WO2013028643A1

WO2013028643A1 - Preparation of polynucleotides on a solid substrate for sequencing

Info

Publication number: WO2013028643A1
Application number: PCT/US2012/051632
Authority: WO
Inventors: Michael Tanner; Stevan B. Jovanovich; Ezra Van Gelder; Dennis Harris; Charles Park; Seth Stern
Original assignee: Integenx Inc.
Priority date: 2011-08-20
Filing date: 2012-08-20
Publication date: 2013-02-28
Also published as: WO2013028643A8

Abstract

The present invention provides methods and devices for performing nucleic acid amplification and sequencing on a solid substrate (e.g., a flow cell), including preparation of libraries of amplified DNA fragments for massively parallel (next-generation) sequencing.

Description

PREPARATION OF POLYNUCLEOTIDES ON A SOLID SUBSTRATE

FOR SEQUENCING

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Patent Application No. 61/602,483, filed on February 23, 2012, and to PCT International Patent Application No.

PCT/US2011/048528, filed on August 20, 2011, both of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] This invention relates to the field of nucleic acid amplification. More specifically, this invention relates to methods, devices, and kits for nucleic acid amplification, such as in preparation for high throughput sequencing. This invention is particularly useful for preparing samples prior to solid phase amplification, including but not limited to cluster or bridge PCR on flow cell surfaces.

[0003] Typically, solid phase amplification of a polynucleotide of unknown sequence is performed by first ligating known adapter sequences to each end of the polynucleotide. The double-stranded polynucleotide is then denatured to form a single-stranded template molecule. The adapter sequence on the 3' end of the template is hybridized to an extension primer that is immobilized on the solid substrate, and amplification is performed by extending the immobilized primer. In what is typically known as bridge PCR, a second immobilized primer, identical to the 5' end of the template, serves as a reverse primer, allowing amplification of both the forward and reverse strands to proceed on the solid substrate.

[0004] One significant disadvantage of this method, however, is the number of steps, including wash steps, that are needed to prepare the target polynucleotide before solid phase amplification can be initiated. As one example, after ligation of the adapter sequences, unused adapter molecules must be separated from the ligated polynucleotides before adding the mixture to the flow cell. Otherwise, the unused adapter molecules can also hybridize to the immobilized primers, preventing efficient hybridization of the primers to the template molecules and subsequent extension. Adapter ligation must also be followed by a separate step to denature and attach the template molecule to the solid surface via hybridization.

[0005] The invention described herein allows simultaneous attachment of the target polynucleotide to an adapter sequence and to the solid substrate by using an adapter sequence that is directly attached to the solid substrate. This reduces the number of steps required to prepare the target polynucleotide for solid phase amplification, which is particularly useful for time-sensitive applications, and for applications that benefit from minimizing reagent or sample loss. Because an adapter can be directly attached to the surface of the solid substrate, they do not compete for hybridization to any primers and therefore do not need to be removed from the mixture. In some embodiments, the adapter molecule can also serve as one of the two

amplification primers, further enhancing efficiency.

[0006] The invention is also particularly suited for devices that integrate sample preparation with analysis, such as fluidically integrated sample-to-sequence devices or lab-on-a-chip devices. The invention as described herein can reduce the footprint of such an integrated device, by allowing adapter ligation and amplification to occur within a single chamber or flow cell.

SUMMARY OF THE INVENTION

[0007] In one aspect, the instant invention provides a method comprising providing a target polynucleotide comprising a 3' and a 5' end; attaching the 3' end of the target polynucleotide to a first adapter and attaching the 5' end of the target polynucleotide to a second adapter, thereby producing a template polynucleotide, wherein the second adapter is attached to a solid substrate; hybridizing a complementary oligonucleotide to at least a portion of the first adapter; and using at least one nucleic acid polymerase to extend the complementary oligonucleotide to produce a product polynucleotide that is complementary to the template polynucleotide.

[0008] In some embodiments, the target polynucleotide is DNA. In some embodiments, the second adapter comprises at the 3' end at least one RNA nucleotide and attaching the 5' end of the target polynucleotide to the second adapter is performed by an RNA ligase. In some embodiments, the 5' end of the target polynucleotide is pre-adenylated, and attaching the 5' end of the target polynucleotide is performed by truncated T4 RNA ligase. In some embodiments, attaching the 5' end of the target polynucleotide occurs before attaching the 3' end of the target polynucleotide. In some embodiments, attaching the 3' end of the target polynucleotide is performed by T4 RNA ligase.

[0009] In some embodiments, the target polynucleotide is RNA. In some embodiments, the 5' end of the first adapter is pre-adenylated, and wherein attaching the 3' end of the target polynucleotide is performed by truncated T4 RNA ligase. In some embodiments, attaching the 3' end of the target polynucleotide occurs before attaching the 5' end of the target polynucleotide. In some embodiments, attaching the 5' end of the target polynucleotide is performed by T4 R A ligase.

[0010] In some embodiments, the at least one nucleic acid polymerase comprises a reverse transcriptase. In some embodiments, the at least one nucleic acid polymerase comprises a DNA polymerase. In some embodiments, the complementary oligonucleotide is attached to the solid substrate. In some embodiments, the complementary oligonucleotide is not attached to the solid substrate. In some embodiments, the first adapter is attached to the solid substrate. In some embodiments, the first adapter is not attached to the solid substrate.

[0011] In some embodiments, the method further comprises cleaving the first adapter, wherein cleaving the first adapter occurs subsequent to extending the complementary oligonucleotide. In some embodiments, the method further comprises amplifying the product polynucleotide. In some embodiments, amplifying the product polynucleotide is performed by polymerase chain reaction. In some embodiments, amplifying the product polynucleotide is performed using a primer pair comprising a first primer and a second primer, wherein both the first primer and the second primer are attached to the solid substrate, and wherein the first primer comprises at least a portion of the sequence of the complementary oligonucleotide and the second primer comprises at least a portion of the sequence of the second adapter. In some embodiments, the first primer and the complementary oligonucleotide have the same sequence. In some embodiments, the second primer and the second adapter have the same sequence. In some embodiments, the first primer comprises a protecting group at the 3 ' end, and the protecting group is removed from the first primer after attaching the 5' end of the target polynucleotide to the second adapter. In some embodiments, the protecting group is a phosphate group.

[0012] In another aspect, the invention provides for a method, comprising providing a target polynucleotide comprising a 3' and a 5' end; attaching the 3' end of the target polynucleotide to a first adapter and attaching the 5' end of the target polynucleotide to a second adapter, thereby producing a template polynucleotide, wherein the first and second adapters are attached to a solid substrate; and cleaving the first adapter to form a cleaved template polynucleotide. In some embodiments, cleaving the first adapter is performed using a restriction enzyme, nicking enzyme, or R ase. In some embodiments, prior to cleaving the first adapter, a complementary

oligonucleotide that is complementary to at least a portion of the first adapter is provided, and cleaving the first adapter is performed using a restriction endonuclease, wherein the recognition site of the restriction endonuclease is formed by the hybridization of the complementary oligonucleotide to the first adapter. In some embodiments, the complementary oligonucleotide is attached to the solid substrate. In some embodiments, the complementary oligonucleotide is not attached to the solid substrate. In some embodiments, the first adapter comprises a photocleavable linkage, and cleaving the first adapter is performed using light-induced cleavage.

[0013] In some embodiments, the target polynucleotide is DNA. In some embodiments, the second adapter comprises at the 3' end at least one RNA nucleotide and attaching the 5' end of the target polynucleotide is performed by an RNA ligase. In some embodiments, the 5' end of the target polynucleotide is pre-adenylated, and attaching the 5' end of the target polynucleotide is performed by truncated T4 RNA ligase. In some embodiments, attaching the 5' end of the target polynucleotide occurs before attaching the 3' end of the target polynucleotide. In some embodiments, attaching the 3' end of the target polynucleotide is performed by T4 RNA ligase.

[0014] In some embodiments, the target polynucleotide is RNA. In some embodiments, the 5' end of the first adapter is pre-adenylated, and attaching the 3' end of the target polynucleotide is performed by truncated T4 RNA ligase. In some embodiments, attaching the 3' end of the target polynucleotide occurs before attaching the 5' end of the target polynucleotide. In some embodiments, attaching the 5' end of the target polynucleotide is performed by T4 RNA ligase.

[0015] In some embodiments, the invention comprises hybridizing the cleaved template polynucleotide to a first primer, wherein the first primer is at least partially complementary to a 3' sequence on the cleaved template polynucleotide and wherein the first primer is attached to a solid substrate; and using at least one nucleic acid polymerase to extend the first primer to produce a product polynucleotide that is complementary to the template polynucleotide. In some embodiments, the first primer comprises a protecting group at the 3' end, and the invention further comprises removing the protecting group from the first primer after attaching the 5 ' end of the target polynucleotide to the second adapter. In some embodiments, the protecting group is a phosphate group.

[0016] In some embodiments, the first adapter is attached to the solid substrate. In some embodiments, the first adapter is not attached to the solid substrate. In some embodiments, the at least one nucleic acid polymerase comprises a reverse transcriptase. In some embodiments, the at least one nucleic acid polymerase comprises a DNA polymerase.

[0017] In some embodiments, the method further comprises amplifying the product polynucleotide. In some embodiments, amplifying the product polynucleotide is performed by polymerase chain reaction. In some embodiments, amplifying the product polynucleotide is performed using a primer pair comprising a first primer and a second primer, and the second primer comprises at least a portion of the sequence of the second adapter. In some embodiments, both the first primer and the second primer are attached to the solid substrate. In some embodiments, the first primer and the complementary oligonucleotide have the same sequence. In some embodiments, the second primer and the second adapter have the same sequence. In some embodiments, the second primer comprises a protecting group at the 3' end, and the protecting group is removed from the second primer after attaching the 5' end of the target polynucleotide to the second adapter. In some embodiments, the protecting group is a phosphate group.

[0018] In some embodiments, the target polynucleotide is single-stranded. In some embodiments, the target polynucleotide comprises a plurality of polynucleotides. The target polynucleotide can comprise a library of polynucleotides isolated from a single sample. In some embodiments, the sample is a forensic, medical, or environmental sample. In some

embodiments, the first adapter is present on the solid substrate at a density less than that of the second adapter. In some embodiments, the first or second adapter is attached to the solid substrate using a crosslinking agent. The crosslinking agent can be selected from the group consisting of EDC, succinic anhydride, maleic anhydride, MBS, SIAB, SMCC, GMBS, or SMPB. In some embodiments, the solid substrate is a particle, a bead, a slide, a surface of an element of a device, a membrane, a flow cell, a well, a chamber, a macrofluidic chamber, a microfluidic chamber, a channel, or a microfluidic channel, preferably a flow cell. In some embodiments, the first adapter or the second adapter comprises a barcode sequence.

[0019] In other aspects, the invention provides for an article comprising a single-stranded template polynucleotide comprising a 5' end and a 3' end, wherein both the 5' and 3' ends are attached to a solid substrate. In some embodiments, the template polynucleotide comprises both R A and DNA nucleotides. In some embodiments, the template polynucleotide comprises a plurality of polynucleotides. In some embodiments, the article further comprises an

oligonucleotide comprising a sequence complementary to a 3' sequence of the template polynucleotide, wherein the oligonucleotide is attached to the solid substrate. In some embodiments, the oligonucleotide is hybridized to the 3' end of the template polynucleotide. In some embodiments, the oligonucleotide is not hybridized to the 3' end of the template polynucleotide.

INCORPORATION BY REFERENCE

[0020] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0022] Figure 1 provides schematics of example methods for attaching a target R A or target DNA sequence to a solid substrate.

[0023] Figure 2 provides schematics of example methods of performing initial extension using a template polynucleotide.

[0024] Figure 3 depicts an example method for attaching a target RNA to a solid substrate and preparing the RNA for bridge PCR amplification.

[0025] Figure 4 depicts another example method for attaching a target RNA to a solid substrate and preparing the RNA for bridge PCR amplification.

[0026] Figure 5 depicts an example method for attaching a target DNA to a solid substrate and preparing the DNA for bridge PCR amplification.

[0027] Figure 6 depicts another example method for attaching a target DNA to a solid substrate and preparing the DNA for bridge PCR amplification.

[0028] Figure 7 depicts examples of how ligation to adapters can be performed with protecting groups or in solution.

[0029] Figure 8 is a schematic for an example method of the invention for attaching and amplifying a double-stranded target polynucleotide.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The invention described herein provides for systems, devices, methods, and kits for amplifying nucleic acids on a solid substrate. The invention allows attachment of a target polynucleotide to an adapter sequence bound to a solid substrate. In preferred embodiments, the target polynucleotide is directly attached to the solid substrate through ligation to the adapter sequence, without the need for any hybridization between the adapter and a capture primer on the substrate.

[0031] It is understood that every embodiment of the invention may optionally be combined with any one or more of the other embodiments described herein which are consistent with that embodiment. [0032] Whenever the term "about" or "approximately" precedes the first numerical value in a series of two or more numerical values or in a series of two or more ranges of numerical values, the term "about" or "approximately" applies to each one of the numerical values in that series of numerical values or in that series of ranges of numerical values. As used herein, the term

"about" or "approximately" refers to values within 10% of the target or specified value.

Oligonucleotides

[0033] Oligonucleotides as referred herein refer to any length polynucleotide, preferably between 6 and 100 bases in length. Oligonucleotides may be single or double-stranded, and may comprise RNA, DNA, synthetic or modified nucleotides, or any combination thereof.

Oligonucleotides as used in this invention may be adapter oligonucleotides (e.g., "adapters"), e.g., for ligation to the target polynucleotide; or priming oligonucleotides (e.g., "primers") for amplification. In some cases, an oligonucleotide can act both as an adapter and as a primer.

[0034] A target polynucleotide can be amplified by the methods of this invention. The target polynucleotide may be single-stranded or double-stranded; if double-stranded, either strand can also be considered the target polynucleotide. The target polynucleotide may have a known or unknown sequence, and can comprise DNA, such as genomic DNA, cDNA, or any form of synthetic or modified DNA; RNA, such as mRNA, miRNA, siRNA, or any form of synthetic or modified RNA, or any combination thereof. A target polynucleotide can vary in length, preferably between about 50 to about 5000 bases in length, more preferably between about 150 to about 2000 bases in length. In some embodiments, the target polynucleotide can be between about 50 and about 200, about 50 and about 300, about 50 and about 500, about 100 and about 300, about 100 and about 500, about 250 and about 500, about 250 and about 750, about 500 and about 1000, about 500 and about 2000, about 500 and about 3000, about 500 and about 4000, about 500 and about 5000 bases in length, or between about 1000 and about 5000 bases in length. A template polynucleotide comprises the target polynucleotide, but may contain one or more additional sequences, such as adapter sequences, primer sequences, or barcode sequences.

[0035] A sample for use in the methods of the invention may comprise multiple target polynucleotides of different sequences or length. For example, a target polynucleotide of the invention may refer to a plurality of polynucleotides of potentially different sequences. In some embodiments, the plurality of polynucleotides may comprise a library, such as an mRNA library, a cDNA library, or a genomic library. In some embodiments, the target polynucleotide comprises multiple sequences from a sample, including but not limited to forensic, environmental, medical or other samples. A sample may comprise polynucleotides from humans, animals, plants, pathogens, viruses, bacteria, or any combination thereof.

[0036] Adapter oligonucleotides refer to oligonucleotides that can be ligated to a target polynucleotide. Adapter oligonucleotides can be of any sequence or length, but preferably contain at least one sequence useful for amplification, for cleavage, or for subsequent steps, such as for sequencing. In some embodiments, adapter oligonucleotides can contain a recognition sequence for an enzyme, such as a restriction endonuclease site, a nicking enzyme recognition site, or a ribozyme cleavage site. In some embodiments, an adapter oligonucleotide can comprise a modified group that allows for induced cleavage, such as by chemical cleavage, photocleavage, UV cleavage, heat-based cleavage or other methods. In some embodiments, adapter oligonucleotides can contain promoter sequences, protein binding sequences, operator sequences, sequences to generate secondary structures such as hairpins, a primer sequence for amplification or sequencing, or barcode sequences, as taught in U.S. Patent Publication No. 2011/0039303, herein incorporated by reference in its entirety. Adapter oligonucleotides can be single-stranded, double-stranded, or partially single-stranded. In some embodiments, an adapter oligonucleotide can comprise both RNA and DNA, and may also include synthetic or modified nucleotides. Adapter and primer oligonucleotides can also contain modifications, including modified nucleotides, that allow covalent or non-covalent attachment to a solid substrate. Such modifications may be at or near the 5' or 3' end of the oligonucleotide. Primer oligonucleotides preferably contain modifications that allow attachment of the primer at or near the 5' end, to allow the 3' end to remain free for extension during amplification. In some embodiments, one or more of the adapter or primer oligonucleotides are not attached to a solid substrate.

[0037] In preferred embodiments, the primers and at least one adapter are attached to the solid substrate, preferably prior to attachment of the target polynucleotide to the adapter. In some embodiments, the primers and the adapters used in the invention are all attached to the solid substrate, preferably prior to attachment of the target polynucleotide to either adapter. In some embodiments, the primers and adapters are attached to a single solid substrate or to a single surface, including but not limited to embodiments wherein the primers and adapters are attached to two or more locations on a single solid substrate or surface. Preferably, one or more of the primers and/or adapters are attached to the solid substrate at a uniform density.

Substrates

[0038] Substrates, or solid substrates, as used herein can refer to any solid surface to which nucleic acids can be covalently or non-covalently attached. Non-limiting examples of solid substrates include particles, beads, slides, surfaces of elements of devices, membranes, flow cells, wells, chambers, macro fluidic chambers, micro f uidic chambers, channels, microf uidic channels, or any other surfaces. Substrate surfaces can be flat or curved, or can have other shapes, and can be smooth or textured. In some embodiments, the substrate can be composed of glass, carbohydrates such as dextrans, plastics such as polystyrene or polypropylene, polyacrylamide, latex, silicon, metals such as gold, or cellulose, and may be further modified to allow or enhance covalent or non-covalent attachment of the oligonucleotides. For example, the substrate surface may be functionalized by modification with specific functional groups, such as maleic or succinic moieties, or derivatized by modification with a chemically reactive group, such as amino, thiol, or acrylate groups, such as by silanization. Suitable silane reagents include

aminopropyltrimethoxysilane, aminopropyltriethoxysilane and 4-aminobutyltriethoxysilane. Glass surfaces can also be derivatized with other reactive groups, such as acrylate or epoxy, using, e.g., epoxysilane, acrylatesilane or acrylamidesilane. The substrate and means for oligonucleotide attachment are preferably stable for the repeated denaturing, annealing and extension cycles necessary for amplification.

[0039] In some preferred embodiments, the solid substrate can be a flow cell, such as that described in U.S. Patent Publication Nos. 2010/0111768 and 2008/0286795, and PCT

Publication Nos. W098/44151 and WO02/46456. The flow cell can be composed of a single layer or multiple layers. For example, a flow cell can comprise a base layer (e.g., of boro silicate glass), a channel layer (e.g., of etched silicon) overlaid upon the base layer, and a cover, or top, layer. When the layers are assembled together, enclosed channels can be formed having inlet/outlets at either end through the cover. The thickness of each layer can vary, but is preferably less than about 1500 μιη. Layers can be composed of any suitable material known in the art, including but not limited to photosensitive glasses (e.g., Foturan®, available from

Mikroglas, Mainz, Germany), borosilicate glass, fused silicate, or silicon. Different layers can be composed of the same material or different materials.

[0040] In some embodiments, flow cells can comprise openings for channels on the bottom of the flow cell. A flow cell can comprise millions of attached target polynucleotides in locations that can be discretely visualized. In some embodiments, various flow cells of use with the invention can comprise different numbers of channels (e.g., 1 channel, 2 or more channels, 3 or more channels, 4 or more channels, 6 or more channels, 8 or more channels, 10 or more channels, 12 or more channels, 16 or more channels, or more than 16 channels). Various flow cells can comprise channels of different depths or widths, which may be different between channels within a single flow cell, or different between channels of different flow cells. A single channel can also vary in depth and/or width. For example, a channel can be less than about 50 μιη deep, about 50 μιη deep, less than about 100 μιη deep, about 100 μιη deep, about 100 μι ίο about 500 μιη deep, about 500 μιη deep, or more than about 500 μιη deep at one or more points within the channel. Channels can have any cross sectional shape, including but not limited to a circular, a semi-circular, a rectangular, a trapezoidal, a triangular, or an ovoid cross-section.

[0041] Oligonucleotides can be attached to a solid surface using any means known in the art, including any chemical or non-chemical attachment method, any covalent or non-covalent bonding method, adsorption, charge affinity, or binding affinity (such as between biotin and avidin, or between an antibody and binding partner). As some non-limiting examples, covalent attachment can be achieved using reactive amine groups, thiol groups, phosphate groups, aldehyde groups, hydroxyl groups or carboxyl groups. Preferably, the attachment is strong enough to keep the oligonucleotide attached to the substrate during sample preparation and amplification.

[0042] In some embodiments, covalent binding of oligonucleotides to a solid support is created by use of a crosslinking agent, such as for example l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide hydrochloride (EDC), succinic anhydride, phenyldiisothiocyanate or maleic anhydride, or a hetero-bifunctional crosslinking agent, such as for example m-maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), N-succinimidyl[4-iodoacethyl]aminobenzoate (SIAB), succinimidyl 4-[N-maleimidomethyl]cyclohexane-l-carboxylate (SMCC), N- maleimidobutyryloxysuccinimide ester (GMBS), succinimidyl-4-[p-maleimidophenyl]butyrate (SMPB) and corresponding sulfo compounds. In some embodiments, oligonucleotides can be attached to the substrate using epoxysilane-amino covalent linkage, or by linking 5 ' carboxylic or aldehyde moieties to hydrazine-derivatized substrates. In some embodiments, an oligonucleotide can be attached to the solid substrate using a polyethylene glycol (PEG) linker, such as a PEG linker with at least 6 PEG units. Additional attachment methods suitable for use with this invention are described in U.S. Publication No. 2005/0079510, herein incorporated by reference in its entirety.

[0043] In some embodiments, the oligonucleotides (e.g., second adapters, primers) can be distributed evenly across the surface of the substrate. Each oligonucleotide can be present on the substrate at the same density or at different densities. As one non- limiting example, the 3' adapter can be present at a lower density than the two oligonucleotides used as amplification primers. In some embodiments, the target polynucleotide can be introduced at a quantity and concentration sufficient for substantially all of the 3' adapter molecules present on the surface of the solid substrate to be able to be ligated to a target polynucleotide. In some embodiments, the target polynucleotide is introduced in a quantity sufficient for at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%), at least about 90%>, at least about 95%, at least about 98%>, at least about 99%, or about 100% of the 3' adapter to be ligated to a target polynucleotide. In some embodiments, the 3' adapters are present in an amount such that at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%), at least about 95%, at least about 98%, at least about 99%, or about 100% of the target polynucleotide is captured on the surface of the solid substrate. In preferred embodiments, the ratio of the adapter oligonucleotides to the priming oligonucleotides is such that, when attached to the solid support, the primers are located at an approximately uniform density over the solid surface, with the adapters immobilized individually at intervals over the surface. Preferably, the target polynucleotide is captured on the surface of the solid substrate at a density low enough that, after amplification, each target polynucleotide results in a single distinct cluster of amplified product on the surface of the solid substrate. A preferred density of primer oligonucleotides is at least about 1 fmol/mm², preferably at least about 10 fmol/mm², more preferably between about 30 and about 60 fmol/mm². The density of adapter oligonucleotides for use in the method of the invention is typically about 10,000/mm² to about 100,000/mm². Higher densities, for example, about 100,000/mm² to about 1,000,000/mm² and about 1,000,000/mm² to about 10,000,000/mm² may be achieved. In embodiments where an adapter oligonucleotide also serves as a primer oligonucleotide, the primer/adapter oligonucleotide is preferably at the higher density intended for primer oligonucleotides. Methods for cluster amplification are described in greater detail in US publication US2008/0160580, herein incorporated by reference in its entirety.

[0044] In some embodiments, two or more of the oligonucleotides attached to the solid surface may have complementary sequences, which could interfere with hybridization while carrying out the methods of the invention as described herein. In some embodiments, during attachment of the oligonucleotides to the solid substrate, various methods may be employed to prevent unwanted oligonucleotide hybridization on the substrate. One non-limiting example is to attach the oligonucleotides at a density sufficiently low that there is a low probability of any two complementary oligonucleotides being attached in close enough proximity on the substrate to hybridize. In some embodiments, oligonucleotides may be pre-hybridized to a complementary sequence that is not able to attach to the substrate before attachment to the solid substrate. After attachment, the substrate can be placed under denaturing conditions and the complementary sequence(s) washed away. [0045] In some embodiments of the invention, components may be included in the device to facilitate the steps described herein. For example, temperature control components can be included to regulate temperature during attachment, extension, amplification, and/or sequencing. The invention can also comprise, e.g., a body or chassis, a flow cell and flow cell holder, one or more manifolds that can be fluidly connected to the flow cell or other solid substrate, reagent storage and waste storage reservoirs (some or all of which optionally can be temperature controlled and some or all of which can be fluidly connected to the manifold/flow cell), sample storage areas, fluidic distribution systems (e.g., tubings, pumps, directional valves, etc.), temperature control components (e.g., for keeping the flow cell isothermal during cluster creation or for keeping reagents at the proper temperature), power supply, computer, etc.

[0046] In some embodiments, one or more pumps can be used to control fluid flow to, through or from the solid substrate or proximity thereto, or to, through or from other components of the device. Examples of pumps suitable for use with devices of the invention include but are not limited to positive/negative displacement pumps, vacuum pumps, peristaltic pumps, hydraulic pumps, and pneumatic pumps. Pumps can be controlled by computer instructions. Pumps can be macro fluidic or micro fluidic.

[0047] In some embodiments, micro fluidic valves and pumps control the flow of reagents to the surface of the solid substrate. In some embodiments, micro fluidic valves used can be diaphragm, pumping, or MOVe (Micro fluidic On-chip Valve) valves, such as those described in U.S. Patent Nos. 7,445,926, 7,745,207, 7,766,033, and 7,799,553; U.S. Publication Nos.

2011/0126911, 2011/0005932, 2007/0248958, 2010/0165784; PCT Publication Nos. WO 2008/115626, WO 2009/108260 and WO 2009/015296; PCT application PCT/US2010/40490; and U.S. provisional applications 61/330,154 and 61/375,791, all herein incorporated by reference in their entirety.

[0048] A diaphragm valve uses a diaphragm to open or close a fluidic path between fluidic conduits. A diaphragm valve typically comprises a valve body having a valve inlet and a valve outlet that communicate with the fluidic conduits entering and exiting the valve. In some embodiments, the body also has a diaphragm disposed within the body and configured to sit against a valve seat to completely or partially close the valve. The valve body also comprises a valve relief, or valve chamber, into which the diaphragm can deflect away from the valve seat. When the diaphragm is deflected away from the valve seat, a space is created, thereby opening the valve. When the valve is open, a continuous flow path is formed through which the valve inlet is in fluid communication with the valve outlet. [0049] In other embodiments, a diaphragm valve is configured as a normally open valve.

Rather than being an interruption in a fluidic conduit, the valve seat takes the form of a recess with respect to the surface of a fluidics layer that contacts an elastic layer, so that the elastic layer does not sit against the recess without application of pressure on the elastic layer, e.g., via an actuation chamber of an actuation layer. The valve seat can have a curved shape that is concave with respect to the surface of the fluidics layer, against which the elastic layer can conform to close the valve. For example, the shape of the valve seat can be a section of a sphere, an inverted dimple or a dome. Such a configuration can decrease the dead volume of the valve, e.g., by not having a valve chamber that contains liquid while the valve is closed. In further embodiments, the concave surface of the valve seat comprises one or more areas having a convex surface, e.g., an inverted dimple comprising an extraverted dimple forming, e.g., a saddle shape. The convex area(s) of the valve seat meet the elastic layer when pressure is applied to the elastic layer, which can seal the valve better.

[0050] Fluidic devices suitable for use with this invention can comprise at least one or a plurality of fluidic conduits in which fluid flows to and away from the solid surface. Flow can be controlled by on-device diaphragm valves and/or pumps actuatable by, for example, pressure, pneumatics, or hydraulics. In some embodiments, the devices comprise a fluidics layer bonded to an elastic layer, wherein the elastic layer functions as a deflectable diaphragm that regulates flow of fluid across interruptions (e.g., valve seats) in the fluidic pathways in the fluidics layer. The elastic layer can comprise an elastomeric polymeric material, such as a polysiloxane (e.g., polydimethylsiloxane (PDMS)). In further embodiments, the devices comprise three layers: a fluidics layer, an actuation layer, and an elastic layer sandwiched therebetween. The actuation layer can comprise actuation conduits configured to actuate or deflect the elastic layer at selected locations, e.g., at diaphragm valves, thereby controlling the flow of fluid in the fluidic conduits. The three layers can be bonded together in a unit. Alternatively, the fluidics layer or the actuation layer can be bonded to the elastic layer to form a unit, and the unit can be reversibly mated with the other layer later. Mating can be accomplished, for example, by applying and releasing pressure, e.g., by clamping.

[0051] Diaphragm valves and pumps can be comprised of functional elements in three layers. A diaphragm valve comprises a body, a seat (optional), a diaphragm and ports configured to allow fluid to flow into and out of the valve. The body is comprised of a cavity or chamber in the actuation layer that opens onto the surface facing the elastic layer ("actuation valve body"). Optionally, the valve body also includes a chamber in the fluidics layer that opens onto a surface facing the elastic layer and which is disposed opposite the actuation layer chamber ("fluidics valve body"). The actuation layer body communicates with a passage, e.g., a channel, through which positive or negative pressure can be transmitted by the actuant. When the actuant is a gas, e.g., air, the actuation layer functions as a pneumatics layer. In other embodiments, the actuant is a liquid, such as water, oil, Fluorinert™, etc., and the actuation layer can function as a hydraulics layer.

[0052] In some embodiments, a diaphragm is formed from a body comprising a chamber in the actuation layer and in the fluidics layer, but without an interruption. In these embodiments, deforming the diaphragm into the actuation chamber creates a volume to accept fluid, and deforming the diaphragm into the fluidics chamber pumps liquid out of the chamber. In this configuration, the position of the diaphragm alters the effective cross-section of the fluidic conduit and, thus, can regulate the speed of flow through the valve. In such a configuration, the valve may not completely block the flow of fluid in the conduit. This type of valve is useful as a fluid reservoir and as a pumping chamber and can be referred to as a "pumping valve."

[0053] When placed in a series of three or more, diaphragm valves can function as a diaphragm pump, which functions as a positive displacement pump. Diaphragm pumps are self-priming and can be made by coordinating the operation of at least three valves (including but not limited to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more valves), and can create flow in either direction. A variety of flow rates can be achieved by the timing of the actuation sequence, diaphragm size, channel widths, and other on-chip dimensions. Routers can similarly be formed from these valves and pumps. The routers can be formed using three or more valves each on a separate channel connecting to a central diaphragm valve. A router can also be made by configuring three channels, each comprising a diaphragm pump, to meet in a common chamber, e.g., a pumping chamber. Bus structures can also be created.

[0054] In some embodiments, a microfluidic or macro fluidic system is used to flow reagents to the surface of the solid substrate, and to wash away leftover reagents and buffers, side products, or other waste products from the surface of the solid substrate. Since the target polynucleotide is attached to the surface of the substrate, each step after attachment can be followed by one or more wash steps to reduce unwanted cross-contamination between steps. In some embodiments, devices for use with the invention can comprise reagent and/or waste reservoirs. Such reservoirs can be included in the device, such as in a closed or open chamber or well, or be located off the device.

[0055] The solid substrate can vary in size, shape and kind. In some embodiments, flow cells are used as the solid substrate. Flow cells or other substrates can be multiplexed, such as by having multiple channels or chambers in a flow cell. For example, particular flow cells can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more channels. Manifolds or other devices can be used to control reagent or buffer flow into or out of each channel of the flow cell

simultaneously.

[0056] Computers or controllers for use with devices of the invention can be programmed to control various components of the device. In some embodiments, computers can be used to analyze images or data. In some embodiments, a computer is included in the device. In some embodiments, the computer is off-board, and may have a specific program to interface with the device. In some embodiments, the computer may be coupled to a display device, which can be used to display the status of the device, the results, analyses, or any other useful information. In some embodiments, a user can input instructions or other information to the computer to control the device. In some embodiments, data, e.g., raw data or analysis results, can be exported from the device, such as through a removable disk, through wireless or internet connections, or by other means known in the art.

Target Preparation

[0057] The target polynucleotide can be attached to a first adapter using ligation. To prevent circularization of the target polynucleotide, and to increase the likelihood that the 5' and 3' ends of the target polynucleotide ligate to the correct adapter, various methods known in the art can be employed. In some embodiments, the adapter molecules can comprise blocking groups on the distal ends. In some embodiments, one end of the target polynucleotide can be blocked during a first ligation step in the presence of only the first adapter, followed by removal of the blocking group and a second ligation step in the presence of only the second adapter.

[0058] In preferred embodiments, a single-stranded target is ligated to a single-stranded end of the first adapter using a single-stranded ligase such as T4 RNA ligase 1 , more preferably truncated T4 RNA ligase 2 or derivatives thereof. Commercially available examples of such ligases include but are not limited to T4 RNA Ligase 2, truncated; or T4 RNA ligase 2, truncated K227Q; both from New England Bio labs (NEB) as M0242 and M0351, respectively. One advantage to using a truncated T4 RNA ligase is that the truncated ligase requires a 5 ' adenylated (App) end and a 3' end comprising a free hydro xyl group. As a result, appropriate ligation of the 5' and 3' ends of the target polynucleotide can be controlled by first performing a

preadenylation-dependent ligation on one end of the target polynucleotide, followed by an adenylation- independent ligation step on the other end, for example, by using T4 RNA ligase 1. If the target polynucleotide is RNA, the 3' end of the target RNA can be ligated to a preadenylated 5' end of a DNA adapter (Figure 1A). Methods of 5' adenylation, referring to addition of a 5',5'-adenyl pyrophosphoryl moiety (App) onto the 5' end of an RNA or DNA molecule, are well-known in the art. If the target polynucleotide is DNA, the 5' end of the target DNA can be pre-adenylated before ligation to the 3' end of an RNA adapter (Figure IB).

[0059] Pre-adenylation of the 5 ' ends of target or adapter DNA can be performed by any 5 ' adenylating enzyme known in the art, such as Mth RNA ligase or modified versions thereof. Adenylation can also be easily performed using commercially available kits, including but not limited to NEB's 5' DNA Adenylation Kit. In some embodiments, adenylation can be generated , such as during generation of an adapter.

[0060] In some embodiments, the RNA adapter also comprises a DNA sequence distal to the 3' end. In some embodiments, the adapter can be single-stranded, double-stranded, or partially double-stranded. The adapter can be attached to a solid substrate or be free in solution.

Preferably, an adapter that is to be ligated to the 5' end of the target polynucleotide is attached to a solid substrate. Preferably, to reduce the number of unique sequences attached to the solid substrate, an adapter that is to be ligated to the 3' end of the target polynucleotide is free in solution. If the adapter is free in solution, the end that is not to be ligated to the target polynucleotide can be blocked to reduce unwanted ligation products.

[0061] In preferred embodiments, the target polynucleotide can be attached to a second adapter using ligation. If the target polynucleotide is single-stranded RNA, the 5' end of the target RNA can be ligated to the 3' end of a DNA adapter using, for example, T4 RNA ligase 1 in the presence of ATP. If the target polynucleotide is single-stranded DNA, the 3' end of the target DNA can be ligated to the 5' end of an oligonucleotide adapter using any suitable single-stranded ligase known in the art. Preferably, ligation to the first adapter occurs before ligation to the second adapter, to ensure that each end of the target polynucleotide is ligated to the correct adapter oligonucleotides. The resulting ligated polynucleotide is attached by at least one end to the solid substrate, and is suitable as a template for the extension step (Fig. 2).

[0062] In other embodiments, the target polynucleotide can be double-stranded, and ligation to a double-stranded adapter can be performed by a double-stranded ligase, such as T4 RNA ligase 2, T4 DNA ligase, Taq DNA ligase, or E. coli DNA ligase. A double-stranded target

polynucleotide may have blunt ends, 5' or 3' overhangs, or combinations thereof. In some embodiments, the target polynucleotide can be extended with a single adenosine, e.g., by A- tailing prior to ligation, as described in greater detail in U.S. Application No. 13/202,884, herein incorporated by reference in its entirety. [0063] In preferred embodiments, at least a third oligonucleotide is attached at the 5' end to the solid substrate, which will serve as one of the primers during subsequent amplification. To prevent unwanted ligation of the target polynucleotide to the third oligonucleotide, said third oligonucleotide can be initially blocked by a removable blocking group at the 3' end to prevent ligation, such as by using a blocking group (B) attached to the terminal hydroxyl group (Fig. 1). The blocking group can be any group known in the art, such as a phosphate group or a ddNTP. After the ligation steps are complete, the blocking group can be removed, for example, by using a phosphatase. Other blocking groups and removal methods are possible and well known in the art, including but not limited to chemical cleavage, photocleavage, UV cleavage, heat-based cleavage, and other methods.

[0064] Initial extension of the resulting template polynucleotide can be performed by a reverse transcriptase, such as AMV or M-MuLV reverse transcriptase. In some embodiments, a reverse transcriptase can be used regardless of whether the target polynucleotide comprises RNA or DNA, as the initial adapter ligation step typically produces an RNA/DNA hybrid template. In some embodiments where the 3' adapter is partially double-stranded, extension can be performed directly from the 3' adapter (Fig. 2A). In other embodiments, a short oligonucleotide

complementary to at least a portion of the 3' adapter can be introduced to serve as a primer (Fig. 2A). In some embodiments, the 3' adapter is ligated in solution, resulting in a free end that allows the template to hybridize to a primer oligonucleotide attached to the substrate. In still other embodiments, the 3' adapter can be cleaved or detached from the solid substrate and the resulting 3' end of the cleaved template hybridized to a primer oligonucleotide to prime the initial extension reaction (Fig. 2B). Such cleavage can be accomplished using restriction endonucleases, nicking enzymes, or other means known in the art, along with suitable design of the 3' adapter as would be obvious to one skilled in the art. In some embodiments, cleavage is preceded by hybridizing the 3' adapter to a complementary sequence to produce a double- stranded recognition sequence, e.g. for a restriction endonuclease.

[0065] Additional details on exemplary methods of the invention are described in detail in the Examples.

Amplification

[0066] After the initial extension step is completed, amplification can occur by any means known in the art, including by polymerase chain reaction (PCR), ligation chain reaction (LCR), transcription amplification, self-sustained sequence replication, RACE, di-oligonucleotide amplification, isothermal PCR, quantitative PCR, fluorescent PCR, multiplex PCR, real time PCR, single cell PCR, restriction fragment length polymorphism PCR, hot start PCR, or picotiter PCR. One exemplary method of amplification is by bridge PCR, well known in the art and described in greater detail in U.S. Publication Nos. 2008/0160580 and 2010/0009871 and in PCT Publication No. WO96/04404 (Mosaic Technologies, Inc., et al). For PCR-based amplification, the substrate and attached oligonucleotides should be stable for the repeated denaturing, annealing and extension cycles necessary for amplification. In some embodiments, denaturation is performed using heat. In other embodiments, amplification is performed isothermally. In one non-limiting example, denaturation can be performed using a chemical denaturant, such as urea or formamide. Denaturing can be followed by a neutralizing/hybridizing buffer to allow hybridization to a primer oligonucleotide for the next round of amplification. Suitable neutralizing/hybridizing buffers are well known in the art (See Sambrook et al, Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, NY; Current Protocols in Molecular Biology, Ausubel et al. Eds., July 31, 2012 Ed., Wiley). Suitable buffers may comprise additives such as betaine or organic solvents to normalize the melting temperate of the different template sequences, and detergents. An exemplary hybridization buffer comprises 2 M betaine, 20 mM Tris, 10 mM ammonium sulfate, 2 mM magnesium sulfate, 0.1% Triton, and 1.3% DMSO, pH 8.8.

[0067] Examples of nucleic acid polymerases which can be used with the invention include without limitation DNA polymerase (e.g. Klenow fragment, T4 DNA polymerase), heat-stable DNA polymerases from a variety of thermostable bacteria (such as Taq, Vent, Deep Vent, Pfu, Tfl DNA polymerases) as well as their genetically modified derivatives (e.g., TaqGold^®, AmpliTaq^®, Vent_R ^®, Vent (exo-), Deep Vent (exo-), Pfu exo). Examples of RNA polymerases that can be used with the invention include without limitation SP6 RNA polymerase and T7 RNA polymerase. Examples of reverse transcriptases that can be used with the invention include without limitation avian myeloblastosis virus (AMV) reverse transcriptase, Moloney murine leukemia virus (M-MuLV, M-MLV or MMLV) reverse transcriptase, HIV-1 reverse

transcriptase, Superscript^® (including Superscript^® II and III) reverse transcriptases (available from Invitro gen/Life Technologies), ThermoScript™ reverse transcriptase (available from Invitro gen/Life Technologies), ArrayScript™ reverse transcriptase (available from

Invitro gen/Life Technologies), and any other commercially available reverse transcriptases and reverse transcription kits, such as ProtoScript™ kits from NEB and Superscript^® kits from Life Technologies. In some embodiments, multiple nucleic acid polymerases can be used for an extension or amplification step. In some embodiments, a combination of DNA polymerase, RNA polymerase, and reverse transcriptase can be used. Preferably the nucleic acid polymerase used for primer extension is stable under PCR reaction conditions, e.g., repeated cycles of heating and cooling. Preferably the DNA polymerase used is Taq DNA polymerase or a derivative thereof.

[0068] Preferably the nucleoside triphosphate molecules used are deoxyribonucleotide triphosphates, for example, dATP, dTTP, dCTP, dGTP, or ribonucleotide triphosphates, e.g., ATP, UTP, CTP, GTP. The nucleoside triphosphate molecules may be naturally or non-naturally occurring.

Detection

[0069] After amplification, the resulting amplified nucleic acids can be detected by any of various methods known in the art. A colony can be prepared for detection by denaturing the amplified nucleic acids to form single-stranded DNA. In some embodiments, one of the two adapter oligos can be cleaved to produce colonies comprising a single single-stranded sequence. In some embodiments, colonies can be screened for a specific sequence using hybridization with a labeled probe. In other embodiments, the amplified DNA can be sequenced using a primer, such as a primer comprising a sequence of one of the adapter oligonucleotides. Sequencing can be performed using any of the methods known in the art, such as by primer extension using fluorescently labeled nucleotides. In some embodiments, sequencing can be performed using real-time sequencing, sequencing by synthesis, sequencing by proton detection, pyrosequencing, superpyro sequencing, sequencing by ligation, Sanger sequencing, or any next generation sequencing technique, next next generation sequencing technique, or future generations of sequencing. In some embodiments, the amplified nucleic acids are sequentially cleaved, such as with an exonuclease. The cleaved nucleotides can be detected, for example, by mass

spectrometry, or by detecting fluorescent labels unique to each type of nucleotide.

[0070] In some embodiments, a sequencing reaction used in this invention includes the amplified target or product polynucleotide, at least one primer, and a polymerase. Nucleotides used for sequencing, such as for sequencing by synthesis, can vary. In some embodiments, nucleotides may be unmodified. In some embodiments, nucleotides may contain an optically detectable label, such as a fluorescent dye. The label can be, for example, attached to the gamma phosphate, the beta phosphate, to the base, to the 2' carbon of the ribose, or to the 3' end of the nucleotide. The label can also include a quenching molecule, which can be similarly attached to the nucleotide. The label or quencher can be attached to the nucleotide by a selectively cleavable bond, such as by a photocleavable or chemically cleavable bond. In some embodiments, the labeled nucleotide contains a FRET pair comprising a fluorophore and a quencher. Upon incorporation of the nucleotide into the elongating strand, the fluorophore is unquenched. The unquenched fluorophore can then be detected to determine the target sequence. In some embodiments, the labeled nucleotide contains a chemiluminescent label. Upon incorporation of the nucleotide into the elongating strand, the label is unquenched and through a chain of reactions or directly light is released and detected to determine the target. In some embodiments, sequencing uses oligonucleotides, such as during sequencing by ligation. Oligonucleotides for use in sequencing by ligation can be less than about five base pairs, less than about 8 base pairs, less than about 10 base pairs, or less than about 20 base pairs. Oligonucleotides for use in sequencing may also be labeled as described for nucleotides.

[0071] In some embodiments, sequencing can be performed on molecules individually immobilized to the bottom of a zero mode waveguide, allowing selective detection of

fluorescently labeled nucleotides present in the active site of the sequencing polymerase. In some embodiments, the solid substrate to which an adapter oligonucleotide is attached is a waveguide for detection. Nucleotides that are incorporated into the elongating strand can be detected and distinguished from nucleotides only transiently present in the active site. After incorporation, the fluorescent label can be removed or destroyed prior to incorporation of the next nucleotide.

[0072] Sequencing of the sample DNA can then be performed by any of the methods described herein or known in the art. In some embodiments, one of the strands of the left or right set of oligonucleotides can be used as a sequencing primer. In other embodiments, a sequencing primer complementary to the primer strand can be added with the sequencing master mix. In some embodiments, one strand of the amplified sample DNA is removed from the solid substrate prior to sequencing. The method described herein has the advantages of combining library

preparation, amplification, and sequencing on one device. In preferred embodiments,

microfluidic pumps can be used to move liquids. In other embodiments, other mechanisms to move fluid are envisioned, such as pistons, air or liquid pressure, hydraulic pumps, macrofluidic pumps, and so on. Similar workflows can be applied to library construction on particles, followed by emPCR or emRCA (Rolling Circle Amplification) or other forms of amplification.

[0073] In embodiments of the invention that use detection of incorporated fluorescent nucleotides for sequencing, if the sequence being determined is unknown, the nucleotides applied to a given colony can be applied in a chosen order which is then repeated throughout the analysis, for example, DATP, dTTP, dCTP, dGTP. If the sequence being determined is known and is being re-sequenced, for example, to analyse whether or not small differences in sequence from the known sequence are present, the sequencing determination process may be made quicker by adding the nucleotides at each step in the appropriate order, chosen according to the known sequence.

[0074] In some embodiments, a charge-coupled device (CCD) camera, a complementary metal-oxide-semiconductor (CMOS) camera, or other imaging device can be used to image the clusters.

Using Double-Stranded Targets

[0075] In some embodiments, the invention comprises methods for attaching a double-stranded target polynucleotide to an adapter molecule on a solid substrate, as described in U.S.

Application No. 13/202,884, herein incorporated by reference. In some embodiments, methods of the invention comprise: providing a nucleic acid sample and a plurality of a first double- stranded oligonucleotide bound to a solid substrate and a plurality of a second double-stranded oligonucleotide bound to said solid substrate; performing a first ligation step that ligates the target polynucleotide to one of the plurality of said first double-stranded oligonucleotide;

performing a second ligation step that ligates said nucleic acid sample to one of the plurality of said second double-stranded oligonucleotide; and amplifying said nucleic acid sample using a strand of said plurality of first and second double-stranded oligonucleotides as primers. In some embodiments, the method further comprises treating the pluralities of first and second double- stranded oligonucleotides such that a portion of said pluralities of first and second double- stranded oligonucleotides not ligated to said nucleic acid sample are modified to form single- stranded oligonucleotides bound to said solid substrate; and wherein said amplifying uses said single-stranded oligonucleotides as primers. In some embodiments, the method further comprises performing an end-repair reaction on said nucleic acid sample to provide at least one blunt end on said nucleic acid sample, and where said first ligation step ligates the blunt end of said nucleic acid sample to the blunt end of said first double-stranded oligonucleotide. In some embodiments, the method further comprises performing a single nucleotide extension on said nucleic acid sample to provide at least one single-base overhang, and where said second ligation step ligates said single-base overhang of said nucleic acid sample to the single base overhang of the second double-stranded oligonucleotide. In some embodiments, said single nucleotide extension is an A-tailing step and said double-stranded oligonucleotide comprises a T overhang.

[0076] In some embodiments, methods of the invention comprise a) providing a substrate having attached thereto first double stranded oligonucleotides, each having a blunt end and optionally at least some of which also have blocked ends, and second double stranded

oligonucleotides, each having an end comprising an overhang, wherein only one strand of each of the first oligonucleotides and the second oligonucleotides is attached to the substrate and only at the 5' end; b) contacting the attached oligonucleotides with target nucleic acids, each of the target nucleic acids having a first blunt end and a second end, and performing a ligation reaction that ligates a blunt end of one of the target nucleic acids with a blunt end of the first oligonucleotide; c) modifying the second end of the ligated target nucleic acids and performing a ligation reaction that ligates the second end of target nucleic acid to the end of the second oligonucleotide; d) optionally, unblocking the blocked first oligonucleotides; e) denaturing double stranded nucleic acid molecules attached to the substrate and removing unbound polynucleotides; f) performing bridge PCR on nucleic acids attached to the surface using attached unligated first

oligonucleotides as extension primers for nucleic acids attached to second oligonucleotides, and using unligated attached second oligonucleotides as extension primers for nucleic acids attached to first oligonucleotides.

[0077] In one non-limiting example, a double-stranded adapter oligonucleotide contains a free blunt end. The other adapter oligonucleotide can comprise a single base overhang, such as a T overhang. In some embodiments, a portion of the blunt ends of the first adapter oligonucleotides are blocked with a removable blocking group. In some embodiments, a portion of the single base overhangs of the second adapter oligonucleotides are blocked with a removable blocking group. The two sets of adapters may be blocked with the same or a different removable blocking group, and the blocking groups can be removed by the same or different means, such as by chemical cleavage, photocleavage, UV cleavage, heat-based cleavage or other methods. In some embodiments, the portion of blocked blunt ends is less than about 5% or at least any of about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99, or 99.99%). In some embodiments, the portion of blocked single base overhangs is less than about 5% or at least any of about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%, or about 100%.

[0078] In some embodiments, to perform the methods described above, a sample DNA, which can optionally be fragmented or generated from reverse transcription, can be introduced. End- repair can be performed to generate blunt ends on the sample DNA, and the blunt ends ligated to any unprotected blunt ends of the left set of oligonucleotides. In another embodiment, end-repair is not performed, and only blunt ended DNA sample molecules will ligate to the blunt, unblocked oligonucleotides.

[0079] Preferably, a single DNA sample molecule is ligated per detectable region, which will allow detection of a single sequence. The detectable region can be, for example, a single well, bead, or site on a solid substrate that can be distinguished from other detectable regions. In the example depicted in Figure 8, a detectable region can be the group of bound oligonucleotides. Preferably, only one end of a single DNA sample molecule is ligated during this ligation step, to avoid situations where the DNA sample molecule is ligated on either end to the two

oligonucleotides of the same sequence, which can reduce sequencing accuracy by resulting in simultaneous elongation of both strands of the DNA sample molecule in subsequent steps. In some embodiments, a removable blocking group can be used to protect a portion of the left set of oligonucleotides from ligation. In some embodiments, the concentration of the sample DNA or of components of the ligation reaction can be controlled to reduce or otherwise adjust ligation rates.

[0080] The ligase can then be washed away. In some embodiments, a second polishing reaction can be performed to create a blunt end on the free end of the ligated sample DNA. In some embodiments, the original end-repair step can be sufficient to generate blunt ends on both ends of the DNA sample molecule. An Ά-tailing' master mix can next be added and any unblocked blunt DNA fragments extended with an A. In some embodiments, removable blocking groups on the right set of oligonucleotides can prevent A-tailing of the single base overhang. After removing the A-tailing mix, ligase in the master mix can again be added. Optionally, any blocking groups on the right set of oligonucleotides can be removed after removing the A-tailing mix. A-tailed overhangs of the ligated sample DNA can base pair with the T overhang of the right set of oligonucleotides to form a ligated sample DNA bridge between the left and right sets of oligonucleotides. The left set of oligonucleotides can then be unblocked. Optionally, the left set of oligonucleotides can be unblocked at the same time as the right set of oligonucleotides.

[0081] Next, PCR bridge amplification can be performed. Figure 8 shows only the first round of amplification. In some embodiments, only one strand of the double-stranded oligonucleotides is linked to the solid substrate, and the left and right sets of oligonucleotides can be treated to remove the unbound strand, leaving single-stranded oligonucleotides suitable for acting as primers for subsequent amplification steps. In some embodiments, both strands of the oligonucleotides are bound to the solid substrate, and a denaturing step can be used to

temporarily form single-stranded primers for amplification. In this method, the left and right sets of primers can include sequences for one or more of: an amplification primer, a sequencing primer, and any quality control sequences, such as barcode sequences, as taught in U.S. Patent Application 12/526,015.

[0082] Sequencing of the sample DNA can then be performed by any of the methods described herein. In some embodiments, one of the strands of the left or right set of oligonucleotides can be used as a sequencing primer. In other embodiments, a sequencing primer complementary to the primer strand can be added with the sequencing master mix. In some embodiments, one strand of the amplified sample DNA is removed from the solid substrate prior to sequencing. The method described herein has the advantages of combining library preparation, amplification, and sequencing on one device. In preferred embodiments, micro fluidic pumps can be used to move liquids. In other embodiments, other mechanisms to move fluid are envisioned, such as other types of micro fluidic pumps, hydraulic pumps, macro fluidic pumps, and so on. Similar workflows can be applied to library construction on particles, followed by emPCR or emRCA or other forms of amplification.

Representative Embodiments

[0083] The following embodiments of the invention are provided by way of example only:

1. A method comprising:

providing a target polynucleotide comprising a 3' end and a 5' end;

attaching the 3' end of the target polynucleotide to a first adapter and attaching the 5' end of the target polynucleotide to a second adapter, thereby producing a template polynucleotide, wherein the second adapter is attached to a solid substrate;

hybridizing a complementary oligonucleotide to at least a portion of the first adapter; and extending the complementary oligonucleotide using at least one nucleic acid polymerase to produce a product polynucleotide that is complementary to the template polynucleotide.

2. The method of embodiment 1, wherein the target polynucleotide is DNA.

3. The method of any one of the preceding embodiments, wherein the second adapter comprises at the 3' end at least one RNA nucleotide, and wherein attaching the 5' end of the target polynucleotide to the second adapter is performed using an RNA ligase.

4. The method of any one of the preceding embodiments, wherein the 5 ' end of the target polynucleotide is pre-adenylated, and wherein attaching the 5' end of the target polynucleotide is performed using a truncated T4 RNA ligase.

5. The method of any one of the preceding embodiments, wherein attaching the 5' end of the target polynucleotide occurs before attaching the 3' end of the target polynucleotide.

6. The method of any one of the preceding embodiments, wherein attaching the 3' end of the target polynucleotide is performed using a T4 RNA ligase.

7. The method of any one of the preceding embodiments, wherein the at least one nucleic acid polymerase comprises a DNA polymerase. 8. The method of embodiment 7, wherein the at least one nucleic acid polymerase further comprises a reverse transcriptase.

9. The method of any one of the preceding embodiments, wherein the complementary oligonucleotide is attached to the solid substrate.

10. The method of any one of the preceding embodiments, wherein the first adapter is not attached to the solid substrate.

11. The method of any one of embodiments 1 to 9, wherein the first adapter is attached to the solid substrate.

12. The method of embodiment 11, further comprising cleaving the first adapter, wherein cleaving the first adapter occurs subsequent to extending the complementary oligonucleotide.

13. The method of any one of the preceding embodiments, further comprising amplifying the product polynucleotide.

14. The method of embodiment 13, wherein amplifying the product polynucleotide is performed using a first primer and a second primer,

wherein the first primer and the second primer are attached to the solid substrate, and wherein the first primer comprises at least a portion of the sequence of the

complementary oligonucleotide and the second primer comprises at least a portion of the sequence of the second adapter.

15. The method of embodiment 14, wherein the first primer and the complementary oligonucleotide have the same sequence, or the second primer and the second adapter have the same DNA sequence, or both.

16. The method of embodiment 14 or 15, wherein the first primer comprises a protecting group at the 3 ' end, and further comprising removing the protecting group from the first primer after attaching the 3' end of the target polynucleotide to the first adapter.

17. The method of any one of embodiments 14 to 16, wherein the second primer comprises a protecting group at the 3 ' end, and further comprising removing the protecting group from the second primer after attaching the 3' end of the target polynucleotide to the first adapter.

18. The method of embodiment 16 or 17, wherein the protecting group is a phosphate group.

19. The method of any one of embodiments 13 to 18, wherein amplifying the product polynucleotide is performed by polymerase chain reaction.

20. The method of embodiment 1, wherein the target polynucleotide is R A. 21. The method of embodiment 20, wherein the 5' end of the first adapter is pre-adenylated, and wherein attaching the 3' end of the target polynucleotide is performed using a truncated T4 R A ligase.

22. The method of embodiment 20 or 21, wherein attaching the 3' end of the target polynucleotide occurs before attaching the 5' end of the target polynucleotide.

23. The method of any one of embodiments 20 to 22, wherein attaching the 5' end of the target polynucleotide is performed using a T4 RNA ligase.

24. The method of any one of embodiments 20 to 23, wherein the at least one nucleic acid polymerase comprises a reverse transcriptase.

25. The method of embodiment 24, wherein the at least one nucleic acid polymerase further comprises a DNA polymerase.

26. The method of any one of embodiments 20 to 25, wherein the complementary oligonucleotide is attached to the solid substrate.

27. The method of any one of embodiments 20 to 26, wherein the first adapter is not attached to the solid substrate.

28. The method of any one of embodiments 20 to 26, wherein the first adapter is attached to the solid substrate.

29. The method of embodiment 28, further comprising cleaving the first adapter, wherein cleaving the first adapter occurs subsequent to extending the complementary oligonucleotide.

30. The method of any one of embodiments 20 to 29, further comprising amplifying the product polynucleotide.

31. The method of embodiment 30, wherein amplifying the product polynucleotide is performed using a first primer and a second primer,

32. The method of embodiment 31 , wherein the first primer and the complementary oligonucleotide have the same sequence, or the second primer and the second adapter have the same sequence, or both. 33. The method of embodiment 31 or 32, wherein the first primer comprises a protecting group at the 3 ' end, and further comprising removing the protecting group from the first primer after attaching the 5' end of the target polynucleotide to the second adapter.

34. The method of embodiment 33, wherein the protecting group is a phosphate group.

35. The method of any one of embodiments 30 to 34, wherein amplifying the product polynucleotide is performed by polymerase chain reaction.

36. A method comprising:

providing a target polynucleotide comprising a 3' end and a 5' end;

attaching the 3' end of the target polynucleotide to a first adapter and attaching the 5' end of the target polynucleotide to a second adapter, thereby producing a template polynucleotide, wherein the first adapter and the second adapter are attached to a solid substrate; and

cleaving the first adapter to produce a cleaved template polynucleotide.

37. The method of embodiment 36, wherein cleaving the first adapter is performed using a restriction enzyme, a nicking enzyme, or an R ase.

38. The method of embodiment 36 or 37, further comprising, prior to cleaving the first adapter, providing a complementary oligonucleotide that is complementary to at least a portion of the first adapter,

wherein cleaving the first adapter is performed using a restriction endonuclease, and wherein the recognition site of the restriction endonuclease is formed by hybridization of the complementary oligonucleotide to the first adapter.

39. The method of embodiment 38, wherein the complementary oligonucleotide is not attached to the solid substrate.

40. The method of embodiment 36, wherein the first adapter comprises a photocleavable linkage, and wherein cleaving the first adapter is performed using light-induced cleavage.

41. The method of any one of embodiments 36 to 40, wherein the target polynucleotide is DNA.

42. The method of embodiment 41, wherein the second adapter comprises at the 3' end at least one R A nucleotide, and wherein attaching the 5' end of the target polynucleotide is performed using an RNA ligase.

43. The method of embodiment 41 or 42, wherein the 5' end of the target polynucleotide is pre-adenylated, and wherein attaching the 5' end of the target polynucleotide is performed using a truncated T4 RNA ligase. 44. The method of any one of embodiments 41 to 43, wherein attaching the 5' end of the target polynucleotide occurs before attaching the 3' end of the target polynucleotide.

45. The method of any one of embodiments 41 to 44, wherein attaching the 3' end of the target polynucleotide is performed using a T4 R A ligase.

46. The method of any one of embodiments 41 to 45, further comprising:

hybridizing the cleaved template polynucleotide to a first primer, wherein the first primer is at least partially complementary to a sequence toward or at the 3' end of the cleaved template polynucleotide, and wherein the first primer is attached to the solid substrate; and

extending the first primer using at least one nucleic acid polymerase to produce a product polynucleotide that is complementary to the template polynucleotide.

47. The method of embodiment 46, wherein the first primer is complementary to at least a portion, or all, of the sequence of the first adapter.

48. The method of embodiment 46 or 47, wherein the first primer comprises a protecting group at the 3 ' end, and further comprising removing the protecting group from the first primer after attaching the 3' end of the target polynucleotide to the first adapter.

49. The method of embodiment 48, wherein the protecting group is a phosphate group.

50. The method of any one of embodiments 46 to 49, wherein the at least one nucleic acid polymerase comprises a DNA polymerase.

51. The method of embodiment 50, wherein the at least one nucleic acid polymerase further comprises a reverse transcriptase.

52. The method of any one of embodiments 46 to 51 , further comprising amplifying the product polynucleotide.

53. The method of embodiment 52, wherein amplifying the product polynucleotide is performed using the first primer and a second primer, and wherein the second primer comprises at least a portion of the DNA sequence of the second adapter.

54. The method of embodiment 53, wherein the second primer and the second adapter have the same DNA sequence.

55. The method of embodiment 53 or 54, wherein the second primer comprises a protecting group at the 3 ' end, and further comprising removing the protecting group from the second primer after attaching the 3' end of the target polynucleotide to the first adapter.

56. The method of embodiment 55, wherein the protecting group is a phosphate group. 57. The method of any one of embodiments 52 to 56, wherein amplifying the product polynucleotide is performed by polymerase chain reaction.

58. The method of any one of embodiments 36 to 40, wherein the target polynucleotide is RNA.

59. The method of embodiment 58, wherein the 5' end of the first adapter is pre-adenylated, and wherein attaching the 3' end of the target polynucleotide is performed using a truncated T4 RNA ligase.

60. The method of embodiment 58 or 59, wherein attaching the 3' end of the target polynucleotide occurs before attaching the 5' end of the target polynucleotide.

61. The method of any one of embodiments 58 to 60, wherein attaching the 5' end of the target polynucleotide is performed using a T4 RNA ligase.

62. The method of any one of embodiments 58 to 61, further comprising:

63. The method of embodiment 62, wherein the first primer is complementary to at least a portion, or all, of the sequence of the first adapter.

64. The method of embodiment 62 or 63, wherein the first primer comprises a protecting group at the 3 ' end, and further comprising removing the protecting group from the first primer after attaching the 5' end of the target polynucleotide to the second adapter.

65. The method of embodiment 64, wherein the protecting group is a phosphate group.

66. The method of any one of embodiments 62 to 65, wherein the at least one nucleic acid polymerase comprises a reverse transcriptase.

67. The method of embodiment 66, wherein the at least one nucleic acid polymerase further comprises a DNA polymerase.

68. The method of any one of embodiments 62 to 67, further comprising amplifying the product polynucleotide. 69. The method of embodiment 68, wherein amplifying the product polynucleotide is performed using the first primer and a second primer, and wherein the second primer comprises at least a portion of the sequence of the second adapter.

70. The method of embodiment 69, wherein the second primer and the second adapter have the same sequence.

71. The method of any one of embodiments 68 to 70, wherein amplifying the product polynucleotide is performed by polymerase chain reaction.

72. The method of any one of the preceding embodiments, wherein the target polynucleotide is single-stranded.

73. The method of any one of the preceding embodiments, wherein providing a target polynucleotide comprises providing a plurality of different target polynucleotides.

74. The method of embodiment 73, wherein the plurality of different target polynucleotides comprises a library of different target polynucleotides isolated from a sample.

75. The method of embodiment 73 or 74, wherein the plurality of different target

polynucleotides is attached to the solid substrate at a density compatible with cluster formation for sequencing.

76. The method of any one of the preceding embodiments, wherein the first adapter is present on the solid substrate at a density substantially equal to, less than or greater than that of the second adapter.

77. The method of embodiment 76, wherein the first adapter is present on the solid substrate at a density less than that of the second adapter.

78. The method of any one of the preceding embodiments, wherein the second adapter is attached to the solid substrate using a crosslinking agent.

79. The method of embodiment 78, wherein the crosslinking agent is selected from the group consisting of EDC, succinic anhydride, maleic anhydride, MBS, SIAB, SMCC, GMBS, and SMPB.

80. The method of any one of the preceding embodiments, wherein the first adapter or the second adapter comprises a barcode sequence.

81. An article comprising a single-stranded template polynucleotide having a 3' end and a 5' end, wherein the 3' end and the 5' end of the template polynucleotide are attached to a solid substrate. 82. The article of embodiment 81 , wherein the 3 ' end or the 5 ' end of the template polynucleotide is not attached to the solid substrate through hybridization of the 3' end or the 5' end of the template polynucleotide to an oligonucleotide that is attached to the solid substrate.

83. The article of embodiment 81 or 82, wherein the template polynucleotide comprises a target polynucleotide having a 3' end and a 5' end,

wherein the 3' end of the target polynucleotide is attached to a first oligonucleotide that is attached to the solid substrate, and

wherein the 5' end of the target polynucleotide is attached to a second oligonucleotide that is attached to the solid substrate.

84. The article of embodiment 83, wherein the template polynucleotide comprises a target DNA polynucleotide or a target RNA polynucleotide.

85. The article of embodiment 83 or 84, wherein:

the template polynucleotide comprises a target DNA polynucleotide;

the first oligonucleotide attached to the 3' end of the target DNA polynucleotide is a DNA oligonucleotide; and

the second oligonucleotide attached to the 5' end of the target DNA polynucleotide is an RNA oligonucleotide or an RNA/DNA oligonucleotide that has one or more RNA nucleotides at the 3 ' end.

86. The article of embodiment 84 or 85, wherein the target DNA polynucleotide is adenylated at the 5 ' end.

87. The article of embodiment 83 or 84, wherein:

the template polynucleotide comprises a target RNA polynucleotide;

the first oligonucleotide attached to the 3' end of the target RNA polynucleotide is a DNA oligonucleotide; and

the second oligonucleotide attached to the 5' end of the target RNA polynucleotide is a DNA oligonucleotide.

88. The article of embodiment 84 or 87, wherein the 5' end of the first oligonucleotide that becomes attached to the 3' end of the target RNA polynucleotide is adenylated.

89. The article of embodiment 83, 84, 87 or 88, wherein:

the template polynucleotide comprises a target RNA polynucleotide;

the article further comprises molecules of the second oligonucleotide that is attached to the 5' end of the target RNA polynucleotide; and the molecules of the second oligonucleotide are attached to the solid substrate and are primers for amplifying a product polynucleotide that is complementary to the template polynucleotide.

90. The article of any one of embodiments 81 to 89, wherein the template polynucleotide comprises R A nucleotides or DNA nucleotides, or both.

91. The article of embodiment 90, wherein the template polynucleotide comprises RNA nucleotides and DNA nucleotides.

92. The article of any one of embodiments 81 to 91, further comprising a third

oligonucleotide comprising a sequence complementary to a sequence toward or at the 3' end of the template polynucleotide, wherein the third oligonucleotide is attached to the solid substrate.

93. The article of embodiment 92, wherein the third oligonucleotide is hybridized to the sequence toward or at the 3' end of the template polynucleotide.

94. The article of embodiment 92, wherein the third oligonucleotide is not hybridized to the sequence toward or at the 3' end of the template polynucleotide.

95. The article of any one of embodiments 92 to 94, wherein the third oligonucleotide is a primer for producing a product polynucleotide that is complementary to the template

polynucleotide.

96. The article of embodiment 95, further comprising molecules of the third oligonucleotide which are attached to the solid substrate, wherein the molecules of the third oligonucleotide are primers for amplifying the product polynucleotide.

97. The article of any one of embodiments 81 to 96, further comprising molecules of a fourth oligonucleotide which are attached to the solid substrate, wherein the molecules of the fourth oligonucleotide are primers for amplifying a product polynucleotide that is complementary to the template polynucleotide.

98. The article of any one of embodiments 81 to 97, further comprising a product polynucleotide that is complementary to the template polynucleotide.

99. The article of embodiment 98, wherein the product polynucleotide is produced according to the method of any one of embodiments 1 to 80.

100. The article of embodiment 98 or 99, further comprising amplification products of the product polynucleotide. 101. The article of embodiment 100, wherein the amplification products of the product polynucleotide are produced by amplifying the product polynucleotide according to the method of any one of embodiments 1 to 80.

102. The article of any one of embodiments 81 to 101, wherein the template polynucleotide is a plurality of different template polynucleotides comprising a plurality of different target polynucleotides.

103. An article comprising a target DNA polynucleotide bound at the 5' end to a first oligonucleotide that is attached to a solid substrate, wherein:

the target DNA polynucleotide is adenylated at the 5' end; and

the first oligonucleotide is an RNA oligonucleotide or an RNA/DNA oligonucleotide comprising one or more RNA nucleotides at the 3 ' end.

104. The article of embodiment 103, wherein the target DNA polynucleotide further is bound at the 3' end to a second oligonucleotide that is attached to the solid substrate, thereby forming a template polynucleotide,

wherein the second oligonucleotide is a DNA oligonucleotide, an RNA oligonucleotide, or an RNA/DNA oligonucleotide comprising one or more RNA nucleotides at the 5' end.

105. The article of embodiment 103, wherein the target DNA polynucleotide further is bound at the 3' end to a second oligonucleotide that is not attached to the solid substrate, thereby forming a template polynucleotide,

wherein the second oligonucleotide is a DNA oligonucleotide, an RNA oligonucleotide, or an RNA DNA oligonucleotide comprising one or more RNA nucleotides at the 5' end.

106. The article of embodiment 105, wherein at least a portion toward or at the 3' end of the template polynucleotide is hybridized to at least a portion of a third oligonucleotide that is attached to the solid substrate.

107. The article of any one of embodiments 104 to 106, further comprising molecules of a third oligonucleotide attached to the solid substrate and molecules of a fourth oligonucleotide attached to the solid substrate, wherein the molecules of the third oligonucleotide and the molecules of the fourth oligonucleotide are primers for amplifying a product polynucleotide that is complementary to the template polynucleotide.

108. The article of any one of embodiments 104 to 107, further comprising a product polynucleotide that is complementary to the template polynucleotide. 109. The article of embodiment 108, further comprising amplification products of the product polynucleotide.

110. The article of any one of embodiments 103 to 109, wherein the target DNA

polynucleotide is a plurality of different target DNA polynucleotides.

111. An article comprising a target DNA polynucleotide bound at the 3 ' end to a first oligonucleotide that is attached to a solid substrate, wherein:

the target DNA polynucleotide is adenylated at the 5' end; and

the first oligonucleotide is a DNA oligonucleotide, an RNA oligonucleotide, or an RNA/DNA oligonucleotide comprising one or more RNA nucleotides at the 5' end.

112. The article of embodiment 111, wherein the target DNA polynucleotide further is bound at the 5' end to a second oligonucleotide that is attached to the solid substrate, thereby forming a template polynucleotide,

wherein the second oligonucleotide is an RNA oligonucleotide or an RNA/DNA oligonucleotide comprising one or more RNA nucleotides at the 3' end.

113. The article of embodiment 112, further comprising molecules of a third oligonucleotide attached to the solid substrate and molecules of a fourth oligonucleotide attached to the solid substrate, wherein the molecules of the third oligonucleotide and the molecules of the fourth oligonucleotide are primers for amplifying a product polynucleotide that is complementary to the template polynucleotide.

114. The article of embodiment 112 or 113, further comprising a product polynucleotide that is complementary to the template polynucleotide.

115. The article of embodiment 114, further comprising amplification products of the product polynucleotide.

116. The article of any one of embodiments 111 to 115, wherein the target DNA

polynucleotide is a plurality of different target DNA polynucleotides.

117. An article comprising a target DNA polynucleotide bound at the 3' end to a first oligonucleotide that is not attached to a solid substrate, wherein:

the target DNA polynucleotide is adenylated at the 5' end;

the first oligonucleotide is a DNA oligonucleotide, an RNA oligonucleotide, or an RNA DNA oligonucleotide comprising one or more RNA nucleotides at the 5' end; and

at least a portion of the first oligonucleotide bound to the target DNA polynucleotide is hybridized to at least a portion of a second oligonucleotide that is attached to a solid substrate. 118. The article of embodiment 117, wherein the target DNA polynucleotide further is bound at the 5' end to a third oligonucleotide that is attached to the solid substrate, thereby forming a template polynucleotide that has a portion toward or at the 3' end hybridized to the second oligonucleotide,

wherein the third oligonucleotide is an R A oligonucleotide or an R A/DNA

oligonucleotide comprising one or more RNA nucleotides at the 3' end.

119. The article of embodiment 118, further comprising molecules of the second

oligonucleotide attached to the solid substrate and molecules of a fourth oligonucleotide attached to the solid substrate, wherein the molecules of the second oligonucleotide and the molecules of the fourth oligonucleotide are primers for amplifying a product polynucleotide that is

complementary to the template polynucleotide.

120. The article of embodiment 118 or 119, further comprising a product polynucleotide that is complementary to the template polynucleotide.

121. The article of embodiment 120, further comprising amplification products of the product polynucleotide.

122. The article of any one of embodiments 117 to 121, wherein the target DNA

polynucleotide is a plurality of different target DNA polynucleotides.

123. An article comprising a target RNA polynucleotide bound at the 3' end to a first oligonucleotide that is attached to a solid substrate, wherein the first oligonucleotide comprises one or more DNA nucleotides at the 5 ' end and is adenylated at the 5 ' end.

124. The article of embodiment 123, wherein the target RNA polynucleotide further is bound at the 5' end to a second oligonucleotide that is attached to the solid substrate, thereby forming a template polynucleotide.

125. The article of embodiment 124, further comprising molecules of the second

oligonucleotide attached to the solid substrate and molecules of a third oligonucleotide attached to the solid substrate, wherein the molecules of the second oligonucleotide and the molecules of the third oligonucleotide are primers for amplifying a product polynucleotide that is

complementary to the template polynucleotide.

126. The article of embodiment 124 or 125, further comprising a product polynucleotide that is complementary to the template polynucleotide.

127. The article of embodiment 126, further comprising amplification products of the product polynucleotide. 128. The article of any one of embodiments 123 to 127, wherein the target R A polynucleotide is a plurality of different target RNA polynucleotides.

129. An article comprising a target RNA polynucleotide bound at the 3' end to a first oligonucleotide that is not attached to a solid substrate, wherein:

the first oligonucleotide comprises one or more DNA nucleotides at the 5' end and is adenylated at the 5 ' end; and

at least a portion of the first oligonucleotide bound to the target RNA polynucleotide is hybridized to at least a portion of a second oligonucleotide that is attached to a solid substrate.

130. The article of embodiment 129, wherein the target RNA polynucleotide further is bound at the 5' end to a third oligonucleotide that is attached to the solid substrate, thereby forming a template polynucleotide that has a portion toward or at the 3' end hybridized to the second oligonucleotide.

131. The article of embodiment 130, further comprising molecules of the second

oligonucleotide attached to the solid substrate and molecules of the third oligonucleotide attached to the solid substrate, wherein the molecules of the second oligonucleotide and the molecules of the third oligonucleotide are primers for amplifying a product polynucleotide that is

complementary to the template polynucleotide.

132. The article of embodiment 130 or 131, further comprising a product polynucleotide that is complementary to the template polynucleotide.

133. The article of embodiment 132, further comprising amplification products of the product polynucleotide.

134. The article of any one of embodiments 129 to 133, wherein the target RNA

polynucleotide is a plurality of different target RNA polynucleotides.

135. A metho d comprising :

providing a target double-stranded DNA (dsDNA);

contacting the target dsDNA with a transposase complex comprising a transposase enzyme and transposon sequences to form dsDNA fragments, wherein the 5 ' end of both strands of the dsDNA fragments is attached to a transposon sequence, and wherein the transposon sequence is appended with an adapter sequence;

denaturing the dsDNA fragments to form transposon-containing single-stranded DNA (ssDNA) fragments; and hybridizing the adapter sequence of the transposon-containing ssDNA fragments to primers attached to a solid substrate.

136. The method of embodiment 135, wherein the transposon-containing ssDNA fragments are not attached to the solid substrate prior to hybridization of the adapter sequence of the transposon-containing ssDNA fragments to primers attached to the solid substrate.

137. The method of embodiment 135 or 136, further comprising, prior to denaturing the dsDNA fragments, performing PCR to add an adapter sequence to the 5' end of both strands of the transposon-containing dsDNA fragments.

138. The method of any one of embodiments 135 to 137, wherein the adapter sequence comprises a barcode sequence.

139. The method of any one of embodiments 135 to 138, wherein the transposon sequence further is appended with a sequencing primer site between the transposon sequence and the adapter sequence.

140. The method of any one of embodiments 135 to 139, wherein the primers are

complementary to at least a portion, or the whole portion, of the adapter sequence of the transposon-containing ssDNA fragments.

141. The method of any one of embodiments 135 to 140, wherein the primers are attached to a cleavable linker that is attached to the solid substrate.

142. The method of any one of embodiments 135 to 141, wherein the number of one or more different primers is equal to the number of one or more different adapter sequences.

143. The method of embodiment 142, wherein the number of different adapter sequences is two, and the number of different primers attached to the solid substrate is two.

144. The method of any one of embodiments 135 to 143, further comprising performing PCR to produce amplification products of the transposon-containing ssDNA fragments.

145. The method of embodiment 144, further comprising releasing the amplification products from the surface of the solid substrate.

146. The method of embodiment 145, wherein releasing the amplification products comprises introducing a cleaving reagent that cleaves the amplification products.

147. The method of embodiment 146, wherein the cleaving reagent is light or a chemical or biochemical cleaving reagent that cleaves the cleavable linker of embodiment 141. 148. The method of any one of embodiments 145 to 147, further comprising sequencing the released amplification products.

149. The method of embodiment 148, wherein sequencing the released amplification products comprises using a sequencing primer that is complementary to at least a portion, or the whole portion, of the transposon sequence or the complementary sequence thereof.

150. The method of embodiment 149, wherein the sequencing primer further is complementary to at least a portion, or the whole portion, of the sequencing primer site of embodiment 139 or the complementary sequence thereof.

151. The method of any one of embodiments 135 to 150, wherein the target double-stranded DNA is genomic DNA.

152. An article comprising a plurality of different single-stranded DNA (ssDNA) fragments hybridized to primers that are attached to a solid substrate,

wherein the 5' end of each ssDNA fragment is attached to a transposon sequence appended with an adapter sequence, and

wherein the adapter sequence of a transposon-containing ssDNA fragment is hybridized to a primer that is attached to the solid substrate.

153. The article of embodiment 152, wherein the transposon-containing ssDNA fragments are not attached at the 3' end to the solid substrate.

154. The article of embodiment 152 or 153, wherein the adapter sequence comprises a barcode sequence.

155. The article of any one of embodiments 152 to 154, wherein the transposon sequence further is appended with a sequencing primer site between the transposon sequence and the adapter sequence.

156. The article of any one of embodiments 152 to 155, wherein the primers are

157. The article of any one of embodiments 152 to 156, wherein the primers are attached to a cleavable linker that is attached to the solid substrate.

158. The article of any one of embodiments 152 to 157, wherein the number of one or more different primers is equal to the number of one or more different adapter sequences. 159. The article of embodiment 158, wherein the number of different adapter sequences is two, and the number of different primers attached to the solid substrate is two.

160. The article of any one of embodiments 152 to 159, further comprising amplification products of the transposon-containing ssDNA fragments.

161. The article of embodiment 160, wherein the amplification products are produced by amplifying the transposon-containing ssDNA fragments according to the method of any one of embodiments 135 to 151.

162. The article of any one of embodiments 152 to 161, wherein the plurality of different transposon-containing ssDNA fragments is produced by:

contacting a target double-stranded DNA (dsDNA) with a transposase complex comprising a transposase enzyme and transposon sequences to form dsDNA fragments, wherein the 5' end of both strands of the dsDNA fragments is attached to a transposon sequence, and wherein the transposon sequence optionally is already appended with an adapter sequence;

optionally performing PCR to add an adapter sequence to the 5' end of both strands of the transposon-containing dsDNA fragments; and

denaturing the dsDNA fragments to form a plurality of different transposon-containing ssDNA fragments.

163. The article of embodiment 162, wherein the target dsDNA is genomic DNA.

164. A metho d comprising :

providing a solid substrate attached to a plurality of different primers, wherein the plurality of different primers comprises a plurality of different pairs of forward and reverse primers for amplifying a plurality of target genetic loci; and

hybridizing to the plurality of different primers attached to the solid substrate a plurality of single-stranded DNA fragments complementary to at least a portion of the sequences of the plurality of different primers.

165. The method of embodiment 164, wherein the plurality of different primers is attached to the solid substrate via a cleavable linker.

166. The method of embodiment 164 or 165, wherein the plurality of different primers is attached to the solid substrate at the 5' end.

167. The method of any one of embodiments 164 to 166, wherein the plurality of different primers is labeled with a fluorescent dye. 168. The method of embodiment 167, wherein each of the plurality of different primers is labeled with a fluorescent dye, and wherein the plurality of different primers is labeled in total with a plurality of different fluorescent dyes.

169. The method of embodiment 168, wherein the plurality of different primers is labeled in total with at least four different fluorescent dyes.

170. The method of any one of embodiments 167 to 169, wherein the plurality of different primers is labeled with a fluorescent dye at the 5 ' end.

171. The method of any one of embodiments 164 to 170, wherein the plurality of target genetic loci comprises a plurality of short tandem repeat (STR) loci.

172. The method of embodiment 171, wherein the plurality of STR loci comprises a plurality of STR loci used in a forensic database.

173. The method of embodiment 172, wherein the plurality of STR loci comprises a plurality of CODIS STR loci selected from the group consisting of CSFIPO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX and vWA.

174. The method of embodiment 173, wherein the plurality of STR loci comprises at least 5 CODIS STR loci.

175. The method of any one of embodiments 172 to 174, wherein the plurality of STR loci comprises all CODIS STR loci.

176. The method of any one of embodiments 172 to 175, wherein the plurality of STR loci further comprises Penta D or Penta E, or both.

177. The method of any one of embodiments 171 to 176, wherein the plurality of target genetic loci further comprises amelogenin.

178. The method of any one of embodiments 164 to 177, wherein the plurality of single- stranded DNA fragments is prepared from genomic DNA obtained from a sample.

179. The method of any one of embodiments 164 to 178, further comprising performing PCR to produce amplification products of the plurality of target genetic loci.

180. The method of embodiment 179, wherein PCR is performed at a substantially constant temperature.

181. The method of embodiment 179 or 180, wherein PCR is performed at a temperature in the range from about 50 °C to about 75 °C. 182. The method of any one of embodiments 179 to 181, wherein PCR is performed using a chemical denaturant.

183. The method of any one of embodiments 179 to 182, further comprising releasing the amplification products from the surface of the solid substrate.

184. The method of embodiment 183, wherein releasing the amplification products comprises introducing a cleaving reagent that cleaves the amplification products.

185. The method of embodiment 184, wherein the cleaving reagent is light or a chemical or biochemical cleaving reagent that cleaves the cleavable linker of embodiment 165.

186. The method of any one of embodiments 183 to 185, further comprising separating the released amplification products by electrophoresis.

187. The method of any one of embodiments 183 to 186, further comprising sequencing the released amplification products.

188. The method of embodiment 186 or 187, further comprising creating a computer file identifying amplification products of the plurality of target genetic loci after separation, or a computer file identifying the sequences of amplification products of the plurality of target genetic loci after sequencing.

189. An article comprising a plurality of different primers attached to a solid substrate, wherein the plurality of different primers:

comprises a plurality of different pairs of forward and reverse primers for amplifying a plurality of target genetic loci; and

is hybridized to a plurality of single-stranded DNA fragments complementary to at least a portion of the sequences of the plurality of different primers.

190. The article of embodiment 189, wherein the plurality of different primers is attached to the solid substrate via a cleavable linker.

191. The article of embodiment 189 or 190, wherein the plurality of different primers is attached to the solid substrate at the 5' end.

192. The article of any one of embodiments 189 to 191, wherein the plurality of different primers is labeled with a fluorescent dye.

193. The article of embodiment 192, wherein each of the plurality of different primers is labeled with a fluorescent dye, and wherein the plurality of different primers is labeled in total with a plurality of different fluorescent dyes. 194. The article of embodiment 192 or 193, wherein the plurality of different primers is labeled in total with at least four different fluorescent dyes.

195. The article of any one of embodiments 192 to 194, wherein the plurality of different primers is labeled with a fluorescent dye at the 5 ' end.

196. The article of any one of embodiments 189 to 195, wherein the plurality of target genetic loci comprises a plurality of short tandem repeat (STR) loci.

197. The article of embodiment 196, wherein the plurality of STR loci comprises a plurality of STR loci used in a forensic database.

198. The article of embodiment 197, wherein the plurality of STR loci comprises a plurality of CODIS STR loci selected from the group consisting of CSFIPO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX and vWA.

199. The article of embodiment 197 to 198, wherein the plurality of STR loci comprises all CODIS STR loci.

200. The article of any one of embodiments 197 to 199, wherein the plurality of STR loci further comprises Penta D or Penta E, or both.

201. The article of any one of embodiments 196 to 200, wherein the plurality of target genetic loci further comprises amelogenin.

202. The article of any one of embodiments 189 to 201, wherein the plurality of single- stranded DNA fragments is prepared from genomic DNA obtained from a sample.

203. The article of any one of embodiments 189 to 202, further comprising amplification products of the plurality of target genetic loci.

204. The article of embodiment 203, wherein the amplification products are produced by amplifying the plurality of target genetic loci according to the method of any one of

embodiments 164 to 188.

205. The method or article of any one of the preceding embodiments, wherein the solid substrate is a particle, a bead, a slide, a surface of an element of a device, a membrane, a flow cell, a well, a chamber, a macro fluidic chamber, a micro fluidic chamber, a channel, or a micro fluidic channel.

206. The method or article of embodiment 205, wherein the solid substrate is a flow cell.

EXAMPLES Example 1. RNA Sequencing Template Preparation on Flow Cell with Initial Extension

[0084] A solid substrate (e.g., a flow cell) containing three different DNA oligonucleotides is prepared. The first oligonucleotide will be the 3' adapter. It can be partially double-stranded with an adenylated 5' overhang on one end, and is attached to the surface of the flow cell via the double-stranded end (Fig. 3A). Alternatively, the first oligonucleotide can be a single-stranded DNA oligonucleotide to which a shorter, complementary single-stranded DNA oligonucleotide hybridizes after ligation of the 3' end or the 5' end of the target RNA. The second

oligonucleotide, the 5' adapter, is single-stranded, attached to the flow cell surface at the 5' end, and contains a hydro xyl group at the free 3 ' end. The third oligonucleotide, which will serve as an additional primer for amplification, is also single-stranded and attached to the flow cell surface at the 5' end, but is initially blocked on the 3' end by a suitable blocking/protecting group, such as a phosphate group. The third oligonucleotide can comprise at least a portion of the sequence of the shorter strand of the 3' adapter, or can have the same sequence as the shorter strand of the 3' adapter.

[0085] Truncated T4 RNA ligase is used to ligate the 3 ' end of the target RNA to the adenylated 5' overhang of the first oligonucleotide (Fig. 3 A). The ligase and remaining buffer are rinsed out of the flow cell, and non-truncated T4 RNA ligase and ATP are added to ligate the 5' end of the target RNA to the free 3' end of the second oligonucleotide. After washing, reverse transcriptase and DNA polymerase are added to extend the shorter strand of the first (3') adapter to create a complementary DNA strand, attached via the first adapter to the surface of the flow cell. The longer strand of the first adapter can optionally be nicked to result in a RNA/DNA hybrid strand containing the target RNA, attached at its 5' end to the flow cell via the second (5') adapter (Fig. 3B). The blocking phosphate on the third oligonucleotide can then be removed using a phosphatase, and bridge PCR is performed using the second and third oligonucleotides as primers, where the bridge PCR comprises alternating cycles of extension of the second and third oligonucleotides, melting of strands, and hybridization of the 3' end of the strands to other molecules of the second and third oligonucleotides (Fig. 3C). The phosphate blocking group on the third oligonucleotide can also be removed earlier in the sequence of steps after the 5' end of the target RNA is ligated to the 5 ' adapter on the flow cell.

[0086] Fig. 7A shows alternative embodiments for RNA sequencing template preparation on a solid substrate (e.g., a flow cell) using an extension- first approach, in which the 3' end of the target RNA is ligated in solution to a single-stranded DNA oligonucleotide that is not attached to the flow cell. In the embodiments of Fig. 7A, the 3' hydro xyl group of Probe 2 is blocked with a suitable blocking/protecting group, such as a phosphate group. The 3' end of the target RNA can be ligated in solution to a 5'-adenylated, single-stranded DNA oligonucleotide that is not attached to the flow cell, the 3' end of the resulting RNA/DNA strand can hybridize to Probe 2 attached to the flow cell, and then the flow cell can be washed before the 5' end of the

RNA DNA strand is ligated to Probe 1 attached to the flow cell and the flow cell is washed again. As a further description, the 3 ' adapter can be single-stranded and unattached to the flow cell. Ligation of the first adapter to the target RNA can then occur in solution, followed by ligation of the target RNA to the second adapter (Probe 1) on the flow cell surface. This will result in a single-stranded RNA/DNA template, the 3' end of which can hybridize to the third oligonucleotide (Probe 2). After phosphatase-dependent removal of the blocking phosphate group, the third oligonucleotide can be extended using reverse transcriptase and DNA

polymerase to form a complementary DNA strand. After extension, the RNA template can optionally be removed entirely, for example, by using an RNase. Subsequent amplification can then be performed using standard bridge PCR, also using the second oligonucleotide (Probe 1) and the third oligonucleotide (Probe 2) as primers, where the bridge PCR comprises alternating cycles of extension of the second and third oligonucleotides, melting of strands, and

hybridization of the 3' end of the strands to other molecules of the second and third

oligonucleotides.

Example 2. RNA Sequencing Template Preparation on Flow Cell with Initial Cleavage

[0087] A solid substrate (e.g., a flow cell) containing three single-stranded DNA

oligonucleotides is prepared. The first oligonucleotide, the 3' adapter, is attached to the flow cell surface via the 3' end, and is adenylated at the 5' end (Fig. 4A). The second oligonucleotide, the 5 ' adapter, is attached to the flow cell surface via its 5 ' end, with a hydro xyl group on the free 3 ' end. The third oligonucleotide, which will serve as an additional primer for amplification, is also single-stranded and attached to the flow cell surface at the 5' end, but is initially blocked at the 3' end by a suitable blocking/protecting group, such as a phosphate group.

[0088] Truncated T4 RNA ligase is used to ligate the 3 ' end of the target RNA to the adenylated 5' end of the 3' adapter (Fig. 4A). After washing, ATP and T4 RNA ligase are added as described in Example 1 to ligate the 5' end of the target RNA to the 5' adapter. Both the 5' and 3' ends of the resulting RNA/DNA hybrid are attached to the surface of the flow cell. The 3' adapter can then be cleaved, for example, by introducing a short complementary oligonucleotide that forms a double-stranded recognition sequence for a restriction enzyme (which cuts both strands) or a nicking enzyme (which cuts one strand). The complementary oligonucleotide can then be washed away, and after cleavage or nicking the free 3' DNA end of the R A/DNA hybrid template is able to hybridize to the third oligonucleotide (Probe 2 in Fig. 4B).

[0089] After phosphatase-dependent removal of the blocking phosphate group on the third oligonucleotide, the third oligonucleotide can be extended using reverse transcriptase and DNA polymerase (to replicate any DNA section at the 3' end of the template polynucleotide) to form a complementary DNA strand (Fig. 4B). After extension, the RNA template can optionally be removed entirely, for example, by using an RNase. Subsequent amplification can then be performed using standard bridge PCR, also using the second oligonucleotide (Probe 1) and the third oligonucleotide (Probe 2) as primers, where the bridge PCR comprises alternating cycles of extension of the second and third oligonucleotides, melting of strands, and hybridization of the 3' end of the strands to other molecules of the second and third oligonucleotides (Fig. 4B). The phosphate blocking group on the third oligonucleotide can also be removed earlier in the sequence of steps after the 5' end of the target RNA is ligated to the 5' adapter on the flow cell.

Example 3. DNA Sequencing Template Preparation on Flow Cell

[0090] Amplifying from a DNA molecule instead of an RNA molecule can be performed similarly to the methods described in the foregoing examples. However, there are some differences. To attach a DNA molecule to a solid substrate (e.g., a flow cell), the 3' adapter is not adenylated. Instead, the 5' end of the target DNA strand can be adenylated, and the 5' adapter can be an RNA oligonucleotide or a DNA/RNA hybrid strand with one or more RNA nucleotides on the free 3' end (Figs. 5 A and 6A). The ligation step using truncated T4 RNA ligase then occurs between the 5' adapter and the 5 '-adenylated target DNA. Ligation of the 3' end of the resulting DNA RNA hybrid strand to the 3' adapter can then be performed using ATP and an RNA ligase (e.g., a T4 RNA ligase). To improve the efficiency of the ligation of the 3' end of the DNA/RNA hybrid strand to the 3' adapter using an RNA ligase, the 3' adapter can be an RNA oligonucleotide or an RNA/DNA oligonucleotide having one or more RNA nucleotides at the 5' end. As an alternative to the order of ligation, the 3' end of the target DNA can be ligated to the 3' adapter prior to ligation of the 5' end of the target DNA to the 5' adapter.

Subsequent steps, including extension and/or cleavage, can be performed similarly as described above and in Figures 5 and 6. Fig. 5 illustrates embodiments of DNA sequencing template preparation on a solid substrate (e.g., a flow cell) using an extension-first approach, and Fig. 6 illustrates embodiments of DNA sequencing template preparation on a solid substrate (e.g., a flow cell) using a cleavage-first approach. In Figures 5 A and 6A, the 3' adapter can be an RNA oligonucleotide or an RNA/DNA oligonucleotide comprising one or more RNA nucleotides at the 5' end to improve ligation of the 3' adapter with the target DNA using an RNA ligase. In Fig. 6A, any potential hybridization between the 3' adapter and Primer 1 can be minimized or avoided by, e.g., controlling the density of the 3' adapter and Primer 1 on the surface of the flow cell.

[0091] Fig. 7B shows alternative embodiments for DNA sequencing template preparation on a solid substrate (e.g., a flow cell) using an extension- first approach, in which the 3' end of the target DNA is ligated in solution to a single-stranded (ss) oligonucleotide (the 3' adapter) that is not attached to the flow cell. In the embodiments of Fig. 7B, the 3' hydro xyl groups of Probe 1 and Probe 2 are blocked with a suitable blocking/protecting group, such as a phosphate group. The adenylated 5' end of the target DNA can be ligated to a flow cell-attached ssRNA oligonucleotide or ssRNA/DNA oligonucleotide having one or more RNA nucleotides at the 3' end (the 5' adapter) and the flow cell can be washed before the 3' end of the resulting DNA/RNA strand is ligated in solution to a flow cell-unattached ssDNA oligonucleotide, ssRNA

oligonucleotide or ssDNA RNA oligonucleotide having one or more RNA nucleotides at the 5' end, and the 3' end of the resulting DNA RNA template hybridizes to Probe 1 attached to the flow cell and the flow cell is washed again (alternatively, the flow cell can be washed after ligation to the 3' adapter and before hybridization to Probe 1). Alternatively, the 3' end of the target DNA can be ligated to the 3' adapter unattached to the flow cell prior to ligation of the 5' end of the target DNA to the 5' adapter attached to the flow cell. The 3' adapter can comprise one or more RNA nucleotides at the 5' end to improve the efficiency of ligation to the 3' end of the target DNA using an RNA ligase. After removal of the phosphate blocking group on Probe 1 and Probe 2, amplification by bridge PCR is performed using Probe 1 and Probe 2 as primers, where the bridge PCR comprises alternating cycles of extension of Probe 1 and Probe 2, melting of strands, and hybridization of the 3' end of the strands to other molecules of Probe 1 and Probe 2.

Example 4. Transposon-Based DNA Sequencing Template Preparation on Flow Cell

[0092] A target double-stranded DNA (e.g., genomic DNA from a sample) is mixed with a transposase complex comprising a transposase enzyme and transposon sequences. As an example for illustration, the transposase complex can be a Transposome™ complex (Epicentre Biotechnologies, Madison, Wisconsin) comprising a transposase enzyme and appended transposon ends. The Transposome™ complex cleaves the target dsDNA into a plurality of dsDNA fragments having staggered cuts at the ends and attaches to the 5' end of both strands of the dsDNA fragments a transposon end oligonucleotide appended with a sequencing primer site. The transposon end oligonucleotide can also be appended with an adapter sequence. If the transposon end is not already appended with an adapter, PCR (e.g. suppression, or limited-cycle, PCR with a, e.g., four-primer reaction) can be performed to add an adapter sequence to the sequencing primer site at the 5' end of the strands of the dsDNA fragments. An adapter can comprise a barcode sequence. The transposon-containing dsDNA fragments are denatured (e.g., by heating at elevated temperature and/or by using a chemical denaturant, such as formamide or urea) to produce single-stranded transposon-containing fragments.

[0093] The single-stranded transposon-containing fragments are attached to a solid substrate, such as a flow cell. For example, the adapter sequence at the 5' end of the transposon-containing fragments can hybridize to a primer oligonucleotide attached to the flow cell. The primer is complementary to at least a portion, or all, of the sequence of the adapter. The number of (one or more) different primers attached to the flow cell matches the number of (one or more) different adapters attached to the 5' end of the transposon-containing fragments. The one or more different primers can be attached to the flow cell, e.g., via a cleavable linker that is attached to the flow cell. The linker can be cleaved in the presence of, e.g., a certain wavelength of light, a chemical cleaving reagent (e.g., ammonia) or a biochemical cleaving reagent.

[0094] After the adapter at the 5' end of a transposon-containing fragment is hybridized to a primer attached to the flow cell, PCR is performed to amplify the fragment. PCR involves alternating cycles of denaturation (or melting) of the transposon-containing fragments from the complementary strands attached to the flow cell, annealing (or hybridization) of the adapter at the 5' end of the transposon-containing fragments to other molecules of the one or more different primers attached to the flow cell, and extension (or elongation) of the primers to form

complementary strands of the transposon-containing fragments. PCR can be performed at two or more different temperatures or two or more different ranges of temperatures (e.g., denaturation at about 90-100 °C, annealing at about 50-65 °C and extension at about 65-80 °C). Alternatively, isothermal PCR can be performed at a substantially constant temperature (e.g., at a temperature in the range of about 50-75 °C or about 50-65 °C) using, e.g., a chemical denaturant (e.g., formamide or urea).

[0095] After completion of PCR amplification, a reagent for cleaving the amplified fragments and releasing them from the surface of the flow cell is introduced to the flow cell. For example, the cleaving reagent can be a certain wavelength of light or a chemical (e.g., ammonia) or biochemical cleaving reagent that cleaves a linker attaching the primers to the flow cell. The released amplified fragments of the plurality of fragments resulting from transposase-induced fragmentation of the target dsDNA can be sequenced using sequencing primers that bind to at least a portion, or the whole portion, of the transposon sequence (or the complementary sequence thereof) and to at least a portion, or the whole portion, of the sequencing primer site (or the complementary sequence thereof) attached to the 5' end of the fragments.

Example 5. PCR on Flow Cell for Chip-Integrated Genetic Analysis

[0096] A plurality of different primers for amplifying a plurality of different target genetic loci is attached at their 5' end or 3' end to a solid substrate, such as a flow cell. The primers can be attached to the flow cell, e.g., via a cleavable linker that is attached to the flow cell. The linker can be cleaved in the presence of, e.g., a certain wavelength of light, a chemical cleaving reagent (e.g., ammonia) or a biochemical cleaving reagent. The primers can be labeled at the 5' or 3' end with a dye (e.g., a fluorescent dye). The primers can be labeled in total with a plurality of different dyes (e.g., with at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different dyes) to improve the identification of amplification products of the target genetic loci after their separation and detection, and/or to improve the identification of the sequences of amplification products of the target genetic loci after their sequencing. The plurality of different primers comprises a plurality of different pairs of forward and reverse primers for amplifying the plurality of different target genetic loci. Each different pair of forward and reverse primers can be dye-labeled at the 5' end or the 3' end of the primers with a different dye (e.g., a different fluorescent dye). The primers can be immobilized on the flow cell in an ordered array - e.g., in a checkerboard fashion, one primer pair for amplifying a different target genetic locus per square, and the number of squares based on the number of target genetic loci. The plurality of different target genetic loci can be, e.g., short tandem repeat (STR) loci used in a forensic database, such as CODIS. The STR loci presently used in CODIS include CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX and vWA. The target genetic loci can also include other STR loci useful for human identification, such as Penta D and Penta E, and a locus useful for sex determination, such as amelogenin (AMEL).

[0097] Fragments of single-stranded DNA (ssDNA), which are prepared from DNA (e.g., genomic DNA) obtained from a sample, are delivered to the flow cell. Single-stranded DNA fragments having sequences complementary to at least a portion, or all, of the sequences of the plurality of different primers immobilized on the flow cell are allowed to hybridize to the primers. The ssDNA fragments hybridized to the primers are not bound to the flow cell at the other end of the fragments. DNA can be extracted from the sample (e.g., by lysing cells in the sample), isolated (e.g., by capturing DNA on capture particles, such as magnetic particles), optionally purified (e.g., by washing captured DNA), and fragmented (e.g., by shearing DNA mechanically or using a chemical or biochemical reagent) using an instrument comprising the flow cell, or the single-stranded DNA fragments can be prepared off such an instrument.

[0098] PCR is performed to amplify the sample ssDNA fragments hybridized to the plurality of different primers immobilized on the flow cell. PCR involves alternating cycles of denaturation (or melting) of the sample ssDNA fragments from the complementary strands attached to the flow cell, annealing (or hybridization) of the sample ssDNA fragments to other molecules of the plurality of different primers immobilized on the flow cell, and extension (or elongation) of the primers to form complementary strands of the sample ssDNA fragments, thereby producing amplification products (also called amplicons) of each of the plurality of different target genetic loci. PCR can be performed at two or more different temperatures or two or more different ranges of temperatures (e.g., denaturation at about 90-100 °C, annealing at about 50-65 °C and extension at about 65-80 °C). Alternatively, isothermal PCR can be performed at a substantially constant temperature using, e.g., a chemical denaturant (e.g., formamide or urea). Isothermal PCR can be performed at a more moderate temperature (e.g., at a temperature in the range of about 50-75 °C or about 50-65 °C) so that a chip comprising the PCR reaction chamber (e.g., the flow cell containing the immobilized primers), or a chip integrated with or in close proximity with a device (e.g., a sample cartridge) comprising the PCR reaction chamber, does not need to be stable at a high temperature (e.g., about 100 °C). The chip can comprise microfluidic channels that connect microfluidic chambers and/or macrofluidic chambers to one another. Valves and pumps, such as normally closed diaphragm valves and pumps and/or normally open diaphragm valves and pumps, can be employed to control fluid flow into and out of the PCR reaction chamber and other chambers. Normally closed diaphragm valves (e.g., MOVe valves) and pumps comprising normally closed diaphragm valves are described in, e.g., US Patent

Application Publication Nos. 2011/0076735 and US 2011/0126911, and normally open diaphragm valves and pumps comprising normally open diaphragm valves are described in, e.g., US 2011/0126911, both publications being incorporated herein by reference in their entirety.

[0099] After completion of PCR cycling, a reagent for cleaving the amplicons and releasing them from the surface of the flow cell is introduced to the flow cell. For example, the cleaving reagent can be a certain wavelength of light or a chemical (e.g., ammonia) or biochemical reagent that cleaves a cleavable linker attaching the primers to the flow cell. The released amplicons can be collected and separated and/or sequenced by an off-instrument separation system and/or sequencing system, or can be delivered to (e.g., injected into) a separation system and/or sequencing system (e.g., a capillary electrophoresis system and/or capillary sequencing system) that is integrated with the instrument comprising the flow cell. The instrument comprising the flow cell can also comprise an analysis system that generates a computer file identifying amplicons of the plurality of different target genetic loci (e.g., a computer-readable profile of amplicons of the target genetic loci) after separation and detection, and/or a computer file identifying the sequences of amplicons of the plurality of different target genetic loci after sequencing. Instruments that can be modified and adapted to perform this Example include those described in, e.g., US Provisional Patent Application No. 61/674,295, which is incorporated herein by reference in its entirety.

[00100] While certain embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will be apparent to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method comprising:

providing a target polynucleotide comprising a 3' end and a 5' end;

2. The method of claim 1, wherein the complementary oligonucleotide is attached to the solid substrate, and wherein the first adapter is not attached to the solid substrate.

3. The method of claim 1, further comprising cleaving the first adapter,

wherein the first adapter is attached to the solid substrate, and

wherein cleaving the first adapter occurs subsequent to extending the complementary oligonucleotide.

4. The method of any one of the preceding claims, further comprising amplifying the product polynucleotide by polymerase chain reaction using a first primer and a second primer, wherein the first primer and the second primer are attached to the solid substrate, and wherein the first primer comprises at least a portion of the sequence of the

5. A metho d comprising :

providing a target polynucleotide comprising a 3' end and a 5' end;

cleaving the first adapter to produce a cleaved template polynucleotide.

6. The method of claim 5, further comprising:

hybridizing the cleaved template polynucleotide to a first primer, wherein the first primer is complementary to a sequence toward or at the 3' end of the cleaved template polynucleotide, and wherein the first primer is attached to the solid substrate; and

7. The method of claim 6, further comprising amplifying the product polynucleotide by polymerase chain reaction using the first primer and a second primer, wherein the second primer comprises at least a portion of the sequence of the second adapter.

8. An article comprising a single-stranded template polynucleotide having a 3' end and a 5' end, wherein the 3' end and the 5' end of the template polynucleotide are attached to a solid substrate.

9. The article of claim 8, wherein the template polynucleotide comprises a target polynucleotide having a 3' end and a 5' end,

10. The article of claim 8 or 9, further comprising a third oligonucleotide comprising a sequence complementary to a sequence toward or at the 3' end of the template polynucleotide, wherein the third oligonucleotide is attached to the solid substrate.

11. An article comprising a template polynucleotide attached to a solid substrate, wherein:

(i) the template polynucleotide comprises a target DNA polynucleotide that is adenylated at the 5' end, bound at the 5' end to a first oligonucleotide that comprises one or more RNA nucleotides at the 3' end and is attached to the solid substrate, and bound at the 3' end to a second oligonucleotide that is not attached to the solid substrate,

wherein the template polynucleotide is hybridized toward or at the 3' end to a third oligonucleotide that is attached to the solid substrate; or

(ii) the template polynucleotide comprises a target RNA polynucleotide that is bound at the 5' end to a first oligonucleotide that is attached to the solid substrate and bound at the 3' end to a second oligonucleotide that is adenylated at the 5' end, comprises one or more DNA nucleotides at the 5' end, and is not attached to the solid substrate, wherein the template polynucleotide is hybridized toward or at the 3' end to a third oligonucleotide that is attached to the solid substrate.

12. The article of any one of claims 8 to 11, further comprising a product polynucleotide that is complementary to the template polynucleotide.

13. The article of claim 12, further comprising amplification products of the product polynucleotide.

14. The method or article of any one of the preceding claims, wherein the target

polynucleotide is DNA or R A.

15. The method or article of any one of the preceding claims, wherein the target

polynucleotide is a plurality of different target polynucleotides, and wherein the plurality of different target polynucleotides is attached to the solid substrate at a density compatible with cluster formation for sequencing.

16. A metho d comprising :

providing a target double-stranded DNA (dsDNA);

denaturing the dsDNA fragments to form transposon-containing single-stranded DNA (ssDNA) fragments; and

hybridizing the adapter sequence of the transposon-containing ssDNA fragments to primers attached to a solid substrate.

17. The method of claim 16, further comprising performing PCR to produce amplification products of the transposon-containing ssDNA fragments.

18. The method of claim 17, further comprising sequencing amplification products released from the solid substrate.

19. An article comprising a plurality of different single-stranded DNA (ssDNA) fragments hybridized to primers that are attached to a solid substrate,

wherein the 5' end of each ssDNA fragment is attached to a transposon sequence appended with an adapter sequence, and wherein the adapter sequence of a transposon-containing ssDNA fragment is hybridized to a primer that is attached to the solid substrate.

20. The article of claim 19, further comprising amplification products of the plurality of different transposon-containing ssDNA fragments.

21. A metho d comprising :

22. The method of claim 21, wherein the plurality of different primers is labeled with one or more fluorescent dyes.

23. The method of claim 21 or 22, wherein the plurality of target genetic loci comprises a plurality of short tandem repeat loci.

24. The method of any one of claims 21 to 23, further comprising performing PCR to produce amplification products of the plurality of target genetic loci.

25. The method of claim 24, further comprising separating by electrophoresis or sequencing, or separating by electrophoresis and sequencing, amplification products of the plurality of target genetic loci released from the solid substrate.

26. An article comprising a plurality of different primers attached to a solid substrate, wherein the plurality of different primers:

27. The article of claim 26, wherein the plurality of different primers is labeled with one or more fluorescent dyes.

28. The article of claim 26 or 27, wherein the plurality of target genetic loci comprises a plurality of short tandem repeat loci.

29. The article of any one of claims 26 to 28, further comprising amplification products of the plurality of target genetic loci.

30. The method or article of any one of the preceding claims, wherein the solid substrate is a particle, a bead, a slide, a surface of an element of a device, a membrane, a flow cell, a well, a chamber, a macro fluidic chamber, a micro fluidic chamber, a channel, or a micro fluidic channel.