115,400, is a so-called "bridging" structure formed by the annealing of a pair of fixed polynucleotide strands and fixed complementary strands, both strands being fixed to the 5' ends on a solid support, preferably via covalent attachment. The cluster amplification method is an example of a method in which a fixed nucleic acid template is used to produce a fixed amplicon. Other suitable methods may also be used to produce a fixed amplicon from a fixed DNA fragment produced according to the method provided herein. For example, one or more clusters or colonies may be formed via solid-phase PCR, whether one or both primers of each pair of amplification primers are fixed.

[0061] In other embodiments, the immobilized DNA fragment is amplified in a soluble state. For example, in some embodiments, the immobilized DNA fragment is cleaved or otherwise released from a solid support, and then an amplification primer is hybridized to the molecule released in a soluble state. In other embodiments, the amplification primer is hybridized to the immobilized DNA fragment for a subsequent amplification step in a soluble state, following one or more initial amplification steps. Thus, in some embodiments, the immobilized nucleic acid template can be used to generate a solution-phase amplicon.

[0062] It will be understood that any amplification method described herein or generally known in the art may be used in combination with general-purpose or target-specific primers to amplify a fixed DNA fragment. Methods suitable for amplification include, but are not limited to, polymerase chain reaction (PCR), strand potential amplification (SDA), transcription-mediated amplification (TMA), and nucleic acid sequence-based amplification (NASBA), as described in U.S. Patent No. 8,003,354, the full text of which is incorporated herein by reference. The amplification methods may be used to amplify one or more nucleic acids of interest. For example, PCR including multiple PCR, SDA, TMA, NASBA, etc., may be used to amplify a fixed DNA fragment. In some embodiments, primers specifically designated for the nucleic acid of interest are included in the amplification reaction.

[0063] Other suitable methods for amplifying nucleic acids may include oligonucleotide extension and ligation, rotational amplification (RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998), incorporated herein by reference) and oligonucleotide ligation assay (OLA) techniques (generally, see U.S. Patents 7,582,420, 5,185,243, 5,679,524 and 5,573,907; EP 0 320 308 B1; EP 0 336 731 B1; EP 0 439 182 B1; WO 90 / 01069; WO 89 / 12696; and WO 89 / 09835, all incorporated herein by reference). It will be understood that these amplification methods may be designed to amplify immobilized DNA fragments. For example, in some embodiments, the amplification method may include a ligation probe amplification or an oligonucleotide ligation assay (OLA) reaction containing a primer specifically designated to the nucleic acid of interest. In some embodiments, the amplification method may include a primer extension-ligation reaction containing a primer specifically designated to the nucleic acid of interest. As non-limiting examples of primer extension and ligation primers that can be specifically designed to amplify the nucleic acid of interest, the amplification may include primers used in the GoldenGate assay (Illumina, Inc., San Diego, CA) as exemplified in U.S. Patents 7,582,420 and 7,611,869, the full text of which is incorporated herein by reference.

[0064] Exemplary isothermal amplification methods that may be used in the methods described herein include, but are not limited to, multiple strand transposition amplification (MDA) as exemplified in the literature [Dean et al., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002)] or isothermal strand transposition nucleic acid amplification as exemplified in, for example, U.S. Patent No. 6,214,587, each of which is incorporated herein by reference in its entirety. Other non-PCR-based methods that may be used in the methods described herein include, for example, the literature [Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995; US Pat. Nos. 5,455,166, and 5,130,238, and Walker et al., Nucl. Acids Res. The isothermal amplification methods include strand-transpotential amplification (SDA) as described in [20:1691-96 (1992)], for example, or overbranched strand potential amplification as described in [Lage et al., Genome Research 13:294-307 (2003)], the full text of each of said references is incorporated herein by reference. The isothermal amplification methods for the random primer amplification of genomic DNA using strand-transpotential Phi 29 polymerase or Bst DNA polymerase large fragments, 5'->3' exo - It can be used. The use of such polymerases has the advantage of their high processability and strand-transition activity. High processability allows the polymerase to produce fragments 10-20 kb in length. As disclosed above, smaller fragments can be produced under isothermal conditions using polymerases having low processability and strand-transition activity, such as Klenow polymerase. Further descriptions of the amplification reaction, conditions, and components are disclosed in detail in U.S. Patent No. 7,670,810, the full text of which is incorporated herein by reference.

[0065] Another nucleic acid amplification useful in this description is tagged PCR using a group of 2-domain primers having an arbitrary 3' region following an invariant 5' region, as described in, for example, [Grothues et al. Nucleic Acids Res. 21(5):1321-2 (1993)], the full text of which is incorporated herein by reference. A first round of amplification is performed to allow multiple initiations on heat-denatured DNA based on individual hybridization from randomly synthesized 3' regions. Due to the nature of the 3' region, the initiation site is considered randomly across the genome. Subsequently, unbound primers can be removed, and further replication using primers complementary to the invariant 5' region may occur.

[0066] sequencing This description further relates to the sequencing of immobilized DNA fragments generated according to the methods provided herein. Immobilized DNA fragments generated by surface-bound transposome-mediated tagging may be sequenced according to any suitable sequencing method, such as direct sequencing, including sequencing by synthesis, sequencing by ligation, sequencing by hybridization, nanopore sequencing, etc. In some embodiments, the immobilized DNA fragments are sequenced on a solid support. In some embodiments, the solid support for sequencing is the same solid support on which surface-bound tagging occurs. In some embodiments, the solid support for sequencing is the same solid support on which amplification occurs.

[0067] One preferred sequencing method is sequencing-by-synthesis (SBS). In SBS, the sequence of nucleotides in a template is determined by monitoring the extension of a nucleic acid primer according to a nucleic acid template (e.g., a target nucleic acid or its amplicon). The underlying chemical process may be polymerization (e.g., catalyzed by a polymerase enzyme). In a specific polymerase-based SBS embodiment, fluorescently labeled nucleotides are added to the primer in a template-dependent manner (thereby extending the primer) so that detection of the sequence and type of nucleotides added to the primer can be utilized to determine the sequence of the template.

[0068] The flurocell provides a solid support convenient for accommodating amplified DNA fragments generated by the method of this description. In such a format, one or more amplified DNA fragments may be processed by SBS or other detection techniques involving the repeated delivery of reagents in a cycle. For example, to initiate a first SBS cycle, one or more labeled nucleotides, DNA polymerase, etc., may flow into or through the flurocell accommodating one or more amplified nucleic acid molecules. Such sites where primer extension causes the incorporation of labeled nucleotides may be detected. Optionally, the nucleotides may additionally possess a reversible termination property that terminates further primer extension once the nucleotides are added to the primer. For example, a nucleotide analog having a reversible termination moiety may be added to the primer to prevent subsequent extension from occurring until a deblocker is delivered to remove the moiety. Accordingly, in the case of a specific embodiment utilizing reversible termination, a deblocking reagent may be delivered to the fluorocell (before or after detection occurs). Washing may be performed between various delivery steps. Subsequently, by repeating the cycle n times to extend the primer by n nucleotides, a sequence of length n can be detected. Exemplary SBS procedures, fluid systems, and detection platforms that can be easily adapted for use with the amplicons produced by the method described herein are, for example, the literature [Bentley et al., each incorporated herein by reference, Nature It is listed in 456:53-59 (2008), WO 04 / 018497; US 7,057,026; WO 91 / 06678; WO 07 / 123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281, and US 2008 / 0108082.

[0069] Other sequencing procedures utilizing cyclic reactions, such as pyrosequencing, may be used. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) following the incorporation of specific nucleotides into the nascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genome Res . 11(1), 3-11 (2001); Ronaghi et al. Science 281(5375), 363 (1998); US 6,210,891; US ​​6,258,568 and US 6,274,320, each incorporated herein by reference). In pyrosequencing, emitted PPi can be detected by immediate conversion to ATP by adenosine triphosphate (ATP) sulfurylase, and the level of the generated ATP can be detected via luciferase-generated photons. Thus, the sequencing reaction can be monitored via a luminescence detection system. The excitation radiation source used in a luminescence-based detection system is not essential to the pyrosequencing procedure. Useful fluid systems, detectors, and procedures that can be adapted for the application of pyrosequencing to amplicons generated according to this description are, for example, the literature [WIPO Pat. App. Ser. No., each incorporated herein by reference]. It is listed in PCT / US11 / 57111, US 2005 / 0191698 A1, US 7,595,883, and US 7,244,559.

[0070] Some embodiments may utilize a method involving real-time monitoring of DNA polymerase activity. For example, nucleotide incorporation may be detected via fluorescence resonance energy transfer (FRET) interactions between the fluorophore-containing polymerase and γ-phosphate-labeled nucleotides, or by a zero-mode waveguide (ZMW). Techniques and reagents for FRET-based sequencing are, for example, described herein by reference [Levene et al. Science 299, 682?686 (2003); Lundquist et al. Opt. Lett . 33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci. USA It is listed in 105, 1176-1181 (2008).

[0071] Some SBS embodiments involve the detection of protons emitted upon the incorporation of nucleotides into the extension product. For example, sequencing based on the detection of emitted protons may utilize electrodetectors and related techniques commercially available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or sequencing methods and systems described in the literature [US 2009 / 0026082 A1; US ​​2009 / 0127589 A1; US ​​2010 / 0137143 A1; or US 2010 / 0282617 A1, each incorporated herein by reference]. The method disclosed herein for amplifying target nucleic acids using kinetic exclusion can be easily applied to the substrate used to detect protons. More specifically, a clonal population of the amplicon used to detect protons can be generated using the method disclosed herein.

[0072] Another useful sequencing technique is nanopore sequencing (see, e.g., Deamer et al. Trends Biotechnol. 18, 147-151 (2000); Deamer et al. Acc. Chem. Res. 35:817-825 (2002); Li et al. Nat. Mater. 2:611-615 (2003), the descriptions of which are incorporated herein by reference). In some nanopore embodiments, the target nucleic acid or individual nucleotides removed from the target nucleic acid pass through the nanopore. As nucleic acids or nucleotides pass through nanopores, each nucleotide type can be identified by measuring fluctuations in the electrical conductivity of the pores (US 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. 130, 818–820 (2008), the descriptions thereof are incorporated herein by reference).

[0073] Exemplary methods of array-based expression and genotyping analysis that can be used for detection according to the present description are described in the literature [US Pat. Nos. 7,582,420; 6,890,741; 6,913,884 or 6,355,431 or US Pat. Pub. Nos. 2005 / 0053980 A1; 2009 / 0186349 A1 or US 2005 / 0181440 A1], each incorporated herein by reference.

[0074] The advantage of the methods disclosed herein is that they provide rapid and efficient simultaneous detection of multiple target nucleic acids. Accordingly, this description provides an integrated system capable of producing and detecting nucleic acids using techniques known in the art, such as those exemplified above. Accordingly, the integrated system of this description may include a system comprising components such as fluid components, pumps, valves, reservoirs, fluid lines, etc., capable of delivering amplification reagents and / or sequencing reagents to one or more immobilized DNA fragments. Fluorocytes may be formed into an integrated system and / or used therein for detecting target nucleic acids. Exemplary fluorocytes are described, for example, in US 2010 / 0111768 A1 and U.S. Provisional Patent Application 13 / 273,666, each incorporated herein by reference. As an example of a fluorocyte, one or more of the fluid components of the integrated system may be used in the amplification method and the detection method. For example, in a specific example of nucleic acid sequencing, one or more of the fluid components of the integrated system may be used for the delivery of the amplification method and sequencing reagent disclosed herein in a sequencing method such as those exemplified above. Alternatively, the integrated system may include separate fluid systems for performing the amplification method and the detection method. Examples of an integrated sequencing system capable of generating amplified nucleic acids and also determining the sequence of nucleic acids include, but are not limited to, the MiSeq™ platform (Illumina, Inc., San Diego, CA) and the apparatus described in U.S. Provisional Patent Application 13 / 273,666 incorporated herein by reference.

[0075] Solid support having immobilized transposome and method of manufacturing

[0076] Other embodiments provided herein comprise a solid support, such as a flurocell, having a transposome complex immobilized thereon. In certain embodiments, the transposome complex comprises a transposase bound to a first polynucleotide, said polynucleotide comprising (i) a 3' portion containing a transposon terminal sequence, and (ii) a first tag comprising a first tag domain. The density of such surface-bound transposoms may vary. For example, in some embodiments, the transposome complex is mm 2 At least 10 per 3 , 10 4 , 10 5 , or at least 10 6 It exists on a solid support at the density of the composite.

[0077] Also provided herein is a method for generating a flurocell for tagging. The method may, for example, include immobilizing a plurality of transposome complexes on a solid support, wherein the transposome complex comprises a transposase coupled to a first polynucleotide, and the first polynucleotide comprises (i) a 3' portion comprising a transposon terminal sequence, and (ii) a first tag comprising a first tag domain.

[0078] Transposome complexes can be immobilized on solid supports in various ways understood by those skilled in the art. In one embodiment, the method comprises providing a solid support on which a plurality of first polynucleotides are immobilized, and bringing the solid support into contact with a transposase holoenzyme and a second polynucleotide, wherein the second polynucleotide comprises a region complementary to the transposon terminal sequence. In some embodiments, the second polynucleotide is hybridized to the immobilized first polynucleotide before the addition of the transposase holoenzyme. In some embodiments, the second polynucleotide and the transposase holoenzyme are provided together. FIG. 5a illustrates one embodiment of such a method for assembling a surface-bound transposome complex. In one method, the transposase holoenzyme is added from the 5' surface-grafted end along with the non-transferred strand of the ME adapter to a flow cell containing an oligonucleotide comprising a cluster amplification primer (A.pr) and a sequencing primer (S.pr) and an ME sequence (Fig. 5a). Alternatively, the non-transferred ME' strand can first be hybridized to the surface-grafted ME strand and then the transposase is added to the flow cell.

[0079] In some embodiments, the transsosome complex is assembled in a soluble state, and immobilization includes an additional step of ligating the first polynucleotide to a splint oligonucleotide coupled to a solid support. Such embodiments are illustrated in FIG. 5b. In some embodiments, the splint oligonucleotide may be extended using a polymerase before the ligation step takes place.

[0080] In some embodiments, the transposome dimer is assembled by hybridizing a loop oligonucleotide to a fixed first polynucleotide. For example, the loop oligonucleotide may comprise a first end and a second end, wherein the first end is complementary to the transposon end sequence of the first polynucleotide. The second end of the loop oligonucleotide may be complementary to the second transposon end sequence. The second transposon end sequence may be, for example, a portion of the solution-phase first polynucleotide. In some embodiments, the fixed first polynucleotide and the solution-phase first polynucleotide may comprise different transposon end sequences or their complements. In some such embodiments, the loop oligonucleotide comprises a sequence complementary to each of the different transposon end sequences at the first and second ends. An example of such an embodiment is illustrated in FIG. 5c. As illustrated in Fig. 5c, a successive adapter pair is generated by hybridizing two oligonucleotides to a surface-bound oligonucleotide. This can be achieved by using two different transposon terminal sequences (ME1 and ME2). The addition of the transposase holoenzyme reconstitutes an active 'loop-type' transposome complex, wherein only one of the two adapters of each transposome is coupled to the surface (Fig. 5c).

[0081] In another embodiment, the transposome complex can be assembled onto a standard pair of terminal flow cells having a fixed amplification primer (e.g., HiSeq flow cells or MiSeq flow cells sold by Illumina Inc, San Diego, CA). This can be achieved, for example, by the hybridization of a 'splint' oligonucleotide annealed to one or both species of the surface-grafted amplification primer. The splint then acts as a template to extend the grafted surface primer with polymerase and dNTPs to form an oligonucleotide duplex containing the transposon terminal sequences of the surface amplification primer, the sequencing primer, and the transposase. The addition of the transposase assembles the transposome on the surface. Such an embodiment is illustrated in FIG. 5d.

[0082] In any embodiment provided herein, the transposome complex may be homodimeric or heterodimeric. For example, as illustrated in FIG. 11, the homodimeric transposome will contain two P5-ME adapters at both sites, or alternatively, two P7-ME adapters. Similarly, the heterodimeric transposome complex will contain both P5-ME and P7-ME adapters as illustrated in FIG. 11.

[0083] Tagging using Transosome Beads

[0084] One embodiment provided herein is a collection of microparticles having immobilized transposome complexes. The use of solid supports, such as beads, can provide several advantages over lysis-based tagging. For example, in standard lysis-based tagging, it is difficult to control the final fragment size of the tagging reaction. Fragment size is a function of the amount and size of DNA and the ratio of transposomes to the duration of the reaction. Even if these variables are controlled, size-selective fractionation is usually required as an additional step to remove excess small fragments shorter than the read length of the combined pair. The method provided herein does not have such disadvantages. Specifically, bead-immobilized transposomes enable the selection of the final fragment size as a function of the spatial separation of the combined transposomes, regardless of the amount of transposome beads added to the tagging reaction. An additional limitation of lysis-based tagging is that purification of some form of the product of the tagging reaction must typically be performed before and after PCR amplification. This typically requires the transfer of some reaction products from tube to tube. In contrast, the tagging product on the bead-based transposome can be released for amplification or other downstream processing after washing, thus avoiding the need for sample transfer. For example, in embodiments where the transposome is assembled on paramagnetic beads, purification of the tagging reaction product can be easily achieved by fixing the beads with a magnet and washing them. Thus, in some embodiments, tagging and other downstream processing, such as PCR amplification, can all be performed in a single tube, vessel, drop, or other container. In some embodiments, the tagging and downstream processing of the sample occur in a microfluidic drop-based device, as exemplified in the description of U.S. Provisional Patent Application No. 13 / 670,318, filed November 6, 2012, titled “INTEGRATED SEQUENCING APPARATUSES AND METHODS OF USE,” the full text of which is incorporated herein by reference.For example, in a microfluidic drop-based device, a drop containing a target nucleic acid, a wash buffer, or other reagent can be passed over a surface containing an immobilized transposome complex. Similarly, a drop containing a bead with a transposome immobilized thereon can be brought into contact with a target nucleic acid, a wash buffer, or other reagent in a microfluidic drop-based device.

[0085] In some embodiments, the immobilized transposome complex comprises a transposase bound to a first polynucleotide and a second polynucleotide; wherein the first polynucleotide is immobilized on the surface of a microparticle at its 5' end and the second polynucleotide is hybridized to the 3' end of the first polynucleotide; and the first polynucleotide comprises (i) a 3' portion comprising a transposon end sequence, and (ii) a first tag comprising a first tag domain.

[0086] FIGS. 9-12 provide examples of transposoms assembled on the surface of paramagnetic beads. FIG. 9 illustrates a bead surface on which two different first polynucleotides are immobilized. The illustrated first polynucleotide includes a transposon terminal sequence (ME). One of the illustrated first polynucleotides includes a tag domain comprising an amplification primer sequence (P5) and a sequencing primer sequence (Read 1). The other polynucleotide illustrated in FIG. 9 includes a different amplification primer sequence (P7) and a sequencing primer sequence (Read 2). The first polynucleotide may also include an index tag. The index tag may be unique to the bead, or it may be shared by one or more other beads in a group. A single bead may have only a single index tag immobilized thereon, or it may have multiple index tags immobilized thereon.

[0087] FIG. 10 illustrates a second polynucleotide hybridized to each first polynucleotide. The second polynucleotide comprises a region complementary to the transposon end sequence of the first polynucleotide. In some embodiments, the second polynucleotide is 15 bp in length. In some embodiments, the second polynucleotide may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 bp, or greater than 20 bp in length. In some embodiments, the second polynucleotide is phosphorylated at its 5' end.

[0088] Fig. 11 illustrates the assembly of transsomes on the surface of a bead, where the transposase enzyme comes into contact with an immobilized oligonucleotide. As shown in Fig. 11, when the transposase is added to the bead, three types of transsomatic complexes are formed: P5:P5, P7:P7, and P5:P7 complexes (Fig. 11).

[0089] When transsomatic beads are added to a solution of target DNA in a tagging buffer, tagging occurs, attaching the DNA to the surface of the beads. An immobilized library of tagged DNA fragments is generated.

[0090] FIG. 12 illustrates a specific example in which successive tagging to DNA generates a bridging molecule between transsomes. When three types of transsomes are present (as in FIG. 11), three types of bridge complexes: P5:P5, P7:P7, and P5:P7 complexes are generated in a ratio of 25:25:50, respectively.

[0091] In some embodiments, the length of the bridged fragment may be determined by the density of the transposome on the surface of the bead. This density may be adjusted through the amount of oligonucleotides on the surface, the amount of duplex transposon terminal complexes on the surface, and the amount of transposase enzyme added during transposome assembly. Once tagging is complete, the P5:P7 tagging product may be released from the surface of the bead using any suitable method. In some embodiments, the tagging product is released from the bead using an amplification method such as inhibition PCR, step-out PCR, etc. In some embodiments, the tagging product is released from the bead by cleavage. Cleaving may be, for example, chemical, enzymatic, photochemical cleavage, or a combination thereof. It will be understood that any suitable method for releasing one or more tagging products from a solid support may be used in the method provided herein.

[0092] DNA can efficiently come into contact with surface-bound transposomes using any suitable method to increase the possibility of contact. For example, in some embodiments, contact between the target DNA and the transposome complex on the solid surface can be increased by utilizing the precipitation of DNA onto a solid surface. Any one of the numerous methods known in the art for contacting DNA with a solid support may be used, as exemplified by the description of WO 2010 / 115122, the full text of which is incorporated herein by reference. As understood by those skilled in the art, DNA may be precipitated onto a solid support by the addition of any one of the various other agents known for precipitating DNA on a surface, including PEG, ethanol, or, for example, any one of the numerous buffers used in solid-phase reversible immobilization (SPRI) techniques.

[0093] In some embodiments, a group of beads having an immobilized transsosome complex may be mixed with an excess of beads not having a transsosome or oligonucleotide to reduce the possibility of tagging across two or more different beads. Another method of reducing the possibility of tagging across two or more different beads includes immobilizing the beads to minimize contact between the beads. Immobilization of the beads may be achieved by any number of techniques known in the art, including, for example, immobilizing the beads on a slide on a solid surface such as a microcentrifuge tube by magnetism, or by any other immobilization technique as exemplified in the combined material of WO 2010 / 115122.

[0094] In some embodiments, transsomatic beads can be used to isolate and identify nucleic acids from a single cell, such as a prokaryotic or eukaryotic cell. For example, in some embodiments, particles such as beads are coated with index transsosomes that share the same index (all transsosomes present on a particular bead have the same index, which differs from the index present on another bead). The beads can then be positioned inside the cell via any of the various methods known in the art. For example, methods for delivering the beads inside the cell include, but are not limited to, gene guns, photothermal nanoblades (Wu et al. Anal Chem. (2011) 4:1321-7), and peptides used in combination with cell-permeable materials (Nitin et al. Ann Biomed Eng. (2009) 37:2018-2027). It will be understood that any suitable method for combining DNA from a single cell with particles containing index transsosomes can be utilized in the method provided herein.

[0095] In some embodiments, the transposome may be covalently attached to the bead as described in detail above. Additionally or alternatively, the transposome may be released from the bead when a chemical or physical stimulus is applied. Some examples of stimuli that may trigger the release of the transposome from the solid support include changes in light and / or temperature. In some embodiments, the transposome is released from the solid support by utilizing the activity of an enzyme such as a restriction endonuclease. In certain embodiments, the transposome may be detached from the bead and may move freely within the cell. Once the bead (or alternatively, the released transposome) comes into contact with chromatin or DNA, tagging may occur. It will be understood that in eukaryotic and prokaryotic systems, not all genomic DNA is always accessible or available for tagging. In some embodiments, 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%, 80%, 85%, 90%, 95%, or less or more than 99% of the total DNA in the cell is tagged by transsomes. The use of uniquely tagged beads enables the identification of reads from the same cell by grouping reads that share the same index together. These reads can be considered to have originated from the same bead (and thus from the same cell). Additionally or alternatively, in some embodiments, a crosslinking step may be performed to crosslink the cell DNA, while the cell DNA remains inside the nucleus of the cell. Such a step ensures that the DNA from a single cell is kept together. After extracting the DNA from the cell, it may then be mixed with index beads and tagged as described in detail above. Additionally or alternatively, a variation of the method includes the tagging of the flow-sorted chromosomes.Flow-classified chromosomes can be mixed with the index beads described above. Specific chromosomes will attach to one bead, and the fragments generated through tagging will contain the same index. This enabled the phasing of SNPs across the entire chromosome (haploid type).

[0096] In some such embodiments, the total number of indices is greater than the total number of successfully tagged cells. In some embodiments, for example, a double index approach may be implemented as a method to increase the number of possible combinations of indices. As an example, the use of two 8-base indices is 4 8 X 4 8 = 4.3X10 9 It will provide the theoretical number of combinations.

[0097] In some embodiments, an approach can be used that ensures individual cells are not tagged by multiple beads. For example, one approach is to use beads of a size similar to that of the cells. This would ensure that the cells are certainly unable to accommodate multiple beads. Additionally, or alternatively, another approach is to use a cell-to-bead ratio that favors single-cell targeting. For example, if there are far more cells than beads, the Poisson distribution of beads within the cells implies that cells adopting a single bead are much more numerically dominant than cells adopting two or more beads.

[0098] In some embodiments, the single-cell approach described above can be used to determine whether two SNPs (or structural rearrangements) exist in the same cell. For example, in the case of a heterogeneous population of cancer cells, knowing whether two SNPs exist in the same cell or in different cells can help in administering the correct cancer therapy.

[0099] In some embodiments, the single-cell approach described above can be used to study RNA. For example, by coating beads with a suitable enzyme (i.e., reverse transcriptase) and an oligonucleotide for the reverse transcription step, gene expression can be analyzed at the single-cell level. In one embodiment, after introducing beads coated with reverse transcriptase, an oligonucleotide, and a transposome, cytoplasmic RNA can be converted into cDNA, tagged, and produced inside the cell.

[0100] Method for assembling long reads using barcodes on a fixed transposome

[0101] Barcode-assisted assembly of DNA fragments enables the separation of individual long DNA molecules within a population of DNA molecules and the conversion of each molecule into a uniquely barcoded fragment library. When the entire population of fragmented DNA molecules is sequenced, the fragments can be reassembled into their original long molecules by reference to the barcodes they contain.

[0102] Various methods for barcoding individual DNA molecules are known. For example, the 'dilution method' separates individual long molecules by extreme dilution and aliquoting into separate compartments (e.g., wells of a plate) so that each well contains only one or a few molecules of DNA. Since each well is physically separated, library preparation can be performed in each well having a unique barcode. The contents of the wells are then pooled and sequenced. Another method utilizes an emulsion, where each spot contains a long DNA molecule, a library preparation reagent, and a barcode unique to each spot. Another approach utilizes a large library of index loop-type transposome complexes to insert multiple twin barcodes along the length of the DNA while preserving the integrity of the molecule. Subsequent cutting between the barcode 'tweens' generates fragments that can be sequenced and reassembled by matching the twin barcodes. Each of the aforementioned barcoding methods has disadvantages that are overcome by the barcoding method provided herein.

[0103] An alternative method to the method described above for separating and barcoding individual long molecules is provided herein. The method provided herein achieves the benefits of physical separation similar to emulsion methods without using emulsions, while providing much greater complexity than that provided by the large number of 'wells' used in dilution methods. The unique barcode of the method is similar to the 'wells' of dilution methods in some methods, except that the number of beads in the bead-based method can often be much higher than the number of wells in the dilution method. An additional advantage over emulsion methods is that, in the bead-based method, the barcodes are deterministically distributed (i.e., one barcode per bead) rather than randomly distributed (i.e., Poisson distributed). The method provided herein also achieves the same initial preservation of molecular integrity and continuity without the need for loop transsosome complexes as used in some other methods. Additionally, the method provided herein does not require a large code space as used in some other methods.

[0104] Accordingly, in some embodiments provided herein, the barcoding method comprises providing a group of microparticles having an immobilized transposome complex, wherein the transposome complex comprises a transposase bound to a first polynucleotide and a second polynucleotide. In some embodiments, the first polynucleotide comprises an index domain bound to the microparticle. In some embodiments, the index domain may be unique to the microparticle. In some embodiments, the group of microparticles comprises at least a plurality of index domains. In some embodiments, the index domain is present on microparticles of more than one of the group of microparticles. In some embodiments, the microparticles of the group of microparticles comprise one or fewer index domains.

[0105] The barcoding method provided herein further comprises applying target DNA to a population of microparticles to produce an immobilized DNA fragment tagged with an index domain. The DNA can efficiently come into contact with surface-bound transsosomes using any suitable method to increase the possibility of contact as discussed above, as exemplified by the integrated data of WO 2010 / 115122.

[0106] The above method may be performed using any one of various known formats, for example, by proceeding with index sequencing and customized data analysis following a combination of a bead array and tagging reagents for library preparation. Any other suitable method of maintaining the beads in a statically separated state from one another may be used for surface tagging and indexing of the sample. For example, physical configurations such as wells or small depressions in the substrate capable of holding the beads so that the microspheres can be retained within the wells, or other forces (magnetic or compressive), or the use of chemically modified or active sites, such as chemically functionalized sites, electrostatically modified sites, hydrophobic and / or hydrophilic functionalized sites, or spots of adhesive.

[0107] In some embodiments, although the well may be further chemically functionalized as generally described below, a crosslinking agent may be used, or a physical barrier, such as a film or membrane, may be used on the bead, the microspheres are non-covalently bonded within the well.

[0108] In certain embodiments, the surface of the substrate is modified to contain chemically modified sites that can be used to attach the microspheres of the present invention covalently or non-covalently to individual sites or locations on the substrate. In this context, “chemically modified sites” include, but are not limited to, the addition of a pattern of chemical functional groups, including amino groups, carboxyl groups, oxo groups, and thiol groups, which can be used to covalently attach microspheres containing corresponding reactive functional groups; the addition of an adhesive pattern that can be used to bond to the microspheres (by prior chemical functionalization for the addition of adhesive or by the direct addition of adhesive); for example, when the microspheres contain charged groups opposite to the sites, the addition of a pattern of charged groups (similar to chemical functional groups) for electrostatic attachment of the microspheres. Under suitable experimental conditions, the addition of a pattern of chemical functional groups that makes the sites differentially hydrophobic or hydrophilic includes the addition of similarly hydrophobic or hydrophilic microspheres to cause the attachment of microspheres to the sites based on hydroaffinity. For example, in an aqueous system, the use of hydrophobic sites by hydrophobic beads performs the preferential attachment of the beads to the sites. As outlined above, the term “pattern” includes, in its meaning, the use of a uniform surface treatment that allows for the attachment of beads at distinct sites, as well as a surface treatment that generates distinct sites. As understood by those skilled in the art, this can be accomplished by various methods.

[0109] In a specific embodiment, a plurality of beads containing surface-bound transposoms are produced, each bead containing many transposoms, but not all transposoms of any given bead contain the same barcode. The production of a group of monoclonal-barcoded transposomal oligonucleotides on the beads may be carried out according to any one of a number of techniques known in the art, as exemplified by the description in U.S. Patent No. 5,604,097, the full text of which is incorporated herein by reference.

[0110] FIG. 13 illustrates a specific example in which surface-bound transsosomes contain long oligonucleotides attached to the surface via their 5' ends. As disclosed in FIG. 13, the 3' end of the oligonucleotide contains the mosaic end (METS) sequence of the Tn5 transposase enzyme. The upstream of this sequence (closer to the 5' end) is typically a barcode sequence of 6-8 bases in length. Further upstream is a primer sequence. Additionally, the oligonucleotide may further include a cleavage site capable of causing the oligonucleotide to be cleaved on the surface: its presence in the oligonucleotide is optional for the entire method. A second short oligonucleotide (non-transferred strand, MENTS) is hybridized to the METS sequence at the 3' end of the long surface-grafted oligonucleotide. Finally, the transposase dimer is assembled at the 3' end of the oligonucleotide to form a functional transsosome.

[0111] FIG. 14 illustrates barcoding on beads in an array of wells. As illustrated in FIG. 14, when a long molecule of dsDNA is added to an array of bead-attached transposoms, the given molecule comes into contact with the beads and is tagged multiple times by the transposoms on the beads. Each fragment is attached to a bead and tagged with a barcode associated with that bead. In a specific embodiment illustrated in FIG. 14, physical separation of the beads in the array chip prevents DNA molecules from reaching between two beads. In other embodiments, the beads are in close contact, and one or more DNA molecules may stretch between two or more beads. In some embodiments, more than one DNA molecule per bead may be tagged. It is unlikely that two alleles will be tagged on the same bead, and this is a function of the concentration of DNA added, the number of beads, and the number of barcodes. For example, to avoid two alleles occurring in the same well, 0.1x haplome equivalents (50,000 genome equivalents) need to be loaded onto 1 million beads, each having a unique barcode.

[0112] FIG. 15 illustrates the transfer of a barcoded tagged molecule to a sequencing reaction. As shown in FIG. 15, once tagging is complete, the DNA is transferred from the bead surface into the solution, whereby individual fragments for sequencing can be pooled and prepared. Barcode-referenced sequencing and assembly can regenerate the sequence of the original long tagged DNA molecule, thereby enabling long or pseudo-long reads and phase analysis of SNPs.

[0113] Release of the barcoded surface-tagged fragment into solution can be achieved using any suitable method known in the art. For example, in some embodiments, the tagged molecule can be cleaved on the surface of a bead through a cleavage moiety present at the 5' end of a surface-bound oligonucleotide (see FIG. 13). The cleavage moiety may be any moiety suitable for cleaving a nucleic acid strand from a solid support. Examples of methods using the cleavage moiety include, but are not limited to, cleavage methods and cleavage moiety disclosed in WO 2006 / 064199, the full text of which is incorporated herein by reference, restriction endonuclease cleavage, chemical cleavage, RNase cleavage, photochemical cleavage, etc.

[0114] Cleavage using a cleavage moiety produces a molecule having the following format:

[0115] 5' - Primer - Barcode - ME - Insert - ME - Barcode - Primer - 3'

[0116] The "primer" region can be used as a hybridization site to hybridize PCR step-out primers that allow additional sequences, such as amplification and sequencing primers, to be added. For example, amplification primers P5 and P7 can be added. Once added, inhibition PCR can be used to enrich a molecule, for example, having a P5 adapter at one end and P7 at the other end.

[0117] In some embodiments, amplification may be performed directly from the beads using step-out primers that add P5 and P7 adapter sequences by inhibition PCR. In another embodiment, each bead may have two types of surface-grafted oligonucleotides, wherein the primer sequence (as in FIG. 13) is a P5-Read1 sequencing primer or a P7-Read2 sequencing primer. This will result in mixed P5-P7 transposomes. These may be treated with inhibition PCR to enrich P5 / P7 molecules after being cleaved from the beads as described above, or may be amplified directly from the beads.

[0118] In some embodiments, a single transposome type (e.g., P5-Read 1-barcode) may be present on the surface of the bead. Once surface tagging is complete, a second transposome having different amplification and / or sequencing primers may be added in a soluble state to cleave the bridged molecule. This results in all molecules having the same adapter format that can be cleaved or amplified from the bead surface. Additional sample-specific barcodes may be added to the solution transposome to allow multiple samples to be pooled by the method. FIG. 16 is an example of such an embodiment. As shown in FIG. 16, a solution transposome having a P7 amplification primer sequence is added to a bead having a fixed bridged tagging product. The fixed fragment is tagged with a polynucleotide sequence comprising a P5 amplification primer sequence and a bead-specific barcode index tag (i9). As shown in FIG. 16, after the second tagging reaction, each fragment has a free end having a P7 primer sequence and a fixed end having a P5 primer sequence.

[0119] Example 1

[0120] Surface tagging on Flurocell

[0121] This embodiment describes an experiment confirming the specific example illustrated in FIGS. 4a and 4b (solution tagging after surface tagging).

[0122] Experiments on an 8-lane flowel were performed using the list of conditions and control means shown in Figures 6a and 6b. Lane 6 represents a complete end-to-end description of the method: unfragmented E. coli ( E. coli Genomic DNA was added to the fluorocells when it was tagged by surface-bound transpososomes. Heat was then applied to the fluorocells to selectively dehybridize the naked ME sequences, thereby destroying them as targets for a second tagging reaction when transpososomes were subsequently added from the solution. After the second tagging reaction, clusters were generated and 'paired-end' sequencing (2x36 base reads) was performed. Sequencing metrics are provided on slide 7, indicating that in lane 6, 73.14% of the clusters passed the filter and 74.69 of them were aligned. This provides sufficient data to obtain a gap size plot of the insert and estimates of library diversity (see Fig. 8).

[0123] Other lanes included the control means listed below:

[0124] Lane 1 It includes PhiX DNA as a positive control to ensure that everything is properly pumped and that cluster generation and sequencing work as expected.

[0125] Lane 2is another control lane and exemplifies tagging to surface-bound transposons in the absence of target DNA. The flow cell (FC) contains a standard paired-terminal FC in which an oligonucleotide containing a tagging primer sequence and an ME' sequence (non-transferred ME' strand) is hybridized to a P7 surface oligonucleotide. These oligonucleotides were added at a saturation concentration. The first extension produced double-stranded transposons with ME ends. P5 transpososomes were assembled in a soluble state and flowed onto the FC (6.25 nM / lane). P5 transpososomes tagged the double-stranded surface-bound transposons. The tagging products were subsequently converted into clusters.

[0126] Lane 3 This exemplifies tagging to a surface-bound transposon in the absence of target DNA, provided that in this case, FC is heated to 75°C before the addition of P5 transposomes from solution to prevent tagging to such constructs, thereby converting the ds surface-bound transposon into a single standard oligonucleotide, because this interferes with the tagging of target DNA.

[0127] Lane 4 ... exemplifies the addition of surface-bound transposons under lane 3 conditions. In these lanes, oligonucleotides containing the tagged primer sequence and the ME' sequence were hybridized to P7 surface oligonucleotides. The first extension produced double-stranded transposons with ME ends. Subsequently, P7 transposons were assembled on the surface of the FC by adding Tn5 enzyme at a concentration of 50X to the lanes and incubating at 30°C for 30 minutes. Then, P5 transposons from the solution were added after heating the FC to 75°C.

[0128] Lane 5It includes the same conditions as Lane 4, except that in this case, instead of adding P5 transsosomes from solution, an E. coli 900 bp library (having P5 / P7 terminals) was added to determine whether P7 surface-bound transsosomes were still active after the heating step.

[0129] Lane 6 The following illustrates an embodiment of the present invention. In this example, the FC comprises a standard paired-terminal FC in which an oligonucleotide containing a tagging primer sequence and an ME' sequence is hybridized to a P7 surface oligonucleotide. A first extension generated a double-stranded transposon having an ME end. P7 transposoms were assembled on the surface of the FC by adding Tn5 enzyme at a concentration of 50X to the lane and incubating at 30°C for 30 minutes. Tagging was induced by adding target DNA (300 ng of unfragmented E. coli genomic DNA) to the FC lane and performing an incubation step at 55°C for 15 minutes. The P7 surface-bound transposoms were washed with PBI (Qiagen), and the FC was heated to 75°C before adding P5 transposoms from the solution. After the addition of P5 transpososomes from the solution and an incubation step of 15 minutes at 55°C, a strand potential extension reaction was performed to fill the 9-bp gap created in the DNA backbone by the translocation reaction. The strand potential extension reaction included the addition of a Bst and dNTP mix and incubation of 5 minutes at 65°C. The P5 transpososomes were washed in the final step. At this point, since all surface-bound molecules would be P5-P7 templates, they could be converted into clusters.

[0130] Lane 7 It includes the same conditions as lane 6, except that in this case, the heating step was omitted to emphasize the heating effect on the number of clusters and % alignment.

[0131] Lane 8It includes a negative control, where P7 transpososomes were assembled on the surface at a 50X concentration in the presence of saturating concentrations of ds surface-bound transposons, but no target DNA was added. This allows for the evaluation of whether surface-bound P7 transpososomes are tagged by their neighboring ds surface-bound transposons.

[0132] Example 2

[0133] E. Preparation of surface-bonded samples from E. coli scrape

[0134] E. E. A E. coli sample (5 mm by scraping from the grass on an agar plate with a 2 mm scraper) was scraped and resuspended in a tube containing water and glass beads. The suspended cells and beads were mixed using a vortex mixer to break down and open the cells, and then centrifuged to form a pellet of cell fragments. The supernatant (containing cell lysates, including proteins and nucleic acids) was removed and added to a genome analyzer Flurocell (Illumina, Inc., San Diego, CA) containing immobilized transpososomes according to the protocol described in Example 1.

[0135] Cluster generation was performed on a Focell using a cluster station sample preparation device (Illumina, Inc., San Diego, CA). After cluster generation, pair-terminal sequencing was performed with 36 bases read in each direction.

[0136] For Read 1, 58.99% of the clusters passed the filter, and 92.16 of them were aligned. For Read 2, 58.99% of the clusters passed the filter, and 55.08 of them were aligned. These data confirm that unrefined cell lysates have surprisingly strong sequencing results and can be directly added to fluorocells containing immobilized transpososomes.

[0137] Example 3

[0138] This embodiment describes a method for avoiding the tagging of surface-bound oligonucleotide duplexes when adding solution-phase transsomes to a flow cell.

[0139] One method for assembling transpososomes on the surface of a flue cell is to adopt a standard paired-terminal flue cell and hybridize a 'splint' oligonucleotide to a P5 and / or P7 surface-grafted oligonucleotide forming an extendable overhang that can be extended by polymerase to produce a duplex containing a double-stranded ME sequence. At this step, a transposase enzyme may be added to form a functional surface-bound transpososome (Fig. 5d). If only a portion of the duplex forms a transpososome, a 'naked' ME duplex may be left to serve as a target for tagging by a nearby surface-bound transpososome or a transpososome added from solution. Furthermore, the fully assembled surface transpososome contains an upstream dsDNA portion of the ME sequence that can also serve as a target for tagging by a nearby surface-bound transpososome or a transpososome added from solution. This undesirable tagging reveals itself in the reduction of the percentage of clusters that pass through the purity filter aligned to the target genome.

[0140] An example of this effect can be shown by comparing lanes 4 and 5 with lanes 6 and 7 in Fig. 18b. In lanes 6 and 7, transsomes were assembled with long splint oligonucleotides as described above, and after elongation and transsome assembly, surface-bonded duplexes having at least 50 double-stranded bases were produced. Upon use in the surface tagging reaction (Fig. 18a) and subsequent sequencing, only 22.59% and 15.52% of the clusters were aligned to the target E. coli, respectively (Fig. 18b).

[0141] To avoid the formation of cluster populations containing sequences not aligned with the target genomic DNA, transposome assembly was performed as directed in FIG. 17. 'Splint' oligonucleotides were first hybridized to P5 and / or P7 surface-grafted oligonucleotides forming an elongable overhang that could be extended by polymerase to produce duplexes containing double-stranded ME sequences. Next, the splint oligonucleotides were removed by de-annealing and washed, and then replaced by hybridizing shorter oligonucleotides that were 12 bases shorter, but preferably 16 bases longer, up to the 3' end of the surface-attached ME oligonucleotides. Transposase was added to assemble transpososomes containing no exposed dsDNA. To selectively de-anneal and remove short duplex DNA, the flow cell was incubated at 60°C, and any 'naked' duplexes not containing the bound transposase were removed by washing under flow conditions.

[0142] The advantageous effect of this approach to assembling transposmes can be seen in lanes 4 and 5, where surface-bound transposmes are assembled by this method and used in the surface tagging reaction (Fig. 18a). The percentage of clusters aligned in the coli increased from 22.59% and 15.52% for lanes 6 and 7, respectively, to 96.47% and 96.41% for lanes 4 and 5, respectively (Fig. 18b).

[0143] Throughout this application, various publications, patents, and / or patent applications have been referenced. The full text of such publications is incorporated herein by reference.

[0144] The term "including" is intended to be without limitation and includes not only the mentioned elements but also any additional elements.

[0145] Numerous 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

Claim 1 (a) a step of providing a solid support having an immobilized transposome complex thereon, wherein the transposome complex comprises a transposase bound to a first polynucleotide, and the first polynucleotide is, (i) a 3' portion containing a transposon terminal sequence, and (ii) a step of including a first tag comprising a first tag domain; and (b) a step of applying the target DNA to a solid support under conditions in which the target DNA is fragmented by a transposome complex, thereby transferring the 3' transposon terminal sequence of the first polynucleotide to the 5' end of at least one strand of the fragment; thereby producing a fixed library of double-stranded fragments, wherein at least one strand is 5'-tagged with the first tag, a method for producing a fixed library of tagged DNA fragments.

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