Preserving genomic linkage information in fragmented genomic DNA samples

Abstract

A method of sequencing a target nucleic acid polymer by (a) modifying a target nucleic acid polymer to generate a modified nucleic acid polymer; (b) generating fragments of the modified nucleic acid polymer, wherein the fragments are attached to positions on a surface of a solid support; (c) determining nucleotide sequences from the fragments at the positions; and (d) generating a nucleotide sequence representation of the target nucleic acid polymer based on relative distances between the nucleotide sequences from the fragments and the positions on the surface of the solid support.

Description

[0001] This application is a divisional application of the invention patent application filed on December 16, 2014, with international application number PCT / US2014 / 070658, national application number 201480073865.9, entitled "Preserving Genomic Linkage Information in Fragmented Genomic DNA Samples".

[0002] This application claims priority to U.S. Application No. 61 / 919,529, filed December 20, 2013, and U.S. Application No. 61 / 935,776, filed February 4, 2014, both of which are in the filing period and are incorporated herein by reference. Background Technology

[0003] This disclosure generally relates to nucleic acid sequencing, and more specifically to phasing, error correction, and assembly of sequence information obtained from nucleic acids.

[0004] The Human Genome Project has opened a wider window into the human genetic code. Further work to unravel the human genome is underway, for example, using high-throughput sequencing technologies. The HapMap (haplotype map) project is a global scientific endeavor aimed at discovering genetic variations that cause diseases by comparing genomic information from individuals with and without the disease. Alleles, as forms of variation in DNA sequences at specific loci, can contain one or more distinct genetic variations. The identification of haplotypes on specific chromosomes or combinations of alleles at different locations or loci is a key focus of the HapMap project. Haplotypes identified that differ between the two population groups may be associated with the location of genetic abnormalities leading to the assessed disease. Thus, HapMap results will help characterize common patterns of human genetic variation and whether these variations are potentially associated with disease.

[0005] The information obtained from these works is expected to provide valuable tools to help decipher the causes or treatments of many diseases and conditions. Unfortunately, performing such large-scale sequencing remains very expensive, and techniques that provide more in-depth information, such as single-chromosome haplotype analysis, allele or target sequence phasing, are difficult to obtain. Therefore, other tools and techniques are still urgently needed to unlock more information from the human genome. This disclosure addresses this need while providing other advantages. Summary of the Invention

[0006] The methods described in this application can be used to determine the proximity of sequence fragments to the larger target nucleic acids that generated these fragments. For example, when the individual sequence reads are shorter than the length of the target nucleic acid being evaluated, these methods can be used to determine phase and identify haplotypes for relatively long target nucleic acid sequences.

[0007] This disclosure provides a method for sequencing a target nucleic acid polymer. The method may include the following steps: (a) modifying the target nucleic acid polymer to generate a modified nucleic acid polymer, wherein the modified nucleic acid polymer includes a plurality of sequence regions derived from the target nucleic acid polymer; (b) generating fragments of the modified nucleic acid polymer in a container having a solid support surface, each fragment including one of the sequence regions; (c) randomly capturing the fragments at locations within a region on the solid support surface; (d) determining the nucleotide sequence of the sequence region by detecting the fragment at said location; and (e) generating a nucleotide sequence characterization of the target nucleic acid polymer based on the relative distance between the nucleotide sequence from the fragment and the location on the solid support surface.

[0008] A method for sequencing a target nucleic acid polymer is also provided, comprising the following steps: (a) adding an insert to the target nucleic acid polymer to form a modified nucleic acid polymer comprising a plurality of internal inserts; (b) generating fragments of the modified nucleic acid polymer in a fluid in contact with a solid support surface, thereby releasing fragments, each fragment comprising at least a portion of the inserts; (c) randomly capturing fragments from the fluid at locations on the solid support surface; (d) determining nucleotide sequences from the fragments by detecting the fragments at the locations; and (e) generating a nucleotide sequence characterization of the target nucleic acid polymer based on the relative distance between the nucleotide sequences from the fragments and the locations on the solid support surface.

[0009] This disclosure also provides a method for sequencing a target nucleic acid polymer, comprising the steps of: (a) modifying the target nucleic acid polymer to generate a modified nucleic acid polymer, wherein the modified nucleic acid polymer includes a plurality of sequence regions derived from the target nucleic acid polymer; (b) attaching the modified nucleic acid polymer to a region on a solid support surface; (c) generating a fragment of the modified nucleic acid polymer attached to the solid support surface, wherein the fragment is attached to a location at the region on the solid support surface; (d) determining a nucleotide sequence from the fragment by detecting the fragment at the location; and (e) generating a nucleotide sequence characterization of the target nucleic acid polymer based on the relative distance between the nucleotide sequence from the fragment and the location on the solid support surface.

[0010] A method for sequencing a target nucleic acid polymer is also provided, comprising the steps of: (a) adding an insert to the target nucleic acid polymer to form a modified nucleic acid polymer comprising a plurality of internal inserts; (b) attaching the modified nucleic acid polymer to a surface of a solid support; (c) generating fragments of the modified nucleic acid polymer attached to the surface of the solid support, wherein the fragments are attached to a location on the surface of the solid support and wherein each fragment comprises at least a portion of the inserts; (d) determining a nucleotide sequence from the fragment by detecting the fragment at the location; and (e) generating a nucleotide sequence characterization of the target nucleic acid polymer based on the relative distance between the nucleotide sequence from the fragment and the location on the surface of the solid support.

[0011] The methods described in this disclosure can also be used to determine the source of obtained sequence reads for mixed samples. For example, when a mixture of target nucleic acids from multiple organisms is treated as a mixture, these methods can be used to identify sequence fragments originating from a common organism. Therefore, these methods can be used to identify individual organisms in metagenomic samples. Other samples containing mixtures of target nucleic acids from different sources can also be used.

[0012] This disclosure provides a method for determining the origin of individual sequences in a mixture of sequences from different sources. The method may include the steps of: (a) providing a mixture of target nucleic acid polymers from a plurality of different sources; (b) modifying the mixture of target nucleic acid polymers to generate a mixture of modified nucleic acid polymers, wherein the mixture of modified nucleic acid polymers includes a plurality of sequence regions from the different sources; (c) generating fragments of the modified nucleic acid polymers in a container having a solid support surface, each fragment including a sequence region from one of the different sources; (d) randomly capturing the fragments at locations on the solid support surface, provided that fragments from a common target nucleic acid polymer are preferentially located at adjacent locations on the solid support surface; (e) determining the nucleotide sequence of the fragment at the location; and (f) identifying nucleotide sequences originating from a common source among the plurality of different sources based on the relative distance between the nucleotide sequence from the fragment and the location on the solid support surface.

[0013] A method for determining the origin of individual sequences in a mixture of sequences from different sources is also provided, wherein the method includes the steps of: (a) providing a mixture of target nucleic acid polymers from a plurality of different sources; (b) adding inserts to the target nucleic acid polymers in the mixture to form a mixture of modified nucleic acid polymers, each polymer including a plurality of internal inserts; (c) generating fragments of the modified nucleic acid polymers in a fluid in contact with a solid support surface, thereby releasing fragments, each fragment including at least a portion of each of the inserts; (d) randomly capturing the fragments from the fluid at locations on the solid support surface; (e) determining the nucleotide sequence from the fragments by detecting the fragments at the locations; and (f) identifying nucleotide sequences originating from a common origin among the plurality of different sources based on the relative distance between the nucleotide sequence from the fragments and the locations on the solid support surface.

[0014] This disclosure also provides a method for determining the origin of individual sequences in a mixture of sequences from different sources, wherein the method includes the steps of: (a) providing a mixture of target nucleic acid polymers from a plurality of different sources; (b) modifying the mixture of target nucleic acid polymers to generate a mixture of modified nucleic acid polymers, wherein the mixture of modified nucleic acid polymers includes a plurality of sequence regions from the different sources; (c) attaching the modified nucleic acid polymers to a surface of a solid support; (d) generating fragments of the modified nucleic acid polymers attached to the surface of the solid support, wherein fragments from a common source among the plurality of sources are attached to adjacent positions on the surface of the solid support; (e) determining the nucleotide sequence of the fragment at the position; and (f) identifying nucleotide sequences from a common source among the plurality of different sources based on the relative distance between the nucleotide sequence from the fragment and the position on the surface of the solid support.

[0015] A method for determining the origin of individual sequences in a mixture of sequences from different sources is also provided, wherein the method includes the steps of: (a) providing a mixture of target nucleic acid polymers from a plurality of different sources; (b) adding inserts to the target nucleic acid polymers in the mixture to form a mixture of modified nucleic acid polymers, each polymer including a plurality of internal inserts; (c) attaching the modified nucleic acid polymers to a surface of a solid support; (d) generating fragments of the modified nucleic acid polymers attached to the surface of the solid support, wherein fragments from a common source among the plurality of sources are attached to adjacent sites on the surface of the solid support; (e) determining the nucleotide sequence from the fragment by detecting the fragment at the site; and (f) identifying nucleotide sequences from a common source among the plurality of different sources based on the relative distance between the nucleotide sequence from the fragment and the site on the surface of the solid support. Brief description of the attached figures

[0016] Figure 1 The illustration shows an insert having two transposon elements connected by a connector (Panel A); the formation of a cyclic complex between the transposase bound to the nucleic acid polymer and the connected transposon elements (Panel B); and a schematic diagram of a modified form of the nucleic acid polymer including the insert containing the connected transposon elements.

[0017] Figure 2 The flow cell (left inset) shows the insertion-modified nucleic acid polymer captured in three separate regions, and the flow cell (right inset) shows the flow cell after cutting and denaturing the insertion-modified nucleic acid polymer.

[0018] Figure 3 The display is generated from what was captured. Figure 2 A schematic diagram of cluster clouds of nucleic acid fragments on a flow cell and the subsequent bridge amplification of the captured fragments.

[0019] Figure 4 Illustrations showing sequence reads aligned to a 50 kb reference sequence in the absence of information on cluster proximity (Illustration A); sequence reads aligned to a 50 kb reference genome sequence, where the reads are from two different genomic DNA molecules (Illustration B); and sequence reads aligned to a 50 kb reference genome sequence from one of the genomic DNA molecules (Illustration C).

[0020] Figure 5 The target nucleic acid attaches to the bead, and then the target nucleic acid is fragmented on the bead.

[0021] Figure 6The modified nucleic acid attaches to the bead, and then the target nucleic acid is fragmented on the bead.

[0022] Figure 7 This demonstrates the modification of the target nucleic acid on the surface of a solid support and the release of the fragment from that surface into solution.

[0023] Figure 8 Displaying a single-terminal transposable to produce a modified nucleic acid with several inserts.

[0024] Figure 9 This diagram shows nucleic acid fragments captured from metagenomic samples on a flow cell and the resulting clusters of clouds produced by subsequent bridging amplification.

[0025] Figure 10 This shows the spatial distribution of sequencing clusters within the flow cell. The control channel (A), prepared without the recommended method, shows no correlation in cluster spatial location; they are uniformly distributed across the entire area of ​​the flow cell. The channel (B), prepared using the recommended method, shows sequencing clusters forming spatially co-located groups, which are identified and assigned to ensembles (C) by an automated algorithm for proximity-based phasing or assembly. All units are in nanometers. X and Y axes are in nm.

[0026] Figure 11 This shows an example of error correction from two sequence reads, A and B. Sequencing reads A and B differ from the reference genome at the same position (C replaces A). The clusters generating reads A and B are 350 μm apart on the flow cell, which is far greater than the distance caused by sequencing human error, although within the average radius (200–300 μm) of nearby groups. Detailed Implementation

[0027] Traditional nucleic acid sequencing methods, as well as so-called next-generation sequencing methods, employ a shotgun approach. Specifically, this involves fragmenting genomic DNA, which is naturally packaged into large, continuous polymers called chromosomes, into smaller fragments that are easier to manipulate and detect in sequencing methods. A challenge with this shotgun approach is that by the time the sequences of each fragment have been read, information about their connections within the chromosome and their proximity to each other has been lost. The process of sequencing these fragments to obtain the chromosome sequence is commonly referred to as "assembly." Assembly is generally time-consuming and requires relatively large computational resources. Sequence and assembly errors can be problematic, depending on the sequencing methodology used and the quality of the genomic DNA sample being evaluated.

[0028] Furthermore, many target genomes contain more than one version of each chromosome. For example, the human genome is diploid, with two sets of chromosomes—one inherited from one parent. Some organisms have polyploid genomes, with more than two sets of chromosomes. Examples of polyploid organisms include animals such as salmon, and many plant species such as wheat, apples, oats, and sugarcane. When diploid and polyploid genomes are fragmented and sequenced using typical shotgun sequencing, phasing information—indicating which fragment comes from which set of chromosomes—is lost. It is difficult or impossible to reconstruct this phasing information using typical shotgun sequencing.

[0029] Evaluating mixed samples presents similar, but often more complex, challenges. Mixed samples can contain nucleic acid molecules from two or more organisms, such as chromosomes, mRNA transcripts, plasmids, etc. Mixed samples from multiple organisms are often referred to as metagenomic samples. Other examples of mixed samples are different cells or tissues that, although originating from the same organism, have different characteristics. Examples include: cancerous tissue that may contain a mixture of healthy and cancerous cells, tissue that may contain precancerous and cancerous cells, and tissue that may contain two or more different types of cancer cells. In fact, there may be multiple different types of cancer cells, as is the case with mosaicity cancer samples. Another example of different cells originating from a single organism is a mixture of maternal and fetal cells obtained from a pregnant woman (e.g., from blood or from tissue). When fragmented mixed nucleic acid samples are sequenced using a typical shotgun sequencing method, information about which fragment comes from which cell, organism, or other source is lost. Using a typical shotgun sequencing method, it is difficult or impossible to reconstruct this source information.

[0030] This disclosure provides modified versions of target nucleic acids that retain the linkage information of the sequence regions present in the source target nucleic acids (from which they are generated). Using the methods described herein, fragments of the modified nucleic acids can be generated and captured on localized regions of a solid support surface. In the case of using mixtures of different modified nucleic acids, fragments generated from each nucleic acid molecule can be localized to corresponding regions of the surface (although each fragment will ultimately be randomly located in a non-predetermined position). Thus, the proximity of different fragments on the surface can be used to determine which fragments originate from a common target nucleic acid molecule. This information can then be used for phasing and haplotype analysis of fragments derived from longer molecules, or for identifying two or more fragments originating from a common cell, organism, or other source.

[0031] The methods disclosed herein can provide benefits such as aiding assembly, improving error correction, identifying nucleic acid sources in mixed samples, and determining phasing information. Specific embodiments provide fragments of genomic DNA (or other target nucleic acids) in an arrangement or combination that correlates with the proximity of the fragment sequences within a specific chromosome (or other nucleic acid). Sequencing of the fragments and assessment of their relative positions within that arrangement or combination can be used to aid assembly, identify and improve errors in assembled sequences, and determine phase or other sequence characteristics for haplotypes. Sequence and relative proximity information can also be used to identify sequences originating from a common source, even when these sequences are derived from multiple fragments obtained from a mixture of sources.

[0032] With a more specific example, a method is provided for processing long target nucleic acid polymers to introduce insert elements that subdivide the polymer into linker segments. These insert elements can provide various capabilities, such as primer sites for amplification and / or sequencing processing, ligands for capture to a surface, and / or cleavage sites for fragmenting the polymer. The polymer with the insert elements can be introduced into a flow cell or other container having a surface for capturing and detecting nucleic acid fragments. Fragments of the polymer can then be generated at or near the surface, such that fragments from adjacent portions of the polymer end up at sites close to each other on the surface. Fragments can optionally be amplified at these sites and then detected using sequencing techniques. Fragments within a given distance of each other will have a high probability of originating from the same target nucleic acid polymer, and this information can be used to aid in assembly, error correction, identification of sources in mixed samples, and phasing.

[0033] As explained in more detail below, the methods described in this paper can be used as improvements to shotgun sequencing methods (such as those commercially recognized as next-generation sequencing methods, or those described in the literature cited in this paper).

[0034] Unless otherwise stated, the terms used herein will take their common meaning in the relevant field. The following explains several terms used herein and their meanings.

[0035] As used herein, the term "active transport" refers to the movement of molecules toward or away from a location due to non-diffusive forces applied to them. This location can be on a surface of a solid support (e.g., on an array). Non-diffusive forces can be provided by external sources, such as those generating electric or magnetic fields, fluid flow, or chemical gradients. Actively transported molecules can move along or against their concentration gradients. Therefore, active transport can include applying energy to move one or more molecules in a desired direction or to a desired location on a solid support.

[0036] As used herein, the term "array" refers to a group of sites that are distinguishable from each other by their relative positions. Different molecules at different sites in an array are distinguishable from each other by their position within the array. Each site in an array may include one or more molecules of a specific type. For example, a site may include a single target nucleic acid molecule having a specific sequence, or a site may include several nucleic acid molecules having the same sequence (and / or its complementary sequence). Sites in an array may be different features or sites on the same substrate. Exemplary sites include, but are not limited to, pores on a substrate, beads (or other particles) within or on a substrate, protrusions from a substrate, ridges on a substrate, or grooves within a substrate. Sites in an array may be separate substrates for each carrying a different molecule. Different molecules attached to separate substrates may be identified by their position on the surface of the substrate (to which the substrate is associated) or by their position within a liquid or gel. Exemplary arrays where separate substrates are located on surfaces include, but are not limited to, arrays with beads in pores.

[0037] As used herein, the term "attached" refers to a state in which two things are joined, secured, adhered, connected, or bound together. For example, an analyte such as a nucleic acid can be bound to a material (such as the surface of a solid support) via covalent or non-covalent bonds. A covalent bond is characterized by a shared pair of electrons between atoms. A non-covalent bond is a chemical bond that does not involve a shared pair of electrons and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions. In some embodiments, the attachment occurs through one or more covalent bonds, thus the attachment is not mediated by any non-covalent bonds.

[0038] In this context, the term "cleavage site" refers to a moiety (such as a connector) within a molecule that can be modified or removed to physically separate two other parts of the molecule. Cleavage sites can be readily modified or removed by biochemical, chemical, physical, or other means.

[0039] When used in this document, the term "adjacent" refers to the covalent bonding of two transposable elements to each other via a connector. The connector may be attached at or near the 5' end of either transposable element (see example). Figure 1 ( ), at or near the 3' end of both transposers, or at or near the 3' end of one transposer and at or near the 5' end of the other transposer. Examples of available adjacent transposer elements are described in PCT Publication No. WO2012 / 061832, U.S. Patent Application Publication No. 2012 / 0208724, U.S. Patent Application Publication No. 2012 / 0208705, and PCT Application Serial No. PCT / US2013 / 031023, each of which is incorporated herein by reference.

[0040] When used in this article, the term "different" in the context of nucleic acids means that nucleic acids have nucleotide sequences that are different from each other. Two or more nucleic acids may have nucleotide sequences that are different along their entire length. Alternatively, two or more nucleic acids may have nucleotide sequences that are different along the major portion of their length. For example, two or more different nucleic acids may have target nucleotide sequence portions that are different from each other, while also having the same common sequence region for both or more different nucleic acids.

[0041] When used in this document to refer to a collection of items, the term "each" is intended to identify an individual item in the collection, but does not necessarily refer to every single item in the collection. Exceptions may be made if explicitly stated otherwise or if the context clearly indicates otherwise.

[0042] When used in this article, the term “extension” is intended to include processes in which one or more nucleotides are added to a primer (e.g., by polymerase activity) or one or more oligonucleotides are added to a primer (e.g., by ligase activity).

[0043] As used herein, the term "flow cell" is intended to refer to a chamber having a surface through which one or more fluid reagents can flow. Typically, a flow cell will have an inlet and an outlet to facilitate fluid flow. Examples of flow cells and associated fluid systems, as well as detection platforms readily used in the methods of this disclosure, are, for example, those by Bentley et al. Nature The references described in 456:53-59 (2008), WO 04 / 018497; US 7,057,026; WO 91 / 06678; WO 07 / 123744; US 7,329,492; US7,211,414; US 7,315,019; US 7,405,281, and US2008 / 0108082 are all incorporated herein by reference.

[0044] As used herein, the term "forked adapter" is intended to refer to a double-stranded nucleic acid molecule having a first end where the two strands are annealed to each other, and a second end where the two strands are not annealed to each other. Examples of forked or Y-shaped adapters are described, for example, in U.S. Patent No. 7,741,463, which is incorporated herein by reference.

[0045] In this context, when referring to the first nucleic acid, the term "fragment" is intended to refer to a segment or portion of a second nucleic acid having the sequence of the first nucleic acid. Typically, the fragment and the first nucleic acid are distinct molecules. The fragment can originate from, for example, physical removal from a larger nucleic acid, replication or amplification of a region of that larger nucleic acid, degradation of other portions of that larger nucleic acid, or a combination thereof. The term can be similarly used to describe sequence data or other characterizations of nucleic acids.

[0046] As used herein, the term "haplotype" refers to a set of alleles at one or more loci inherited by an individual from one of their parents. A haplotype may include two or more loci from all or part of a chromosome. Alleles include, for example, single nucleotide polymorphisms (SNPs), short tandem repeats (STRs), gene sequences, chromosomal insertions, chromosomal deletions, etc. The term "phased alleles" refers to the distribution of specific alleles from a particular chromosome or a portion thereof. Accordingly, the "phase" of two alleles may refer to the characteristic or characterization of the relative positions of two or more alleles on one or more chromosomes.

[0047] When used herein, when referring to the use of a polymer, the term "insertion" is intended to refer to a molecule that has been or will be attached to the polymer. The polymer can be, for example, a nucleic acid such as DNA, RNA, or an analogue thereof. The molecule can be, for example, a nucleic acid, an analogue thereof, or a molecule having a nucleic acid moiety or an analogue thereof.

[0048] When used herein, when referring to a polymer having two ends, the term "internal" is intended to refer to a location within the polymer between the two ends of the polymer. For example, an insert internal to the polymer may be located at a position at least 1, 2, 3, 4, 5, 10, 100, 200, 500, or 1000 monomer subunits from the polymer ends.

[0049] When used in this document, the terms “ligand” and “receptor” are intended to refer to components that specifically bind to each other to form a complex. Examples of ligands and receptors include, but are not limited to, polyhistidines (e.g., pentahistidine and hexahistidine) and nickel; avidin (or its analogues, such as streptavidin) and biotin (or its analogues, such as 2-iminobiotin, desulfobiotin, NeutrAvidin (Molecular Probes, Eugene, Oreg.), CaptAvidin (Molecular Probes), etc.); binding proteins and their substrates (e.g., maltose and maltose-binding protein (MBP), calcium and calcium-binding protein / peptide (CBP); antibodies and antigens such as c-MYC, HA, VSV-G, HSV, V5, and FLAG Tag™); aptamers and their corresponding targets; fluorophores and antifluorophore antibodies; nucleic acids and their complements, etc. For the purposes of this disclosure, the terms “ligand” and “receptor” are used interchangeably unless the context or explicit disclosure otherwise indicates. Thus, for example, an antibody may be considered a receptor or a ligand relative to an antigen.

[0050] As used herein, the term "connector" is intended to refer to a chemical bond or portion that covalently bridges two other parts. A connector can be, for example, a sugar-phosphate backbone linking a nucleotide in a nucleic acid moiety. Connectors can include, for example, one or more of a nucleotide moiety, a nucleic acid moiety, a non-nucleotide chemical moiety, a nucleotide analog moiety, an amino acid moiety, a polypeptide moiety, or a protein moiety. Connectors can be non-amplifiable, for example, due to the presence of a non-nucleic acid moiety. Exemplary connectors are described in more detail below and in PCT Publication No. WO 2012 / 061832, U.S. Patent Application Publication No. 2012 / 0208724, U.S. Patent Application Publication No. 2012 / 0208705, and PCT Application Serial No. PCT / US2013 / 031023, each of which is incorporated herein by reference.

[0051] As used herein, the term "nucleic acid" can refer to at least two nucleotide monomers linked together. Examples include, but are not limited to, DNA, such as genomic or cDNA; RNA, such as mRNA, sRNA, or rRNA; or hybrids of DNA and RNA. As will be apparent from the examples below and elsewhere herein, nucleic acids can have naturally occurring nucleic acid structures or non-naturally occurring nucleic acid analog structures. Nucleic acids can contain phosphodiester bonds; however, in some embodiments, nucleic acids can have other types of backbones, including, for example, phosphoramide, thiophosphate, dithiophosphate, O-methylphosphamide, and peptide nucleic acid backbones and bonds. Nucleic acids can have positive backbones, nonionic backbones, and non-ribosyl backbones. Nucleic acids can also contain one or more carbon-cyclic sugars. Nucleic acids used in the methods or compositions herein can be single-stranded, or alternatively double-stranded, as indicated. In some embodiments, nucleic acids can contain both double-stranded and single-stranded sequence portions, for example, as demonstrated by fork-shaped linkers. Nucleic acids may contain any combination of deoxyribonucleotides and ribonucleotides, as well as any combination of bases (including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine) and base analogues (such as nitropyrrole (including 3-nitropyrrole) and nitroindole (including 5-nitroindole)). In some embodiments, nucleic acids may include at least one promiscuous base. The promiscuous base may pair with more than one base of a different type and may be useful, for example, when included in oligonucleotide primers or inserts for random hybridization in complex nucleic acid samples (such as genomic DNA samples). Examples of promiscuous bases include inosine, which can pair with adenine, thymine, or cytosine. Other examples include hypoxanthine, 5-nitroindole, acyclic 5-nitroindole, 4-nitropyrazole, 4-nitroimidazole, and 3-nitropyrrole. Mixed bases capable of base pairing with at least two, three, four, or more types of bases may be used.

[0052] When used in this paper, the term "nucleotide sequence" is intended to refer to the order and type of nucleotide monomers within a nucleic acid polymer. A nucleotide sequence is a characteristic of a nucleic acid molecule and can be represented in any of a variety of forms, including, for example, descriptions, images, electronic media, series of symbols, series of numbers, series of letters, series of colors, etc. This information can be represented, for example, at single nucleotide resolution, higher resolution (e.g., indicating the molecular structure of nucleotide subunits), or lower resolution (e.g., indicating chromosomal regions such as haplotype domains). The letter series "A", "T", "G", and "C" are well-known sequence characterizations used for DNA, which can be correlated to the actual sequence of a DNA molecule at single nucleotide resolution. Similar characterization is used for RNA, except that in this series, "T" is replaced by "U".

[0053] In this context, the term "passive diffusion" is intended to refer to the movement of molecules along their concentration gradient.

[0054] As used herein, the term "random" can be used to refer to the spatial arrangement or combination of positions on a surface. For example, there are at least two types of orders used in the arrays described herein: the first involves the spacing and relative positions of features (also called "sites"), and the second involves the identity or predetermined knowledge of the specific molecular species present at a particular feature. Thus, features in an array can be randomly positioned such that the closest features are randomly spaced apart from each other. Alternatively, the spacing between features can be ordered, for example, forming a regular pattern, such as a linear grid or a hexagonal grid. On the other hand, the features in an array can be random relative to the identity or predetermined knowledge of the species of analyte (e.g., a specific sequence of nucleic acid) occupying each feature, regardless of whether the spacing produces a random or regular pattern. Arrays described herein can be ordered on one hand and random on the other. For example, in some embodiments described herein, a surface is in contact with said group of nucleic acids attached to certain sites, said sites being ordered in terms of the relative positions of the sites, but random in terms of the knowledge of the sequence of the nucleic acid species present at any particular site. The mention of “randomly” capturing different nucleic acids at certain locations on a surface is intended to refer to a lack of understanding or pre-determinism regarding which nucleic acid will be captured at which location (regardless of whether the locations are arranged in an ordered pattern).

[0055] In this context, when referring to the use of the term "region," it means a surface extent smaller than the entire surface area. A region can be smaller than the entire surface area exposed to or accessible to fluids. Generally, the term "region" is used to refer to a continuous, uninterrupted extent of a surface, regardless of whether the region encompasses surface features, sites, contours, etc. A region may encompass the location where one or more nucleic acids are attached or will be attached.

[0056] As used herein, the term "single species" is intended to refer to essentially one and only one species within a particular major class. This term is not necessarily intended to limit the number of single species that can be characterized. For example, a group of nucleic acid molecules, each having the same nucleotide sequence, comprises a single species of nucleic acid. In this context, the term "single" is not intended to exclude the presence of other substances not within the relevant major class. For example, a surface location containing a single species of nucleic acid from a library may include multiple nucleic acids having the same sequence, excluding other target nucleic acids from that library, but not necessarily any other non-nucleic acid components. It is to be understood that an apparent single-species population may have a small amount of another species present at a level that is considered by those skilled in the art to be negligible impurities or artificial factors for the specific use of the population. For example, if the amount of any nucleic acid molecule having a second sequence is low enough that it is undetectable or negligible when the first sequence is detected, then a cluster of nucleic acids derived from a single template having the first sequence would be considered to have an apparent single species. Alternatively, an absolutely single-species population would have one and only one species.

[0057] As used herein, the term "solid support" refers to a rigid substrate that is insoluble in aqueous liquids. The substrate may be non-porous or porous. The substrate may optionally be capable of absorbing liquids (e.g., due to its porosity), but typically is rigid enough that the substrate does not significantly expand upon absorbing liquids and does not significantly shrink upon drying to remove the liquids. Non-porous solid supports are generally impermeable to both liquids and gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylic plastics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutene, polyurethane, Teflon™, cycloolefins, polyimide, etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, fiber bundles, and polymers. For some embodiments, particularly useful solid supports are located within a flow cell apparatus. Exemplary flow cells are described in more detail below.

[0058] As used herein, the term "source" is intended to include the origin of nucleic acid molecules, such as tissues, cells, organelles, compartments, or organisms. This term can be used to identify or distinguish the origin of a specific nucleic acid in a mixture that includes several other nucleic acid sources. A source can be a specific organism in a metagenomic sample having several different species of organisms. In some embodiments, the source will be identified as a single source (e.g., a single cell or organism). Alternatively, the source can be identified as a species encompassing several individuals of the same type in a sample (e.g., bacteria or other organisms in a metagenomic sample having several individual members of that species as well as members of other species).

[0059] When used herein, the term "surface" is intended to refer to the outer portion or outer layer of a material when referring to its use. This surface may be in contact with another material, such as a gas, liquid, gel, polymer, organic polymer, a second surface of a similar or different material, metal, or coating. The surface or an area thereof may be substantially flat. The surface may have surface features such as holes, depressions, grooves, ridges, raised areas, posts, columns, etc. The material may be, for example, a solid support, a gel, etc.

[0060] When used in this article, the term "target" is intended to distinguish the nucleic acid, for example, from other nucleic acids, modified forms of nucleic acids, fragments of nucleic acids, etc., when referring to nucleic acid polymers. Any of the various nucleic acids described in this article can be identified as a target nucleic acid, examples of which include genomic DNA (gDNA), messenger RNA (mRNA), copy DNA (cDNA), and derivatives or analogs of these nucleic acids.

[0061] As used herein, the term "transposase" is intended to refer to an enzyme capable of forming a functional complex with a composition containing transposon elements (e.g., a transposon, a transposon terminus, or a transposon terminus composition) and, for example, catalyzing the insertion or transposition of the composition containing transposon elements into a target DNA incubated therewith, for example, in an in vitro transposition reaction. The term may also include integrases derived from retrotransposons and retroviruses. Transposases, transposomers, and transposon complexes are well known to those skilled in the art, as illustrated in the disclosure of U.S. Patent Application Publication No. 2010 / 0120098, which is incorporated herein by reference. Although many embodiments described herein refer to Tn5 transposases and / or highly active Tn5 transposases, it should be understood that any transposition system capable of inserting transposon elements with sufficient efficiency to label target nucleic acids is available. In specific embodiments, preferred transposition systems are capable of inserting transposon elements to label target nucleic acids in a random or near-random manner.

[0062] As used herein, the term "transposon" is intended to refer to a transposase bound to a nucleic acid. This nucleic acid is typically double-stranded. For example, the complex may be the product of incubating a transposase with double-stranded transposon DNA under conditions supporting the formation of a non-covalent complex. Transposon DNA may include, but is not limited to, Tn5 DNA, a portion of Tn5 DNA, a composition of transposon elements, a mixture of transposon element compositions, or other nucleic acids capable of interacting with a transposase, such as a highly active Tn5 transposase.

[0063] As used herein, the term "transposon element" is intended to refer to a nucleic acid molecule or a portion thereof comprising a nucleotide sequence that forms a transposon with a transposase or integrase. Typically, the nucleic acid molecule is a double-stranded DNA molecule. In some embodiments, the transposon element is capable of forming a functional complex with a transposase in a transposition reaction. As a non-limiting example, the transposon element may include a 19-bp outer-terminal ("OE") transposon end, inner-terminal ("IE") transposon end, or "mosaic-terminal" ("ME") transposon end recognized by wild-type or mutant Tn5 transposases, or the R1 and R2 transposon ends set forth in U.S. Patent Application Publication No. 2010 / 0120098, which is incorporated herein by reference. The transposon element may include any nucleic acid or nucleic acid analogue suitable for forming a functional complex with a transposase or integrase in an in vitro transposition reaction. For example, the transposon end may include DNA, RNA, modified bases, non-natural bases, a modified backbone, and may include a gap in one or both strands.

[0064] In this context, the term "universal sequence" refers to a sequence region shared by two or more nucleic acid molecules, which also possess sequence regions that differ from each other. A universal sequence present in different members of a molecule set can allow the use of a set of universal capture nucleic acids complementary to the universal sequence to capture a variety of different nucleic acids. Similarly, a universal sequence present in different members of a molecule set can allow the use of a set of universal primers complementary to the universal sequence to replicate, amplify, or sequence analyze a variety of different nucleic acids. Therefore, universal capture nucleic acids or universal primers include sequences that specifically hybridize to the universal sequence.

[0065] The embodiments described below and listed in the claims can be understood with reference to the above definitions.

[0066] This disclosure provides a method for sequencing a target nucleic acid polymer. The method may include the following steps: (a) modifying the target nucleic acid polymer to generate a modified nucleic acid polymer, wherein the modified nucleic acid polymer includes a plurality of sequence regions derived from the target nucleic acid polymer; (b) generating fragments of the modified nucleic acid polymer in a container having a solid support surface, each fragment including one of the sequence regions; (c) randomly capturing the fragments at locations within a region on the solid support surface; (d) determining the nucleotide sequence of the sequence region by detecting the fragment at the location; and (e) generating a nucleotide sequence characterization of the target nucleic acid polymer based on the relative distance between the nucleotide sequence from the fragment and the location on the solid support surface.

[0067] This disclosure also provides a method for sequencing a target nucleic acid polymer, comprising the steps of: (a) modifying the target nucleic acid polymer to generate a modified nucleic acid polymer, wherein the modified nucleic acid polymer includes a plurality of sequence regions derived from the target nucleic acid polymer; (b) attaching the modified nucleic acid polymer to a region on a solid support surface; (c) generating a fragment of the modified nucleic acid polymer attached to the solid support surface, wherein the fragment is located at the region on the solid support surface; (d) determining a nucleotide sequence from the fragment by detecting the fragment at the location; and (e) generating a nucleotide sequence characterization of the target nucleic acid polymer based on the relative distance between the nucleotide sequence from the fragment and the location on the solid support surface.

[0068] This disclosure provides a method for determining the origin of individual sequences in a mixture of sequences from different sources. The method may include the steps of: (a) providing a mixture of target nucleic acid polymers from a plurality of different sources; (b) modifying the mixture of target nucleic acid polymers to generate a mixture of modified nucleic acid polymers, wherein the mixture of modified nucleic acid polymers includes a plurality of sequence regions from the different sources; (c) generating fragments of the modified nucleic acid polymers in a container having a solid support surface, each fragment including a sequence region from one of the different sources; (d) randomly capturing the fragments at locations on the solid support surface, provided that fragments from a common target nucleic acid polymer are preferentially located at proximate locations on the solid support surface; (e) determining the nucleotide sequence of the fragment at the location; and (f) identifying nucleotide sequences originating from a common source among the plurality of different sources based on the relative distance between the nucleotide sequence from the fragment and the location on the solid support surface.

[0069] A method is also provided for determining the origin of individual sequences in a mixture of sequences from different sources, wherein the method includes the steps of: (a) providing a mixture of target nucleic acid polymers from a plurality of different sources; (b) adding inserts to the target nucleic acid polymers in the mixture to form a mixture of modified nucleic acid polymers, each polymer including a plurality of internal inserts; (c) generating fragments of the modified nucleic acid polymers in a fluid in contact with a solid support surface, thereby releasing fragments, each fragment including at least a portion of each of the inserts; (d) randomly capturing fragments from the fluid at locations on the solid support surface; (e) determining the nucleotide sequence from the fragments by detecting the fragments at the locations; and (f) identifying nucleotide sequences originating from a common source among the plurality of different sources based on the relative distance between the nucleotide sequence from the fragments and the locations on the solid support surface.

[0070] The target nucleic acid that can be used in the methods or compositions described herein may have the structure and / or origin set forth elsewhere herein. Exemplary types of nucleic acids that can be targeted herein include, but are not limited to, DNA, RNA, peptide nucleic acids, morpholinonucleotides, locked nucleic acids, glycol nucleic acids, threonine nucleic acids, mixtures thereof, and hybrids thereof. In a preferred embodiment, a fragment of genomic DNA or an amplified copy thereof is used as the target nucleic acid. In another preferred embodiment, mitochondrial or chloroplast DNA is used. In still some embodiments, RNA or its derivatives, such as mRNA or cDNA, are targeted. In some embodiments, the target nucleic acid may be derived from a single cell. In some embodiments, the target nucleic acid may be derived from noncellular fluids, such as cell-free plasma or sputum. In some embodiments, the target nucleic acid may be derived from circulating tumor cells.

[0071] In some implementations, the target nucleic acid may include ribosomal RNA (rRNA) or a sequence derived from it. For example, the rRNA sequence can be used in particular to distinguish different organisms in a metagenomic sample.

[0072] The target nucleic acid can have any of a variety of nucleotide sequences. In some embodiments, the target nucleic acid includes a homopolymer sequence. The target nucleic acid may also include a repeating sequence. The repeating unit may be, for example, at least 2, 5, 10, 20, 30, 40, 50, 100, 250, 500, 1000 nucleotides or more. The repeating sequence may be repeated a plurality of times in a continuous or discontinuous manner, including, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 times or more. The methods described herein are particularly useful for analyzing and evaluating target nucleic acids having homopolymer and / or repeating sequences because the actual length of these sequence regions can be determined from sequence assembly, which is at least partially derived from proximity information obtained from these methods.

[0073] Some embodiments described herein may utilize a single target nucleic acid species existing in one copy (i.e., a single molecule) or, alternatively, in multiple copies (i.e., all nucleic acid molecules having the same sequence). Other embodiments may utilize multiple different target nucleic acid species (i.e., multiple nucleic acid molecules with different nucleotide species). Thus, multiple target nucleic acids may include multiple identical target nucleic acids, multiple different target nucleic acids in which some target nucleic acids are the same, or multiple target nucleic acids in which all target nucleic acids are different.

[0074] Implementation schemes utilizing multiple target nucleic acids can be carried out in multiple ways, such that reagents are delivered to multiple target nucleic acids simultaneously, or multiple target nucleic acids are manipulated simultaneously (e.g., one or more steps of the methods described herein can be performed simultaneously on multiple target nucleic acids). Target nucleic acids or derivatives of target nucleic acids (e.g., fragments and / or amplicones) can be provided in one or more chambers, or on an array surface, to facilitate multiplexing and / or evaluation.

[0075] In some embodiments, multiple target nucleic acids may comprise substantially the entire genome of a particular organism. These multiple target nucleic acids may comprise at least a portion of the genome of the particular organism, including, for example, at least 1%, 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 99% of the genome. In specific embodiments, this portion may have an upper limit of at most about 1%, 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 99% of the genome. Exemplary genomes from which the target nucleic acids can be obtained include, but are not limited to, prokaryotes (e.g., *Escherichia coli* spp.). Escherichia ), Bacillus spp. ( Bacillus ), Serratia ( Serratia Salmonella ( Salmonella Staphylococcus spp. Staphylococcus Streptococcus ( Streptococcus Clostridium ( Clostridium Chlamydia ( ) Chlamydia ), Neisseria spp. Neisseria ), genus *Treponema* ( Treponema Mycoplasma genus Mycoplasma ), genus *Borghex* ( Borrelia Legionella ( ) Legionella ), Pseudomonas spp. Pseudomonas Mycobacterium, Helicobacterium Helicobacter Erwinia ( ) Erwinia ), Agrobacterium, Rhizobium ( Rhizobium ), and Streptomyces ( Streptomyces ); Archaea, such as the phylum Isoarchaea ( crenarchaeota ), Narcobacteria ( nanoarchaeota ) or Archaea ( euryarchaeotia ); or eukaryotes, such as fungi (e.g., yeast), plants, protozoa, and animals (including insects (e.g., fruit flies)). Drosophila spp.), nematodes (e.g., Caenorhabditis elegans (spp.)), and nematodes (e.g., Caenorhabditis elegans (spp.)). Caenorhabditis elegans (), as well as mammals (e.g., rats, mice, monkeys, non-human primates, and humans).

[0076] Multiple target nucleic acids may have a complexity equal to at least about 3 gigabases (the size of a haploid human genome), at least about 2 gigabases (representing at least about 60% of the human genome), or at least about 1 gigabase (representing at least about 30% of the human genome). The complexity of multiple target nucleic acids may be greater or less than that of the human genome, and is, for example, at least about 0.1 gigabases, 0.2 gigabases, 0.5 gigabases, 0.8 gigabases, 1 gigabase, 1.5 gigabases, 2 gigabases, 2.5 gigabases, 3 gigabases, 3.5 gigabases, 4 gigabases, 4.5 gigabases, 5 gigabases or more. Alternatively or additionally, the complexity of the multiple nucleic acids used in the embodiments herein may not exceed approximately 5 gigabases, 4 gigabases, 3 gigabases, 2 gigabases, 1 gigabases, 0.5 gigabases, 0.1 gigabases or less.

[0077] In some embodiments, the plurality of target nucleic acids comprises RNA from a desired organism, examples of which include, but are not limited to, those listed above. The target nucleic acid sample may comprise substantially all or a portion of a complete complement of RNA present in the organism. The plurality of target nucleic acids may comprise at least a portion of the transcriptome of a particular organism, including, for example, at least 1%, 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 99% of the transcriptome. Alternatively or further, the portion may have an upper limit of at most about 1%, 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 99% of the transcriptome.

[0078] In some implementations, the target nucleic acid, or the modified nucleic acid generated by integrating an insert into the target nucleic acid, may have a molecular weight of at least 0.1 kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75 kb, 80 kb, 85 kb, or 90 kb. b. Lengths of 95kb, 100kb, 150kb, 200kb, 250kb, 300kb, 350kb, 400kb, 450kb, 500kb, 550kb, 600kb, 650kb, 700kb, 750kb, 800kb, 850kb, 900kb, 950kb, 1000kb, 5000kb, 10000kb, 20000kb, 30000kb, or at least 50000kb. As an alternative or additional, the length of the target nucleic acid or modified nucleic acid does not exceed 0.1kb, 1kb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 15kb, 20kb, 25kb, 30kb, 35kb, 40kb, 45kb, 50kb, 55kb, 60kb, 65kb, 70kb, 75kb. 80kb, 85kb, 90kb, 95kb, 100kb, 150kb, 200kb, 250kb, 300kb, 350kb, 400kb, 450kb, 500kb, 550kb, 600kb, 650kb, 700kb, 750kb, 800kb, 850kb, 900kb, 950kb or not exceeding 1000kb.

[0079] Target nucleic acids can be prepared from nucleic acid molecules obtained from a single organism or from a group of nucleic acid molecules obtained from natural sources including more than one organism. Target nucleic acids can originate from a single cell; from multiple cells, one or more tissues or body fluids of a single organism; from cells, tissues or body fluids of several organisms of the same species; or from multiple species, such as metagenomic samples, or from environmental samples. The sources of nucleic acid molecules include, but are not limited to, organelles, cells, tissues, organs, or organisms.

[0080] In some implementations, the target nucleic acid sample may be treated before the addition of the insert or before any other modifications described herein. For example, the target nucleic acid sample may be amplified before the addition of the insert, before attachment to a bead, before attachment to a surface of a solid support, or before binding to a transposase. Amplification is particularly useful when the sample is of low abundance or when a small amount of target nucleic acid is provided. Methods for amplifying the vast majority of sequences within a genome are called “whole-genome amplification” methods. Examples of these methods include multiple substitution amplification (MDA), strand substitution amplification (SDA), or hyperbranched strand substitution amplification, each of which is performed using degenerate primers. Particularly useful methods are those suggested by providers of whole-genome sequencing platforms, such as Illumina Inc., San Diego, and Life Technologies Inc., Carlsbad, for use during sample preparation. Other useful methods are described in U.S. Patent No. 7,670,810, which is incorporated herein by reference.

[0081] Targeted amplification can be used to selectively amplify only a subset of nucleic acids, whether they are the target nucleic acid, modified nucleic acid, nucleic acid fragments, or other nucleic acids described herein. Examples of targeted amplification techniques include, but are not limited to, multiplex PCR, GoldenGate assay (Illumina Inc., San Diego), rolling circle amplification, and other methods known in the art, such as those described in U.S. Patent Nos. 7,670,810, 6,355,431, or 7,582,420, each of which is incorporated herein by reference. Other methods that can be used for the selective enrichment and optional amplification of target regions of the genome include targeted probe pull-out techniques, such as those commercially available from Illumina Inc. (TruSeq™ brand), NimbleGen Inc. (SeqCap EZ™ brand), or Agilent Technologies, Inc. (SureSelect™ brand).

[0082] In some embodiments, the target nucleic acid sample may be fragmented before the addition of the insert or before any other modifications described herein. In some embodiments, fragmentation is inherently generated by amplification, for example, in cases where portions of the template appearing between flanking primer hybridization sites are selectively copied. In other cases, fragmentation may be achieved using chemical, enzymatic, or physical techniques known in the art. Fragments within the desired size range may be separated using methods known in the art, such as gel electrophoresis or SPRI. ®The target DNA polymers are purified from beads (AgenCourt, Beverly, MA). Accordingly, fragmentation can be performed before adding the insert to obtain target DNA polymers with lengths up to about 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.8 kb, 0.6 kb, 0.5 kb, 0.4 kb, 0.2 kb, or 0.1 kb or shorter. Alternatively, or additionally, size selection can be used to obtain target nucleic acid polymers with maximum sizes selected from those exemplified above and minimum sizes of at least about 0.1 kb, 0.5 kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 10 kb or longer.

[0083] The methods disclosed herein may include the step of modifying a target nucleic acid polymer to generate a modified nucleic acid polymer, wherein the modified nucleic acid polymer includes a plurality of sequence regions derived from the target nucleic acid polymer. Exemplary modifications include, but are not limited to, binding a transposase to the target nucleic acid to form a transposon, adding an insert to the target nucleic acid, attaching the target nucleic acid to a bead or other carrier, or attaching the target nucleic acid to a substrate surface and subsequently capturing fragments thereon.

[0084] In specific embodiments, the target nucleic acid polymer can be modified by adding one or more inserts to the polymer. Therefore, the methods of this disclosure may include the step of adding inserts to the target nucleic acid polymer, thereby preparing a modified nucleic acid. Some insertion methods involve contacting the insert having transposon elements with the target nucleic acid in the presence of an enzyme (such as a transposase or integrase) under conditions sufficient to allow the insert to integrate into the target nucleic acid. In some embodiments, the addition of the insert to the target nucleic acid may be non-random. In some embodiments, the insert having transposon elements may contact the target nucleic acid in the presence of one or more proteins that inhibit integration at certain sites. For example, integration of the insert into genomic DNA comprising proteins, genomic DNA comprising chromatin, genomic DNA comprising nucleosomes, or genomic DNA comprising histones may be inhibited.

[0085] For example, the step of adding the insert to the target nucleic acid in solution can be performed, such as... Figure 1 As shown, and as described in more detail below, the solution-phase target nucleic acid can be used to generate fragments that will subsequently bind to the surface of a solid support. In an alternative embodiment, the target nucleic acid can be captured on beads or other solid-phase supports so that an insert can be added thereto while the target nucleic acid is attached to the solid-phase support. Figure 5The image shows beads 100 with attached transposase, similar beads 110 with a solution phase of target nucleic acid present, and beads 120 with the target nucleic acid bound to the transposase on the bead surface. The target nucleic acid can be tagged and fragmented on the surface of the beads. The solid support can be prepared from a solid support material (i.e., rigid) or from other insoluble materials, whether rigid or compressible (e.g., hydrogel beads).

[0086] Another embodiment that can be used to add inserts to target nucleic acids on a solid phase has been shown. Figure 6 In this example, bead 200 has attached nucleic acid probes that are complementary to an insert added to the modified nucleic acid. The insert has been added to the modified nucleic acid via a transposase, and the modified nucleic acid is bound to bead 210 by annealing the probes and inserts to form a solid-phase modified nucleic acid. When bound to the bead, transposons may be present on the modified nucleic acid (e.g., ...). Figure 6 (As shown in the diagram), or the transposons can be removed before this binding. The solid-phase, modified nucleic acid can be fragmented on a bead to generate beads 220 with solid-phase nucleic acid fragments.

[0087] Any of the target nucleic acids or modified nucleic acids described herein can be attached to beads or other solid-phase carriers. Therefore, the steps described herein for preparing and using target nucleic acids, modified nucleic acids, or nucleic acid fragments can be performed before or after solid-phase attachment. For example, the target nucleic acid can be attached to beads or other solid-phase carriers and then treated with a transposase, integrase, or other reagent that introduces an insert. Furthermore, the insert need not be added to the solid-phase target nucleic acid; instead, the solid-phase target nucleic acid can be fragmented without the addition of an insert. Beads or other solid-phase carriers to which the target nucleic acid, modified nucleic acid, or fragments thereof are bound can be delivered to a container having a surface where the nucleic acid fragment will be sequenced or otherwise detected.

[0088] Other examples of adding inserts to solid-phase target nucleic acids have been shown. Figure 7 As shown in Illustration A, the target nucleic acid is attached to the surface via a bond at or near its terminal. If desired, this attachment can occur at other sites on the target nucleic acid. In Illustration B, a transposase is bound to the solid-phase target nucleic acid to form multiple solid-phase transposons. The target nucleic acid can be tagged and fragmented so that multiple distinct fragments are released from the surface, as shown in Illustration C. The released fragments are then recaptured onto a region of the surface adjacent to the site where the target nucleic acid was previously captured. The captured fragments can then be manipulated and detected using the methods described herein.

[0089] Regardless of whether the target nucleic acid is in solution or solid phase, the insert can have any of a variety of groups or portions. In specific embodiments, the insert can be associated with an affinity tag to integrate the transposon element of the insert into a specific sequence in the target nucleic acid. For example, the insert can be associated with a protein that targets a specific nucleic acid sequence, such as histones, chromatin-binding proteins, transcription factors, promoters, etc., and with an antibody or antibody fragment that binds to a specific sequence-specific nucleic acid-binding protein. In an exemplary embodiment, the insert can be associated with an affinity tag (such as biotin); and this affinity tag is associated with a nucleic acid-binding protein.

[0090] It is important to understand that during the integration of some insert carrying the transposon element, several consecutive nucleotides at the integration site of the target nucleic acid are replicated in the integration product. Therefore, the integration product may include repetitive sequences at each end of the insert region in the resulting modified nucleic acid, as described, for example, in PCT Publication No. WO 2012 / 061832, U.S. Patent Application Publication No. 2012 / 0208724, U.S. Patent Application Publication No. 2012 / 0208705, and PCT Application Serial No. PCT / US2013 / 031023, each of which is incorporated herein by reference. These repetitive sequences can be used as host tags. For example, the Tn5 transposase binds to the target nucleic acid and creates two gaps in the DNA backbone, one in each strand, separated by nine bases. This nine-base region provides a host tag that will be present in adjacent fragments. This tag can be used to confirm that fragments that are superficially close and also have the same tag sequence indeed originate from the same target nucleic acid. Therefore, this tag can be used for error checking, aiding in the assembly of fragment sequences, or determining haplotype phase.

[0091] In some embodiments, multiple inserts provided herein are added to a specific target nucleic acid polymer. Some embodiments include selecting conditions sufficient to achieve the integration of multiple transposon sequences into the target nucleic acid polymer such that the average distance between each integrated transposon sequence in the polymer is a specific number of consecutive nucleotides in the target nucleic acid. In some embodiments, conditions may be selected such that the distance or average distance between the inserts in the target nucleic acid is at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more consecutive nucleotides. In some embodiments, the distance or average distance between the inserts in the target nucleic acid is at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more consecutive nucleotides. In some embodiments, the distance or average distance between the inserts in the target nucleic acid is at least about 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 90 kb, 100 kb or more consecutive nucleotides. In some embodiments, the distance or average distance between inserts in the target nucleic acid is at least about 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb or more consecutive nucleotides. Alternatively or as an alternative to the thresholds described above, the distance or average distance between inserts in the target nucleic acid may be up to about 1000 kb, 500 kb, 100 kb, 10 kb, 5 kb, 1 kb, 500 bases, 100 bases, 50 bases or 10 bases.

[0092] Some embodiments may include copying all or part of a sequence in a nucleic acid. For example, some embodiments include hybridizing a primer to a primer site of an insert integrated into a modified nucleic acid or a fragment thereof. In some such embodiments, the primer may hybridize to the primer site and extend. The copied sequence may include at least a portion of the target nucleic acid or a fragment thereof. In some embodiments, the primer may be a sequencing primer. In some embodiments, the sequencing primer is used to obtain sequencing data. Some embodiments of the methods described herein may include amplifying a sequence comprising at least a portion of one or more inserts and at least a portion of the target nucleic acid or a fragment thereof. In some embodiments, a primer hybridizing to a primer site of an insert integrated into a target nucleic acid polymer is used to amplify at least a portion (or a fragment thereof) of the target nucleic acid.

[0093] In a specific embodiment, the insert is added to the target nucleic acid polymer via a transposase. The transposase can form a functional complex with the insert, which includes transposon elements, thereby forming a transposome. The transposome formed in this manner can catalyze a transposition reaction to integrate the insert into the target nucleic acid polymer. In some such insertion events, one strand of the transposase recognition site can be transferred into the target nucleic acid.

[0094] Some embodiments may include the use of highly active Tn5 transposases and Tn5-type transposase elements (see, for example, Goryshin and Reznikoff). J. Biol. Chem (Journal of Biochemistry), 273:7367 (1998), the reference of which is incorporated herein by reference), or MuA transposases and Mu transposase elements including R1 and R2 terminal sequences (see, for example, Mizuuchi, Cell (Cell), 35: 785, (1983) and Savilati et al., EMBO J (European Organization for Molecular Biology Journal), 14: 4893, (1995), each of which is incorporated herein by reference. Exemplary transposase elements that form complexes with highly active Tn5 transposases (e.g., EZ-Tn5™ transposase from Epicentre Biotechnologies, Madison, Wisconsin) are described in PCT Publication No. WO 2012 / 061832, U.S. Patent Application Publication No. 2012 / 0208724, U.S. Patent Application Publication No. 2012 / 0208705, and PCT Application Serial No. PCT / US2013 / 031023, each of which is incorporated herein by reference.

[0095] Further examples of transposable systems that can be used with some of the embodiments provided herein include Staphylococcus aureus Tn552 (Colegio et al., J. Bacteriol. (Journal of Bacteriology), 183: 2384-8 (2001); Kirby et al. Mol. Microbiol. (Molecular Microbiology), 43: 173-86 (2002)), Tyl (Devine & Boeke, Nucleic Acids Res (Nucleic Acid Research), 22: 3765-72 (1994) and PCT Publication No. WO 95 / 23875), transposon Tn7 (Craig, Science (Science) 271: 1512 (1996); Craig, Curr Top Microbiol Immunol (Special Issues in Microbiology and Immunology), 204:27-48 (1996)), Tn / O and IS10 (Kleckner et al., Curr Top Microbiol Immunol (Special Issues in Microbiology and Immunology), 204:49-82 (1996)), Mariner transposases (Lampe et al., EMBO J (European Organization for Molecular Biology Journal), 15: 5470-9, (1996)), Tcl (Plasterk, Curr. Topics Microbiol. Immunol (Special Issues in Microbiology and Immunology), 204: 125-43, (1996)), P Element (Gloor, Methods Mol. Biol. (Molecular Biology Methods), 260: 97-114, (2004)), Tn3 (Ichikawa & Ohtsubo, J Biol. Chem. (Journal of Biochemistry) 265:18829-32, (1990)), bacterial insertion sequence (Ohtsubo & Sekine, Curr. Top. Microbiol. Immunol (Special Issues in Microbiology and Immunology) 204: 1-26, (1996)), retroviruses (Brown et al., Proc Natl Acad Sci USA (Proceedings of the National Academy of Sciences of the United States of America), 86:2525-9, (1989)), and yeast retrotransposons (Boeke & Corces, Annu Rev Microbiol (Annual Review of Microbiology) 43:403-34, (1989)). More examples include IS5, Tn10, Tn903, IS911, and engineered versions of transposase family enzymes (Zhang et al., PLoS Genet. (PLOS Genetics) 5:el000689. Epub 2009 Oct 16; and Wilson et al., J. Microbiol. Methods (Journal of Microbiological Methods) 71:332-5 (2007)). Each of the references listed in this paragraph is incorporated into this paper by way of citation.

[0096] Some embodiments provided herein include inserts having transposon elements, modified nucleic acid polymers having transposon elements, or nucleic acid fragments having transposon elements. In some embodiments, the transposon elements are present in the insert along with other portions set forth herein, such as adapters to second transposon elements, primer sites for amplification, primer sites for primer extension-based detection (e.g., SBS detection), binding portions, and / or cleavage sites. Figure 1A schematic diagram describing the insert, including the transpose element and other parts.

[0097] Transposon elements may include two nucleic acid strands containing specific binding sites for transposases or integrases. These strands may be completely complementary along their length (e.g., double-stranded nucleic acids), or complementary along at least a portion of their length (e.g., forked connectors, connectors with non-complementary protrusions, etc.). Exemplary embodiments of double-stranded transposon elements having a complementary strand in an annealed portion and a non-complementary strand forming a non-annealed portion are shown. Figure 1 middle.

[0098] In specific embodiments, the insert may include two transposon elements connected to each other. A connector may be included in the insert so that the first transposon element and the second transposon element are continuous. Particularly useful inserts are those that form “circular” complexes, as set forth in PCT Publication No. WO 2012 / 061832, U.S. Patent Application Publication No. 2012 / 0208724, U.S. Patent Application Publication No. 2012 / 0208705, and PCT Application Serial No. PCT / US2013 / 031023, each of which is incorporated herein by reference. In these structures, a single insert having continuous transposon elements binds to two transposase subunits to form a “circular” complex. This circular complex can be used to place the insert into a target nucleic acid while maintaining the original sequence information of the target nucleic acid and without fragmenting the resulting modified nucleic acid polymer. The insertion of circular transposon elements provides a method for adding inserts to target nucleic acids without having to fragment the target nucleic acid. As will be explained in more detail below, the resulting modified nucleic acid can be fragmented in subsequent steps.

[0099] Useful adapters may have portions including, but not limited to, one or more nucleotides, nucleic acids, chemical moieties containing non-nucleotides, nucleotide analogs, amino acids, polypeptides, or proteins. In a preferred embodiment, the adapter includes a nucleic acid moieties. The adapter may include at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides. Alternatively or additionally, the adapter may include up to about 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide. In a preferred embodiment, the adapter is a single strand between the ends of a double-stranded ME, for example, to prevent self-insertion.

[0100] Linkers can be, for example, non-amplifiable due to the presence of one or more non-nucleic acid moieties. In some cases, linkers will not contain any nucleic acid material and will be completely devoid of nucleotides. Examples of non-amplifiable linkers include synthetic linkers, such as those with moieties selected from alkyl, propyl, and PEG; linkers with non-natural bases (such as IsoC, isoG); or linkers with any groups that are not amplified in DNA-based amplification protocols. For example, transposons containing isoC, isoG pairs can be amplified using a mixture of dNTPs lacking complementary isoG and isoC, ensuring that amplification across the inserted transposon does not occur.

[0101] In a specific embodiment, a non-amplifiable adapter may be introduced between the mosaic end (ME) regions. For example, the insert may have the following configuration: ME-primer site-adaptor-primer site-ME. The primer sites may be the same or different. For example, as... Figure 1 The primer sites shown in the examples are all P5 primer sites. However, it should be understood that... Figure 1 The structure can be modified so that the two P7 primer sites are connected by a connector, or that a P5 primer site on one fork connector is connected to a P7 primer site on another fork connector. The P5 and P7 primer sites are described in U.S. Patent No. 8,563,477, which is incorporated herein by reference.

[0102] Another method for adding inserts to target nucleic acids without fragmenting the target nucleic acids is one-ended transposition. One-ended transposition can be used to create a gap at the transposition site and directly attach the insert to only one strand of the target DNA. Figure 8 Illustrative examples are shown in the figure. Various enzymes, including transposases, can be used for single-terminal transposition. For example, Mu, Tn5, and Rag-like transposases have been shown to exhibit single-terminal transposition. Certain mutants of Mu (e.g., MuE392Q) show a preference for single-terminal transposition over dipeptid transposition (see Haapa et al., Nucl. Acids Res (Nucleic Acid Research) 27:2777 (1999), the referenced document is incorporated herein by reference.

[0103] An exemplary method for achieving single-terminal transposition is to employ a transposon dimer as a product of a mixture of active and inactive transposon monomers. The inactive monomers may be generated by mutagenesis, chemical modification, or both. The appropriate ratio of active and inactive monomers for obtaining suitable levels of single-terminal transposition can be determined based on statistical evaluation (e.g., Poisson distribution) and / or titration. In a specific embodiment, the transposon dimer may be formed from a mixture of active and inactive monomers containing up to 50%, 25%, 10%, 5%, 1%, 0.1%, or 0.01% active monomers.

[0104] Another method for obtaining a single-terminal transposon is to employ a mixture of reactive and non-reactive transposon elements and an active transposase dimer. Exemplary non-reactive transposon elements include those that are blocked at the 3' end (e.g., by a dideoxynucleotide at the 3' end or an extended blocking portion at the 3' end). In a specific embodiment, the transposon dimer may be formed from a mixture of reactive and non-reactive transposon elements containing up to 50%, 25%, 10%, 5%, 1%, 0.1%, or 0.01% reactive elements.

[0105] Another approach considered for obtaining single-terminal transposons is to employ monomeric transposons. It is envisioned that monomeric transposons are generated by modifying or removing dimerizing contacts on the transposase protein. Removal can be achieved by mutagenesis of the monomer to delete a portion of the protein structure forming the dimerizing contact and / or by point mutation to modify the amino acids involved in dimerization. Alternatively, or additionally, the site of the dimerizing contact can be modified by chemical modification (e.g., at native amino acids present in the dimerizing contact region or at mutant amino acids introduced into the dimerizing contact region, such as reactive cysteine ​​or lysine).

[0106] Intercalations used in the methods or compositions described herein may include one or more primer sites. In some embodiments, the intercalation contains a single type of primer site. Alternatively, the intercalation may include at least one, two, or more primer sites. The orientation of the primer sites in these embodiments may be such that primers hybridizing to a first primer site and primers hybridizing to a second primer site are in the same or different orientations. In one embodiment, the primer site sequences in the intercalation may be complementary to the primers used for amplification. Alternatively or additionally, the primer site sequences may be complementary to primers used for sequencing or other primer extension-based detection techniques. In other embodiments, two primer sites may be present in the intercalation, with the first primer site complementary to the amplification primer and the second primer site complementary to the primers used for sequencing or other primer extension-based detection techniques.

[0107] In some embodiments, the insert includes a first primer site and a second primer site, with one or more other portions situated between the primer sites, such as cleavage sites, binding portions, adapters, or other portions described below or elsewhere herein. Some embodiments may employ fork-shaped or Y-shaped adapter designs suitable for direct sequencing, such as those described in U.S. Patent No. 7,741,463. Figure 1 An example is shown in the image.

[0108] In some embodiments, it is advantageous to employ inserts having universal primer sites. For example, the target nucleic acid may be modified to include inserts comprising paired universal primer sites (one per strand). Universal primer sites can have various applications, such as hybridization sites for amplifying, sequencing, and / or recognizing primers in the target nucleic acid. When two primer sites are used, the first and second universal primer sites may be identical, substantially similar, analogous, or different. In some embodiments, to prepare a target nucleic acid comprising a first universal primer site (complementary to the first primer) and a second universal primer site (complementary to the second primer), the transposon element of the insert will comprise a first transposase recognition site and a second transposase recognition site separated by a linker. The first primer site may comprise a sequence that is the reverse complement of a sequence within the second primer. In some embodiments, the first primer site comprises a sequence doubly symmetrical to a sequence within the second primer. In some embodiments, the first primer site comprises a sequence symmetrical to the C2 sequence within the second primer. Multiple inserts can be inserted into the target nucleic acid via transposition in the presence of a transposase. The incorporated sequence can be cleaved to produce multiple target nucleic acid fragments, each including a first primer site and a second primer site.

[0109] The first and second universal primer sites can be incorporated into each target nucleic acid by various methods. For example, the first and second primer sites can be used to amplify the target nucleic acid using tailed-oligonucleotides. As is known in the art, the tailed-oligonucleotide may include a sequence complementary to the primer site and an additional sequence. In an exemplary embodiment, the first tailed-oligonucleotide includes a sequence complementary to the first primer site and a sequence for the first universal primer site, and the second tailed-oligonucleotide includes a sequence complementary to the second primer site and a sequence for the second universal primer site. Further examples are set forth in PCT Publication No. WO 2012 / 061832, U.S. Patent Application Publication No. 2012 / 0208724, U.S. Patent Application Publication No. 2012 / 0208705, and PCT Application Serial No. PCT / US2013 / 031023, each of which is incorporated herein by reference. It is to be understood that nucleic acid sequences including the first and second universal primer sites can be used in other sequencing methods.

[0110] The insert used in the methods or compositions of this disclosure may include one or more cleavage sites. The modified nucleic acid polymer including the insert may then be cleaved at the cleavage site to generate fragments of the modified nucleic acid polymer. Cleavage sites sensitive to biochemical, chemical, physical, or other cleavage mechanisms may be used. In some embodiments, the fragmentation site may include a nucleotide or nucleotide sequence that can be fragmented by various means. For example, the fragmentation site may be a substrate of an enzyme, such as a nuclease. Restriction endonuclease sites having nucleotide sequences particularly sensitive to the corresponding restriction endonucleases may be particularly useful.

[0111] In another embodiment, the fragmentation site may contain at least one ribonucleotide in a nucleic acid that would otherwise include a deoxyribonucleotide, and may be cleaved with RNase. Chemical cleavage agents capable of selectively cleaving the phosphodiester bond between the deoxyribonucleotide and the ribonucleotide may be used, including, for example, metal ions such as rare earth metal ions (e.g., La). 3+ Especially Tm 3+ Yb 3+ Or Lu 3+ (Fe(3) or Cu(3)), or exposure to elevated pH.

[0112] In another example, the cleavage site may include one or more recognition sequences for a nicking enzyme (i.e., a nicking endonuclease that breaks one strand of a double-stranded nucleic acid). Therefore, the fragmentation site may include a first nicking enzyme recognition sequence and optionally a second nicking enzyme recognition sequence. The first and second nicking enzyme recognition sequences may be the same as or different from each other.

[0113] In another example, the cleavage site may comprise one or more nucleotide analogs including a base-free site and allow cleavage at the fragmentation site in the presence of certain chemical reagents (such as polyamine, N,N'-dimethylethylenediamine (DMED)) (see, for example, U.S. Patent Application Publication No. 2010 / 0022403, which is incorporated herein by reference). In some embodiments, the base-free site may be generated by, for example, modifying the uracil nucleotide within the cleavage site with a uracil DNA glycosylase (UDG). The polynucleotide chain including the base-free site may then be cleaved at the base-free site by an endonuclease (e.g., Endo IV endonuclease, AP lyase, FPG glycosylase / AP lyase, Endo VIII glycosylase / AP lyase), heating, or alkaline treatment. The base-free site may also be generated at a nucleotide analog other than deoxyuridine and cleaved in a similar manner by an endonuclease, heating, or alkaline treatment. For example, 8-oxoguanine may be converted to a base-free site by exposure to FPG glycosylase. By exposure to AlkA glycosylation enzyme, deoxyinosine can be converted into a debasement site. The resulting debasement site can typically be cleaved by treatment with a suitable endonuclease, such as Endo IV or AP lyase (see, for example, U.S. Patent Publication No. 2011 / 0014657, which is incorporated herein by reference).

[0114] In another example, the fragmentation site may include a diol bond, which allows cleavage by treatment with periodate (e.g., sodium periodate). In yet another example, the fragmentation site may include a disulfide group, which allows cleavage with a chemical reducing agent, such as tris(2-carboxyethyl) phosphate hydrochloride (TCEP).

[0115] In some embodiments, the fragmentation site may include a photocleavage portion. Photochemical cleavage can be performed by any of a variety of methods that utilize light energy to break covalent bonds. The photochemical cleavage site may be provided by a non-nucleotide chemical portion of the nucleic acid, such as phosphoramidamide [4-(4,4'-dimethoxytriphenylmethoxy)butamidomethyl)-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidamide (Glen Research, Sterling, Va., USA, Cat No. 10-4913-XX).

[0116] In some embodiments, the fragmentation site may include a peptide, for example, wherein the peptide molecule is linked to a coupling structure of a nucleic acid. The peptide molecule may then be cleaved by a peptidase of appropriate specificity or by a suitable non-enzymatic chemical or photochemical cleavage method. In some embodiments, the coupling between the peptide and the nucleic acid is formed by covalently linking the peptide to a nucleic acid (e.g., one strand of a double-stranded nucleic acid). The coupling between the peptide and the nucleic acid can be prepared using techniques known in the art. In one such technique, the peptide and nucleic acid components of the desired amino acid and nucleotide sequences can be, for example, synthesized separately using standard automated chemical synthesis techniques and then coupled in an aqueous / organic solution. For example, the OPeC™ system, available from Glen Research, is based on the natural linking of an N-terminal thioester-functionalized peptide to a 5'-cysteine ​​oligonucleotide.

[0117] In specific embodiments, fragments of the modified nucleic acid polymer can be generated by amplifying discontinuous portions of the polymer. For example, in some embodiments, primer sites are inserted into the modified nucleic acid polymer, and the regions sandwiched between the primer sites can be amplified, for example, using primers hybridized to the primer sites via PCR amplification or similar amplification methods. It should be understood that in some embodiments, such as those where fragments are generated by amplification techniques, the modified nucleic acid polymer need not include cleavage sites.

[0118] In cases where modified nucleic acids are attached to beads or other solid-phase supports, fragments can be generated by disrupting the beads. For example, gel beads can be melted or dissolved to release the fragments attached to them.

[0119] The insert may include one or more ligands. The ligands present in the insert may be binding partners specific to a particular receptor. For example, the ligand may be specific to a receptor present on the surface of a solid support. Thus, the receptor-ligand binding may facilitate surface capture of modified nucleic acids or nucleic acid fragments containing a portion of the insert having the ligand. Examples of ligands and receptors include biotin or polyhistidine, which may bind streptavidin or nickel, respectively. Other examples include paired ligands and their receptors known in the art, such as avidin-biotin, streptavidin-biotin, and biotin, streptavidin, or derivatives of avidin, including but not limited to 2-iminobiotin, desulfobiotin, NeutrAvidin (Molecular Probes, Eugene, Oreg.), CaptAvidin (Molecular Probes, Eugene, Oreg.), etc. Probes, etc.; binding proteins / peptides, including maltose-maltose binding protein (MBP) and calcium-calcium binding protein / peptide (CBP); antigen-antibody pairs, including epitope tags (including c-MYC, HA, VSV-G, HSV, V5, and FLAGTag™) and their corresponding anti-epitaxy antibodies; haptens (e.g., dinitrophenyl and digoxigenin) and their corresponding antibodies; aptamers and their corresponding targets; polyhistidine tags (e.g., pentahistidine and hexahistidine) and their binding partners, including corresponding immobilized metal ion affinity chromatography (IMAC) materials and anti-polyhistidine antibodies; fluorophores and anti-fluorophore antibodies; nucleic acid chains and their complementary chains; etc.

[0120] In some embodiments, the insert may include a reporting portion. Useful reporting portions include any of a variety of identifiable tags, markers, or groups known in the art. In some embodiments, the reporting portion may emit a signal. Examples of signals include those that are fluorescence, chemiluminescence, bioluminescence, phosphorescence, radioactivity, calorimetry, or electrochemiluminescence. Exemplary reporting portions include fluorophores, radioactive isotopes, chromogens, enzymes, antigens including epitope tags, semiconductor nanocrystals (such as quantum dots), heavy metals, dyes, phosphorescent groups, chemiluminescent groups, electrochemical detection portions, binding proteins, phosphors, rare earth chelates, transition metal chelates, near-infrared dyes, electrochemiluminescent markers, and mass spectrometry-compatible reporting portions such as mass tags, charge tags, and isotopes. Further reporting portions that can be used with the methods and compositions described herein include spectral markers such as fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, etc.); radioactive markers (e.g., 3 H, 125 I, 35 S, 14 C 32 P, 33P, etc.); enzymes (e.g., horseradish peroxidase, alkaline phosphatase, etc.); spectral colorimetric markers, such as colloidal gold, colored glass, or plastics (e.g., polystyrene, polypropylene, latex, etc.); beads; magnetic markers; electrical markers; thermal markers; and quality labels.

[0121] In some embodiments, multiple different inserts may be used, each of which can be distinguished by the presence of a unique barcode sequence. Therefore, multiple barcode-containing inserts can be added to the target nucleic acid to generate a modified nucleic acid polymer that has multiple unique barcodes throughout. Exemplary barcodes and methods for their preparation and use are described in PCT Publication No. WO 2012 / 061832, U.S. Patent Application Publication No. 2012 / 0208724, U.S. Patent Application Publication No. 2012 / 0208705, and PCT Application Serial No. PCT / US2013 / 031023, each of which is incorporated herein by reference. In alternative embodiments, it is to be understood that these barcodes are not necessary. This is possible, for example, when the linkage or proximity information retained by the methods described herein is sufficient to obtain an accurate nucleotide sequence characterization of the target nucleic acid polymer without the need for the phenotypic methods described in the above documents. Therefore, in specific embodiments, the multiple inserts used in the methods described herein will not include sequences (e.g., barcodes) that are unique to each other. Conversely, all inserts used in the methods described herein, present in the modified nucleic acid polymers of this disclosure, or present in the multiple nucleic acid fragments of this disclosure, may have the same sequence (i.e., a universal series). Therefore, some embodiments of the methods described herein will be carried out without distinguishing the sequences of individual inserts (i.e., differentiating one insert from another), which may or may not be present in the modified nucleic acid polymer or fragments thereof.

[0122] The method disclosed herein may include the step of releasing fragments of a modified nucleic acid polymer into a fluid. In some embodiments, each of the fragments will include at least a portion of an insert previously added to the nucleic acid polymer. In a particular embodiment, the step may include (i) contacting the modified nucleic acid polymer with a solid support surface, and (ii) releasing fragments of the modified nucleic acid polymer into a fluid. When multiple different modified nucleic acid polymers are contacted with a solid support surface, it is generally advantageous to do so with the individual modified nucleic acid polymers spatially separated from each other. For example, the polymer may be delivered at a relatively diluted concentration to a container having a surface (e.g., a flow cell with a detection surface) such that the average distance between the closest adjacent polymers in the container or on the surface is within the same relative range as the expected average distance between the closest adjacent fragments when captured on the surface. Similar spatial separation can be achieved by delivering the modified nucleic acid polymer attached to a bead. In cases where the released fragments are used as templates for amplification to form clusters (or clones) on the surface, once the fragments are amplified on the surface, the distance between the closest adjacent polymers may be within the range of the expected average spacing of the clusters.

[0123] In a specific embodiment, multiple different modified nucleic acid polymers are delivered to a flow cell (or other container) and allowed to diffuse (actively or passively) through a fluid in contact with a surface (on which fragments will subsequently be attached). Thus, the polymer can be delivered under stopped-flow conditions, wherein the polymer-loaded solution first flows into the flow cell and then the flow is stopped to allow diffusion. Figure 2 The diagram illustrates an embodiment in which three different modified nucleic acid polymers are allowed to diffuse to locations along the surface of a flow cell. As illustrated above, the solid support surface may optionally include a capture portion for binding to a binding chaperone present on the polymer. Any of the various receptors and ligands set forth herein or otherwise known in the art may be used for this purpose. Thus, the insert may include a ligand, and the solid support surface may include a receptor for that ligand, such that contact between the modified nucleic acid polymer and the solid support surface will attach the modified nucleic acid polymer to the surface.

[0124] The modified nucleic acid can be attached to a solid support (such as a bead) and the bead can be delivered to a container with a solid surface. Figure 5 Exemplary modified target nucleic acids are shown on the beads. Figure 6 Another example of a modified target nucleic acid displayed on a bead. A solid-phase carrier having a target nucleic acid, a modified nucleic acid, or a fragment thereof may be allowed to contact a solid support surface (e.g., by gravity sedimentation) and, in some cases, may be attached to that surface using the receptors and ligands or other means described herein.

[0125] Once one or more distinct modified nucleic acids are present in or near corresponding regions on the surface of a solid support, fragments of the modified nucleic acids can be generated. In some embodiments, the distinct modified nucleic acid polymers can be cleaved to generate fragments of the polymers. Cleavage can occur at cleavage sites present in the inserts that have been integrated into the modified nucleic acid polymers. Alternatively or additionally, fragments can be generated by amplifying the sequence regions of the modified nucleic acids. For example, amplification can be performed using primers annealed to primer sites that have been added to the inserts in the modified nucleic acids. Denaturation can optionally be performed to generate single-stranded versions of the fragments. Fragments generated from one or more modified nucleic acid polymers can be randomly captured at locations on the surface of the solid support, as described in more detail below.

[0126] As an alternative to passive diffusion of modified nucleic acid polymers or fragments thereof, active transport techniques can be used to move molecules to desired locations or influence the spatial configuration of molecules. A particularly useful active transport technique is electric field-assisted (e-field-assisted) transport. For example, one or more regions of an array can be electrically connected to a power source to generate charges that attract the target nucleic acid polymer or fragments thereof. In some configurations, a positive charge can attract nucleic acids via a negatively charged sugar-phosphate backbone. Exemplary methods and apparatus for employing e-field assistance to attract nucleic acids to surfaces are described in U.S. Patent Application Publication No. 2009 / 0032401 A1 or U.S. Patent Application Serial No. 13 / 783,043, each of which is incorporated herein by reference. Other techniques that can be used for the active transport of nucleic acids include, but are not limited to, fluid flow under positive or negative pressure, gravity induced by centrifugation, movement along chemical or temperature gradients, and so on.

[0127] The spatial configuration of modified nucleic acid polymers can be controlled using the methods described herein. In specific embodiments, the polymer can be folded into a relatively compact configuration, for example, using chemical, temperature, or electrical conditions. Exemplary conditions for producing compact structures include those for producing DNA nanospheres, as described in U.S. Patent Application Publication No. 2008 / 0242560 A1 or U.S. Patent Application Publication No. 2008 / 0234136 A1, each of which is incorporated herein by reference. Alternatively, one or more modified nucleic acid polymers can be stretched or otherwise configured into elongated configurations. For example, the polymer can be attached to a surface (e.g., at or near the ends of the polymer) and elongated along a region of the surface. Fragments derived from the polymer can ultimately be attached to a region of the surface according to the methods described herein. Exemplary techniques for elongating nucleic acid polymers include, but are not limited to, fluid flow stretching, weak force stretching, e-field assisted stretching, or other methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0129704 A1, which is incorporated herein by reference. Therefore, this The method of the invention may include steps (i) stretching the modified nucleic acid polymer along the surface of a solid support, and (ii) releasing fragments of the modified nucleic acid polymer into a fluid.

[0128] Conditions in the methods of this disclosure can be selected to obtain fragments of desired lengths. For example, this length can be influenced by the conditions used to attach the insert to the target nucleic acid polymer. In cases where the insert contains cleavage sites, the length of the resulting fragment will be related to the average distance between the inserts in the polymer. The average size of the fragment can also be affected by the degree of completion of the allowed cleavage reaction. A cleavage reaction run to substantially completion will produce fragments related to the average distance between cleavage sites in the modified nucleic acid polymer, while a cleavage reaction run to partial completion will produce fragments with an average length greater than the average distance between cleavage sites.

[0129] In some embodiments, the absolute or average fragment length may be at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides. In some embodiments, the absolute or average fragment length may be at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides. In some embodiments, the absolute or average fragment length may be at least about 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 90 kb, 100 kb or more nucleotides. In some embodiments, the absolute or average fragment length may be at least about 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb or more nucleotides. As an alternative to the thresholds described above, or otherwise, the absolute or average fragment length may be up to about 1000 kb, 500 kb, 100 kb, 10 kb, 5 kb, 1 kb, 500 bases, 100 bases, 50 bases, or 10 bases or less.

[0130] In some embodiments, target nucleic acids, modified nucleic acids, or nucleic acid fragments may be attached to the surface of a solid support. The solid support may be two-dimensional or three-dimensional and may be a flat surface (e.g., a glass slide) or may be shaped. Useful materials include glass (e.g., controlled-aperture glass (CPG)), quartz, plastics (such as polystyrene (low-crosslinked and high-crosslinked polystyrene), polycarbonate, polypropylene, and poly(methyl methacrylate)), acrylic copolymers, polyamides, silicon, metals (e.g., alkylthiol-derived gold), cellulose, nylon, latex, dextran, gel matrices (e.g., silica gel), polyacrylaldehyde, or complexes. Suitable three-dimensional solid supports include, for example, spheres, microparticles, beads, membranes, glass slides, plates, micromechanical processing chips, tubular structures (e.g., capillaries), micropores, microfluidic devices, grooves, filters, or any other structure suitable for anchoring nucleic acids. The solid support may include a planar microarray or matrix capable of having regions comprising nucleic acid or primer groups. Examples include nucleoside-derived CPG and polystyrene slides; derived magnetic slides; polystyrene grafted with polyethylene glycol, etc.

[0131] Various methods can be used to attach, anchor, or fix nucleic acids to the surface of a solid support. This attachment can be achieved through direct or indirect binding to the surface. This binding can be via covalent bonds. See Joos et al. (1997). Analytical Biochemistry (Analytical Biochemistry), 247:96-101; Oroskar et al. (1996) Clin. Chem(Clinical Chemistry), 42:1547-1555; and Khandjian (1986) Mol. Bio. Rep (Molecular Biology Research), 11:107-11, each of which is incorporated herein by reference. Preferred attachment is a direct amine bond between the terminal nucleotide of the nucleic acid and the epoxide integrated onto the surface. This bond can also be non-covalent. For example, biotin-streptavidin (Taylor et al. (1991)). J. Phys. D: Appl. Phys. (Applied Physics), 24:1443, the referenced document is incorporated herein by reference) and digoxin and anti-digoxin (Smith et al., Science (Science), 253:1122 (1992), the reference of which is incorporated herein by reference) is a common tool for anchoring nucleic acids to surfaces.

[0132] Nucleic acids can be attached to a surface via intermediate structures such as beads, particles, or gels. Examples of attaching nucleic acids to an array via gels include flow cells available from Illumina Inc. (San Diego, CA), or described in U.S. Patent Application Publication Nos. 2010 / 0111768 A1 or 2012-0270305 A1; or WO 05 / 065814, each of which is incorporated herein by reference. Exemplary gels that may be used in the methods and apparatus described herein include, but are not limited to, gels having a colloidal structure, such as agarose; gels having a polymeric network structure, such as gelatin; or gels having a crosslinked polymeric structure, such as polyacrylamide, SFA (see, for example, U.S. Patent Application Publication No. 2011 / 0059865 A1, which is incorporated herein by reference) or PAZAM (see, for example, U.S. Provisional Patent Application Serial No. 61 / 753,833 and WIPO Application Serial No. PCT / US2013 / 044305, each of which is incorporated herein by reference).

[0133] Although this disclosure describes several embodiments of modifying the target nucleic acid before it is attached to the surface of a solid support, it should be understood that modification of the target nucleic acid before attachment is necessary. For example, the target nucleic acid may be attached to the solid support before modification by the methods described herein. Transposase treatment may occur after the target nucleic acid has been attached to the surface of the solid support. Exemplary embodiments are illustrated in Figure 7In this embodiment, the target nucleic acid is attached to a surface (Illustration A), and treated with a transposase via tag-fragmentation to generate multiple fragments (Illustration B), resulting in the release of several fragments containing the insert into the solution (Illustration C). These fragments can be randomly captured at sites within regions of the solid support surface. In embodiments where multiple different target nucleic acids are attached to different regions of the surface, fragments can be released and captured under conditions where fragments from each target nucleic acid are ultimately randomly located at different positions within their respective regions. As described in more detail herein, the captured fragments can optionally be amplified and / or detected.

[0134] In embodiments where an insert is attached to a target nucleic acid polymer using a transposase, the transposase may be present when the nucleic acid polymer modified with the insert comes into contact with a substrate surface, or the transposase may be removed from the nucleic acid polymer modified with the insert prior to such contact. In specific embodiments, the transposase may include receptors or ligands that bind to corresponding ligands or receptors on a solid support surface. Alternatively or additionally, the ligand or receptor mediating binding to the solid support may be present in the insert of the modified nucleic acid polymer. Thus, the insert can mediate binding to the surface regardless of whether the transposase is attached to the modified nucleic acid polymer. The transposase can be removed from the nucleic acid using methods known in the art, such as thermal denaturation, chemical denaturation (e.g., surfactant treatment), or treatment with one or more proteases.

[0135] In many embodiments, the solid support to which nucleic acids are attached in the methods described herein will have a continuous or monolithic surface. Therefore, fragments can be attached at spatially random locations, where the distance between the closest adjacent fragments (or the closest adjacent clusters derived from fragments) is variable. The resulting array will have a variable or random spatial pattern of features. Alternatively, the solid support used in the methods described herein may comprise an array of features present in a repeating pattern. In these embodiments, the features provide locations where the modified nucleic acid polymer or fragments thereof can be attached. Particularly useful repeating patterns are hexagonal patterns, linear patterns, grid patterns, patterns with reflection symmetry, patterns with rotational symmetry, etc. The features to which the modified nucleic acid polymer or fragments thereof are attached may each have a size of less than about 1 mm. 2 500 μm 2 100 μm 2 25 μm 2 10 μm 2 5 μm 2 1 μm 2 500 nm 2 or 100 nm 2 The area. Alternatively, or additionally, each feature may have an area greater than approximately 100 nm.2 250 nm 2 500 nm 2 1 μm 2 2.5μm 2 5 μm 2 10 μm 2 100 μm 2 or 500 μm 2 The area of ​​a cluster or clone generated by fragment amplification on an array (whether patterned or spatially random) can similarly have an area greater than or between the upper and lower limits selected from the examples above.

[0136] In embodiments comprising an array of features on a surface, the features may be discrete, i.e., separated by gap regions. Alternatively, some or all of the features on the surface may be adjacent (i.e., not separated by gap regions). Regardless of whether the features are discrete or adjacent, the average size of the features and / or the average distance between the features may vary, such that the array may be high-density, medium-density, or low-density. A high-density array is characterized by features having an average spacing of less than about 15 μm. A medium-density array has an average feature spacing of about 15 to 30 μm, while a low-density array has an average feature spacing of greater than 30 μm. Arrays that can be used in the present invention may have, for example, feature spacing of less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm. Alternatively or additionally, the feature spacing may be, for example, greater than 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, or 100 μm.

[0137] Several commercially available sequencing platforms rely on well-drained substrates that provide a diffusion barrier to detection reagents (e.g., pyrophosphates available from 454 LifeScience (a subsidiary of Roche, Basel, Switzerland) or protons available from Ion Torrent (a subsidiary of Life Technologies, Carlsbad, California)) during the sequencing step. The methods described herein can be advantageously used to deliver fragments of modified nucleic acids into the wells in a manner that preserves phase or proximity information.

[0138] The method disclosed herein may include the step of capturing fragments from a modified nucleic acid polymer onto a surface of a solid support. Typically, the fragments are first released from the polymer into a fluid and then captured from the fluid, such that the fragments are ultimately randomly located at specific sites on the surface. These sites may be on a monolithic or continuous surface, or alternatively, these sites may be prefabricated features on a patterned array. Thus, the location of fragment attachment can be random with regard to the predictability or knowledge of where any particular fragment will attach (i.e., what nucleic acid sequence will be present at a particular site), regardless of whether the spatial pattern of the features is random.

[0139] Fragments in a fluid can be randomly transported to locations on a surface via passive diffusion or active transport. Exemplary conditions and techniques that can be used for such transport are known in the art, or are exemplified herein in the context of transporting modified nucleic acids to surfaces.

[0140] The method disclosed herein may also include the step of amplifying fragments at a location on the surface to generate an amplified fragment. For example, the amplified fragment can be detected by nucleic acid sequencing techniques described below. In a specific embodiment, these fragments may be amplified using at least one primer attached to the surface. The primers used for amplification are hybridizable to primer sites on the insert, at least in some configurations. Extendable primers generate an amplified fragment attached to the surface (e.g., at a specific location). Solid-phase extension methods may be used, as described in more detail below.

[0141] The methods disclosed herein may include the steps of amplifying a target nucleic acid, a modified nucleic acid, or a portion thereof. Any suitable amplification methodology known in the art may be employed. In some embodiments, the nucleic acid fragment is amplified on a solid support. For example, in some embodiments, a bridging amplification methodology is used to amplify the nucleic acid fragment, as illustrated in U.S. Patent Nos. 5,641,658; 2002 / 0055100; 7,115,400; 2004 / 0096853; 2004 / 0002090; 2007 / 0128624; and 2008 / 0009420, each of which is incorporated herein by application. The bridging amplification method allows the amplification products to be immobilized on a solid support to form an array comprising immobilized clusters (or “clones”) of nucleic acid molecules. Each cluster or clone on such an array is formed by a plurality of identical immobilized polynucleotide chains and a plurality of identical immobilized complementary polynucleotide chains. The array thus formed is referred to herein as a "cluster array". When formed by annealing pairing of an immobilized polynucleotide chain and an immobilized complementary chain, both chains are immobilized at the 5' end on a solid support, preferably by covalent linkage, and the product of the solid-phase amplification reaction is a so-called "bridged" structure. The bridged amplification methodology is an example of a method for using an immobilized nucleic acid template to generate immobilized amplicons. Other suitable methodologies can also be used to generate immobilized amplicons from immobilized nucleic acid fragments generated according to the methods provided herein. For example, one or more clusters or clones can be formed by solid-phase PCR, solid-phase MDA, solid-phase RCA, etc., regardless of whether one or both primers in each amplification primer pair are immobilized.

[0142] In other embodiments, the target nucleic acid, the modified nucleic acid, or fragments thereof are amplified in solution. For example, in some embodiments, amplification primers are hybridized to primer sites of the insert in solution. In other embodiments, amplification primers are hybridized to the insert when the modified nucleic acid or fragment thereof is attached to a solid support.

[0143] It is to be understood that any amplification methodology described herein or known in the art can be used with universal or target-specific primers to amplify immobilized DNA fragments. Suitable methods for amplification include, but are not limited to, polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), and nucleic acid sequence-dependent amplification (NASBA), such as that described in U.S. Patent No. 8,003,354, which is incorporated herein by reference. The above amplification methods can be used to amplify one or more target nucleic acids. For example, PCR, multiplex PCR, SDA, TMA, NASBA, etc., can be used to amplify immobilized nucleic acid fragments. In some embodiments, primers specifically targeting the target nucleic acid are included in the amplification reaction.

[0144] Other suitable methods for amplifying nucleic acids may include oligonucleotide extension and ligation, rolling circle amplification (RCA) (Lizardi et al., Nat. Genet (Nature Genetics) 19:225-232 (1998), the references of which are incorporated herein by reference) and Oligonucleotide Linking Detection (OLA) (generally see U.S. Patent Nos. 7,582,420, 5,185,243, 5,679,524 and 5,573,907; EP 0 320 308 B1; EP 0 336 731 B1; EP 0 439 182 B1; WO 90 / 01069; WO 89 / 12696; and WO 89 / 09835, each of which is incorporated herein by reference). It is to be understood that these amplification methodologies can be designed to amplify immobilized nucleic acid fragments. For example, in some embodiments, the amplification method may include linker probe amplification or an oligonucleotide linking detection (OLA) reaction containing primers specifically targeting the target nucleic acid. In some embodiments, the amplification method may include a primer extension-linking reaction containing primers specifically targeting the target nucleic acid. As a non-limiting example of primer extension and linking primers that can be specifically designed to amplify the target nucleic acid, the amplification may include primers for the GoldenGate® assay (Illumina, Inc., San Diego, CA), as illustrated in U.S. Patent Nos. 7,582,420 and 7,611,869, each of which is incorporated herein by reference in its entirety.

[0145] Isothermal amplification techniques can be used in the methods disclosed herein. Exemplary isothermal amplification methods include, but are not limited to, those described by Dean et al. Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America) Multiple displacement amplification (MDA) illustrated in, for example, 99:5261-66 (2002), or isothermal strand displacement nucleic acid amplification illustrated in, for example, U.S. Patent No. 6,214,587, each of which is incorporated herein by reference. Other non-PCR-based methods that may be used in this disclosure include, for example, strand displacement amplification (SDA), as described in, for example, Walker et al. Molecular Methods for Virus Detection (Molecular Biological Methods for Virus Detection), Academic Press, Inc., 1995; US Patent Nos. 5,455,166 and 5,130,238, and Walker et al. Nucl. Acids Res The amplification is described in *Nucleic Acid Research* 20:1691-96 (1992); or hyperbranched strand displacement amplification, which is described, for example, in Lage et al., *Genome Research* 13:294-307 (2003), each of which is incorporated herein by reference. Further descriptions of amplification reactions, conditions, and components are set forth in U.S. Patent No. 7,670,810, which is incorporated herein by reference. Other useful isothermal amplification techniques include recombinase-assisted amplification techniques, such as those commercially available by TwistDx (Cambridge, UK) as the TwistAmp™ kit. Useful components of recombinase-assisted amplification reagents and reaction conditions are set forth in US 5,223,414 and US 7,399,590, each of which is incorporated herein by reference. Helicase-dependent amplification can also be used, for example, as described in Xu et al. EMBO Rep The reference described in (European Organization for Molecular Biology Reports) 5:795-800 (2004) is incorporated herein by reference.

[0146] In some embodiments, a re-seeding step may be desired. For example, a nucleic acid fragment may be captured at a location within a surface region, replicated over one or more cycles of an amplification method, the original fragment and / or its replicons may be released from that location, the released nucleic acid may be captured at other locations within the same region, and the newly captured nucleic acid may be amplified. In a particular example, a single-cycle bridging amplification may be performed on the fragment seeded on the surface, and instead of washing away the original template fragment after release from the surface, the template fragment is re-seeded at a new location on the surface near its original seeding location. Subsequent rounds of bridging amplification will allow clusters to grow at both the original seeding location and the re-seeding location. Using these methods, replicative clones can be generated at the surface region to provide technical replicates. Sequence analysis of technical replicates can provide the benefit of error checking. For example, sequence variations observed only in a subset of neighboring clusters (identified as technical replicates) may be identified as amplification errors, while sequence variations appearing in all clusters identified as technical replicates are more likely to be true variations.

[0147] The methods described herein may include steps for sequencing fragments derived from target nucleic acids. One example is sequencing-by-synthesis (SBS). In SBS, the extension of a nucleic acid primer along a nucleic acid template (e.g., a fragment of the target nucleic acid or its replicons) is monitored to determine the nucleotide sequence in the template. The primer may hybridize to primer sites present in the insert, as described above. The underlying chemical process may be a polymerization reaction (e.g., catalyzed by a polymerase). In a specific polymerase-based SBS embodiment, fluorescently labeled nucleotides are added to the primer in a template-dependent manner (thereby extending the primer), such that the sequence and type of nucleotides added to the primer can be used to determine the sequence of the template. SBS technology can be used on multiple different nucleic acid fragments that have been attached to different positions in the array using the steps described herein, provided that events occurring on different templates can be distinguished based on their positions in the array.

[0148] A flow cell provides a convenient form for containing arrays of nucleic acid fragments generated by the methods of this disclosure and subjected to SBS or other detection techniques involving repeated delivery of reagents in cycles. For example, to initiate a first round of SBS cycling, one or more labeled nucleotides, DNA polymerases, etc., can flow into and / or through the flow cell containing the array of nucleic acid fragments. Those sites in the array that can be detected, where primer extension (e.g., by hybridization of the primer with a primer site located on an insert linked to a nucleic acid fragment) results in the incorporation of a labeled nucleotide. Optionally, these nucleotides may further include reversible termination properties, terminating further primer extension once the nucleotide has been added to the primers. For example, nucleotide analogs with reversible termination portions can be added to the primers such that subsequent extensions cannot occur until a deblocking agent is delivered to remove the portion. Thus, in embodiments using reversible termination, a deblocking agent can be delivered to the flow cell (before or after detection occurs). Washing can be performed between delivery steps. This cycle can then be repeated n times to extend the primer with n nucleotides, thereby detecting sequences of length n. For example, in Bentley et al., Nature Exemplary SBS operations, fluid systems, and detection platforms are described in Nature 456:53-59 (2008), WO 04 / 018497; US 7,057,026; WO 91 / 06678; WO 07 / 123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281, and US2008 / 0108082, which can be readily modified for use with arrays generated by the methods of this disclosure, each of which is incorporated herein by reference.

[0149] Other sequencing operations employing cyclic reactions, such as pyrosequencing, can be used. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as specific nucleotides are incorporated into the nascent nucleic acid chain (Ronaghi et al.). Analytical Biochemistry (Analytical Biochemistry) 242(1), 84-9 (1996); Ronaghi, Genome Res. (Genome Research) 11(1), 3-11 (2001); Ronaghi et al., Science ("science") US Patent 281(5375), 363 (1998); US 6,210,891; US ​​6,258,568 and US 6,274,320, each of which is incorporated herein by reference. In pyrosequencing, the released PPi is detected by being converted to adenosine triphosphate (ATP) by ATP sulfatase, and the level of ATP produced can be detected by photons generated by luciferase. Therefore, the sequencing reaction can be monitored by a luminescent detection system. The excitation radiation source used in a fluorescence-based detection system is not necessary for the pyrosequencing operation. Useful fluid systems, detectors and operations that can be used to apply pyrosequencing to the methods of this disclosure are described, for example, in WIPO patent application serial numbers PCT / US11 / 57111, US 2005 / 0191698 A1, US 7,595,883 and US 7,244,559, each of which is incorporated herein by reference.

[0150] Ligation-while-sequencing reactions are also useful, including, for example, those described by Shendure et al. Science (Science) 309:1728-1732 (2005); US 5,599,675; and those described in US 5,750,341, each of which is incorporated herein by reference. Some embodiments may include hybridization-while-sequencing operations, for example, in Bains et al. Journal of Theoretical Biology (Journal of Theoretical Biology) 135(3) 303-7 (1988); Drmanac et al., Nature Biotechnology (Nature Biotechnology) 16, 54-58 (1998); Fodor et al., Science References 251(4995), 767-773 (1995); and WO 1989 / 10977, each of which is incorporated herein by reference. In both ligation-while-sequencing (SBS) and hybridization-while-sequencing (HBS) operations, the target nucleic acid fragment (or its amplicon) present at the array site undergoes repeated cycles of oligonucleotide delivery and detection. Fluid systems used in the SBS methods described herein or cited herein can be readily modified for delivery of reagents in ligation-while-sequencing or HBS-while-sequencing operations. Typically, the oligonucleotides are fluorescently labeled and can be detected using fluorescent detectors similar to those described herein when referring to SBS operations or cited herein.

[0151] Some embodiments may utilize methods involving real-time monitoring of DNA polymerase activity. For example, nucleotide incorporation can be detected via fluorescence resonance energy transfer (FRET) interaction between a fluorophore-containing polymerase and a γ-phosphate-labeled nucleotide, or via zero-mode waveguides (ZMWs). Methods and reagents for FRET-based sequencing are, for example, described by Fevene et al. Science (Science) 299, 682-686 (2003); Fundquist et al. Opt. Lett (Optics Letters) 33, 1026-1028 (2008); and Korlach et al. Proc. Natl. Acad. Sci. USA As described in the Proceedings of the National Academy of Sciences of the United States of America (PNAS) 105, 1176-1181 (2008), each of which is incorporated herein by reference.

[0152] Some SBS embodiments include the detection of protons released when nucleotides are incorporated into the extension product. For example, sequencing based on the detection of released protons may employ electrical detectors and related technologies available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or the sequencing methods and systems described in US 2009 / 0026082 A1; US ​​2009 / 0127589 A1; US ​​2010 / 0137143 A1; or US 2010 / 0282617 A1, each of which is incorporated herein by reference.

[0153] The sequencing steps in this method may include nanopore sequencing technology, such as Deamer & Akeson. Trends Biotechnol (Trends in Biotechnology) 18, 147-151 (2000); Deamer & Branton, Acc. Chem. Res (Chemical Research Review) 35:817-825 (2002); and Li et al., Nat. Mater. Those described in (Nature Materials) 2:611-615 (2003), each of which is incorporated herein by reference. In these embodiments, the target nucleic acid fragment passes through a nanopore. The nanopore may be a synthetic pore or a biomembrane protein, such as α-hemolysin. As the target nucleic acid passes through the nanopore, each base pair can be identified by measuring the conductivity fluctuations of the pore. (US Patent No. 7,001,792; Soni & Meller) Clin. Chem.(Clinical Chemistry) 53, 1996-2001 (2007); Healy, Nanomed. (Nanomedicine) 2:459-481 (2007); and Cockroft et al., J. Am. Chem. Soc. (Journal of the American Chemical Society) 130:818-820 (2008, each of which is incorporated herein by reference). In some embodiments, the location of the individual nanopores is analogous to sites or features on the array illustrated herein. The proximity of the nanopores to each other may be correlated with the proximity of their read length fragment sequences in order to, for example, facilitate the assembly of these fragments into a larger sequence (from which the fragments originate).

[0154] The sequencing steps described herein can advantageously be performed in multiplexed forms, allowing for the simultaneous manipulation of multiple different target nucleic acids. In certain embodiments, different target nucleic acids may be processed in a common reaction vessel or on the surface of a specific substrate. This allows for convenient delivery of sequencing reagents, removal of unreacted reagents, and detection of incorporation events in multiplexed manner. In embodiments employing surface-bound target nucleic acids or fragments thereof, the target nucleic acids or fragments thereof may be in array form. In array form, fragments of the target nucleic acid may typically be bound to the surface in a spatially distinguishable manner, for example, using the attachment techniques described herein. The array may include a single copy of the target nucleic acid fragment at each site (also referred to as a feature), or multiple copies having the same sequence may be present at each site or feature. Multiple copies may be generated by amplification methods, such as bridging amplification or emulsion PCR as described in more detail herein.

[0155] The method described herein can employ an array of features, the density of which is any of a variety of densities, including, for example, at least about 10 features / cm². 2 100 features / cm 2 500 features / cm 2 1,000 features / cm 2 5,000 features / cm 2 10,000 features / cm 2 50,000 features / cm 2 100,000 features / cm 2 1,000,000 features / cm 2 5,000,000 features / cm 2 10 7 Features / cm 2 5x10 7 Features / cm 2 10 8 Features / cm2 5x10 8 Features / cm 2 10 9 Features / cm 2 5x10 9 Features / cm 2 , or higher.

[0156] The methods disclosed herein may include the step of determining haplotype phases for polymorphisms appearing in the nucleotide sequences of different fragments released from the modified nucleic acid polymer. Therefore, nucleotide sequence characterization of the target nucleic acid polymer may include haplotype phase characterization of alleles appearing in the nucleotide sequences of at least two different fragments released from the modified nucleic acid polymer.

[0157] The method disclosed herein may include the step of comparing complementary sequences determined at adjacent locations on a solid support surface to identify sequence errors.

[0158] According to the methods described herein, the relative proximity of any two fragment types on a solid support can provide useful information for aligning sequence information obtained from the two fragments. Specifically, the distance between clusters on a solid support originating from any two given fragments can be positively correlated with the probability that the two clusters originate from the same target polynucleotide molecule, as described in more detail in WO 2012 / 025250, which is incorporated herein by reference. Similarly, the distance between clusters on a solid support originating from any two given fragments can be positively correlated with the probability that the two clusters originate from a common source (since all fragments of a particular target polynucleotide will originate from the source of that target polynucleotide).

[0159] For example, in some embodiments, fragments of long nucleic acid molecules captured at the surface of the flow cell appear in a straight line across the flow cell surface (e.g., if the nucleic acid is stretched before fragmentation or amplification), or appear in clouds on the surface (e.g., if the nucleic acid is stretched). Figure 2 (As shown in the clusters). Additionally, a physical map of the immobilized nucleic acids can then be generated. This physical map is thus related to the physical relationships between the clusters of immobilized nucleic acids after amplification. Specifically, the physical map is used to calculate the probability of linkage between sequence data obtained from any two clusters, as described in the incorporated WO 2012 / 025250 material. Alternatively, or additionally, the physical map can indicate the genome of a specific organism in a metagenomic sample. In the latter case, the physical map can indicate the order of sequence fragments in the organism's genome; however, it is not necessary to specify this order, and conversely, for a physical map characterizing a mixed sample and one or more organisms therein, the mere presence of two or more fragments in a common organism (or other source) is sufficient.

[0160] In some embodiments, the physical map is generated by imaging a solid support to establish the position of the immobilized nucleic acid molecules across the surface. In some embodiments, the immobilized nucleic acids are imaged by adding an imaging agent to the solid support and detecting the signal from the imaging agent. In some embodiments, the imaging agent is a detectable marker. Suitable detectable markers include, but are not limited to, protons, haptens, radionuclides, enzymes, fluorescent markers, chemiluminescent markers, and / or chromogenic agents. For example, in some embodiments, the imaging agent is an intercalating dye or a non-intercalating DNA binder. Any suitable intercalating dye or non-intercalating DNA binder known in the art may be used, including but not limited to those set forth in US2012 / 0282617, which is incorporated herein by reference.

[0161] In some embodiments, multiple modified nucleic acid molecules flow into a flow cell comprising multiple nanogrooves. As used herein, the term nanogroove refers to a narrow groove into which a long linear nucleic acid molecule is stretched. In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or no more than 1000 individual long-chain nucleic acids are stretched along each nanogroove. In some embodiments, the individual nanogrooves are separated by a physical barrier that prevents individual long-chain target nucleic acids from interacting with the multiple nanogrooves. In some embodiments, the solid support comprises at least 10, 50, 100, 200, 500, 1000, 3000, 5000, 10000, 30000, 50000, 80000, or at least 100000 nanogrooves.

[0162] In some embodiments, nucleic acids are modified to include inserts with cleavage sites, and the cleavage sites are cleaved once the nucleic acids are extended along the nanogrooves. The resulting fragments may optionally be amplified to form clusters along the groove surface. Then, for example, adjacency mapping can be performed by the clusters along the length direction of one of these grooves. For example, a flow cell having 1000 or more nanogrooves (with mapped, immobilized fragment products in the nanogrooves) can be used to sequence the genome of an organism at short, “positioning” reads. In some embodiments, the mapped, immobilized fragment products in the nanogrooves can be used to resolve haplotypes. In some embodiments, the mapped, immobilized fragment products in the nanogrooves can be used to resolve phasing problems.

[0163] In some embodiments, the methods described above in this invention can be used for error correction of sequencing read lengths. Figure 11An example of this error correction is shown. Two sequencing reads, A and B, differ from the reference genome at the same position (C replaces A). The clusters that produced reads A and B are 350 μm apart on the flow cell. This distance is far greater than the distance caused by sequencing human error, although it is within the average radius (200–300 μm) of nearby groups. Therefore, combined with the fact that the two reads have the same start-stop position and are in opposite directions, it can be inferred that they started from complementary fragments, and that the C mutation shown here is not a human error in sample preparation or sequencing.

[0164] In some embodiments, the methods described above can be used for gene expression analysis. In some embodiments, the target mRNA originates from a single cell. In some embodiments, the mRNA can be captured on beads comprising oligomeric d(T) probes.

[0165] The various combinations of components described above with respect to exemplary reaction mixtures and reaction methods can be provided in the form of a kit. Such a kit may include individual components separate from each other, for example, contained in separate containers or packages. The kit may include one or more sub-combinations of the components described herein, which are separate from other components of the kit. These sub-combinations are combinable to produce the reaction mixture described herein (or combined to implement the methods described herein). In certain embodiments, sub-combinations of components present in individual containers or packages are insufficient to perform all steps of the methods described herein. However, the kit as a whole may include a collection of containers or packages whose contents can be combined to implement the methods described herein.

[0166] The kit may include suitable packaging materials to contain the kit contents. This packaging material may be constructed using known methods, preferably to provide a sterile, uncontaminated environment. Packaging materials used herein may include, for example, those commonly used in commercial kits sold for use with nucleic acid sequencing systems. Exemplary packaging materials include, but are not limited to, glass, plastic, paper, cardboard, foil, etc., capable of containing, within a defined range, the components described herein.

[0167] The packaging material may include a label indicating the specific use of the component. The use of the kit indicated by the label may be one or more methods described herein, depending on the specific combination of components present in the kit. For example, the label may indicate that the kit can be used to add inserts to nucleic acid polymers, cleave modified nucleic acid polymers to generate fragments, or determine the sequence of nucleic acids.

[0168] Instructions for use of the packaged reagents or components may also be included in the kit. These instructions typically include detailed descriptions of reaction parameters, such as the relative amounts of kit components to the sample to be mixed, the holding time of the reagent / sample mixture, temperature, buffer conditions, etc.

[0169] It is important to understand that not all components required for a particular reaction need to be present in a specific kit. Instead, one or more additional components may be available from other sources. The instructions provided with the kit will indicate one or more additional components to be provided and where they can be obtained.

[0170] The following examples are intended to illustrate, not limit, the invention.

[0171] Example I Connected transposons are used to integrate inserts into genomic DNA to preserve connectivity information within cluster arrays. The two transposable components are connected together, such as Figure 1 As shown in Figure A. Each transposon element forms a forked linker structure having a double strand forming an annealed double-stranded portion and a non-annealed portion. The annealed portion includes a complementary portion of each strand, which forms a mosaic element (ME). The non-annealed portion includes a P5 primer site near the 5' end of one strand and a P7 primer site near the 3' end of the other strand. The two transposons are identical and linked together by the 5' end of the non-annealed portion (i.e., the 5' end of the strand containing the P5 primer site). The linked transposon elements bind to the corresponding transposase subunit to form a transposon complex in the form of a circular complex, and several circular complexes bind to the target genomic DNA polymer (…). Figure 1 B). This adapter prevents the target DNA from fragmenting during transposition (i.e., it inhibits "tag-based fragmentation," despite the insertion of linked transposon elements, such as...). Figure 1 (As shown in C). As a result of this 5' to 5' connection, the modified nucleic acid polymer will include sequence portions of alternating strands from the target nucleic acid polymer, which are concatenated into a single polymer chain. The insert includes cleavage sites present in the adapter, and optionally also contains binding portions in the adapter.

[0172] The target nucleic acid polymer, modified to include the insert, is then loaded into a flow cell and captured on the surface of the flow cell by receptors specific to the binding site. For example, in cases where the adapter contains a specific nucleic acid sequence, the flow cell may include capture probes having a sequence complementary to that specific sequence; alternatively, the adapter may include biotin analogs that bind to streptavidin attached to the surface of the flow cell. The genomic DNA modified with the insert is attached to the surface of the flow cell in double strand form, as shown below. Figure 2As shown. Under non-flowing conditions, the individual insert-modified genomic DNA polymers will bind to localized regions on the surface. The adapters can then be cleaved and the strands denatured to allow the individual fragments (e.g., in the range of 200b to 100kb) to diffuse and separate from each other and be seeded onto a flow cell, as shown. Figure 2 As shown in the right-hand illustration. Diffusion conditions are selected to allow these fragments to be inoculated at locations close to the localized region to which the insert-modified genomic DNA polymer is attached. Diffusion conditions are selected to allow this localization while preventing the fragments from being too close together; instead, they are inoculated as templates that can form micron-sized clusters of monoclonal antibodies in the subsequent bridging amplification step. Once the template is inoculated and bridging amplified, as... Figure 3 As shown, sequencing can be performed using standard procedures on the HiSeq or MiSeq platform (Illumina Inc., San Diego, CA).

[0173] If Figure 3 The positions of the three clusters in the middle and Figure 2 As illustrated by comparing the regions where the three insert-modified genomic DNA polymers bound to the surface of the flow cell reside, clusters from adjacent or overlapping genomic regions form cloud-like clusters. Some cloud-like clusters can be distinguished from others, such as cloud α, which is distinct from clouds β and γ. Cloud-like clusters can mix together without problem, such as clouds β and γ. Therefore, the flow cell can achieve a fairly high density of clusters.

[0174] Sequence analysis is then performed. During assembly, adjacent reads are grouped based on a distance metric (e.g., normalized physical spacing between clusters in a flow cell) to assess whether two reads should be assembled together, considered phased, or used to correct each other's errors. Nearby reads can even be assembled and phased across corresponding regions, and adjacent complementary reads can be compared for robust error correction.

[0175] Typically, it's unknown which sequence reads obtained from different clusters in a flow cell originate from a common original molecule. For example, in... Figure 4 In A, a monochromatic collection of reads with a length of 20 kb is aligned with a 50 kb reference sequence 10. However, the distance information on the flow cell generated using the method described above can group reads into sets, and their distances can be compared with those from the first genomic DNA molecule ( Figure 4 The light gray fragment 21 in B is consistent with, or consistent with, a molecule from the second genomic DNA. Figure 4(B, dark gray fragment 22). Therefore, the collection of fragment 22 from the second genomic DNA molecule can assemble into a phase-consolidation group, such as... Figure 4 As shown in C.

[0176] Using the methods illustrated above, homologous and heterologous sites between sets can be used to assemble phase contigs; matching the overlap of reads from the same molecule helps with assembly from scratch; and complementary reads from the same molecule can be used for read error correction and to identify rare variants.

[0177] Example II Algorithm for determining the phase of sequenced fragments The target nucleic acid can be processed to generate an array of fragments, wherein the proximity of the fragments to each other on the array is directly related to the probability that these fragments were generated from the same target nucleic acid molecule. This processing can be performed as described in Example I or elsewhere herein.

[0178] In one embodiment, the process begins with the isolation of target nucleic acids from a biological source using a mild preparation method to minimize damage to genomic DNA (gDNA) in order to preserve the length of the target gDNA molecule to the greatest extent possible. Each target gDNA is modified to add an insert. A library of the modified gDNA polymers is delivered to a MiSeq flow cell (Illumina, Inc., San Diego, CA) and allowed to diffuse to achieve separation between library members. The modified gDNA is fragmented in the flow cell such that each modified gDNA polymer produces a sub-library of gDNA fragments randomly captured at locations on the flow cell surface. Conditions are employed to allow fragments from each modified gDNA to be captured within close proximity to each other. Clusters grow from each fragment at the corresponding location. The clusters are sequenced using a standard MiSeq procedure (Illumina, Inc., San Diego, CA).

[0179] The total amount of sequences present in the gDNA fragment sub-library may constitute a proportion of the gDNA sequence of the said organism, or may at least 1x cover the entire gDNA sequence. Typically, the entire set of gDNA fragments will cover the entire gDNA sequence by, for example, at least 10x or more. When aligned with the entire gDNA sequence, the sequences of the gDNA fragments in the entire set may be adjacent, overlapping, or gapped.

[0180] The proximity of surface-attached fragments (or clusters derived from fragments) can be treated as a characterization of the physical distance between sublibrary members. Although proximity is a measure of “closeness,” surface-attached fragments (or clusters) should be far enough apart that they can be resolved by the sequencing platform detector. This resolution is required for fragments from different gDNA fragment sublibraries (i.e., between) and fragments from the same sublibrary (i.e., within).

[0181] For analytical purposes, members of a given sub-library can be considered superficially "close" if the spatial distance between members is much smaller than the spatial distance to the nearest sub-library from an adjacent genomic region. See, for example, [link to relevant documentation]. Figure 3 Clusters in the γ-sub-library are considered to be "close" to each other, although some clusters in the γ-sub-library (compared to their distance from clusters in themselves and their own sub-libraries) are closer to clusters in the nearby β-sub-library. Clusters in the γ-sub-library are considered "close" because, although they appear to be physically close to the γ-sub-library on the surface, the β-sub-library can be identified by sequence analysis as regions originating from distant gDNA sequences (i.e., the α and γ-sub-libraries originate from regions in the target genome that are far apart from each other).

[0182] Algorithms for determining the sequence of a ligated fragment in a target gDNA may include the following steps: (a) Obtain sequence read lengths for clusters on the surface; (b) Align these sequence reads with the reference genome and identify variations; (c) Use a sliding window along the genome (e.g., 100kb) to reduce the number of reads to be analyzed; (d) Use density-based spatial clustering algorithms to identify cloud-like structures (i.e. regions) of “proximate” clusters. (e) Assign a virtual barcode to each cloud (i.e., reads originating from the same cloud have the same barcode, and barcodes are unique between different clouds); and (f) The barcode reads were analyzed using ReFHap software (Duitama et al., Proceeding of the First ACM International Conference on Bioinformatics and Computational Biology, pp. 160-169 (2010), the reference of which is incorporated herein by reference) to determine the phase of the identified variants.

[0183] Alternative algorithms for determining connectivity may include steps (a) and (b) of the above algorithm, followed by ReFHap software modified to employ a distance metric. An exemplary distance metric assigns greater weight to two SNPs that are closer to each other (i.e., more distant) compared to SNPs that are far apart.

[0184] Example III Metagenomics Applications In this embodiment, proximity mapping is used to attribute sequence reads to individual organisms in a mixed sample of various organisms. Therefore, the mixed sample can be considered as a mixture of parent and paternal haplotypes that are distinct in haplotype analysis or phasing applications.

[0185] The workflow is as follows: (a) Extracting DNA from organisms in a mixed sample.

[0186] (b) Optionally, the desired target may be enriched or “known and / or non-target” organisms may be excluded from the sample. This can be accomplished, for example, by employing targeted amplification methods to selectively amplify only a portion of the nucleic acid sample, as described earlier in this document.

[0187] (c) Prepare DNA from the transposon complex as described in Example I or elsewhere herein.

[0188] (d) As described in Example I or elsewhere herein, the DNA prepared in step (c) is seeded into a flow cell.

[0189] (e) Clusters / sequence reads that are close to each other will have a certain probability of originating from the same starting organism.

[0190] (f) Optionally, reads that match “known and non-target” organisms (i.e. during the second sequence analysis) are filtered out, thereby reducing the effective “density” of noise around the cloud of adjacent fragments.

[0191] (g) This information can then help construct the assembly framework of the genome of each organism in the sample.

[0192] An alternative workflow can be used to capture an organism at a location within a flow cell and then perform sample preparation as described in steps (b) through (d) in situ. In this way, essentially all recyclable DNA from a living organism is located at a given spatial location within the flow cell, rather than just long segments thereof.

[0193] A diagram showing the results is displayed. Figure 9The image shows three distinct cloud-like clusters originating from three different organisms in a metagenomic sample. These clouds are identified as α, β, or γ. For example, with α and β clouds, when these two clouds overlap, fragments matching known organisms (e.g., β organisms) can be subtracted or removed from the analysis to more definitively identify sequences originating from α organisms.

[0194] Example IV Generate near-read length on the flow cell 10 μl of buffer containing 50 ng of genomic DNA was used to generate near-close reads on the flow cell. A transposon mixture (Tn5 complex, MgCl₂, Tris buffer) was added to the DNA to obtain a final 20 μl reaction mixture, and the mixture was heated to 55 °C for 10 min. The DNA was diluted to a concentration of 50 pM (based on average fragment size), and the DNA still bound to the transposons was loaded onto the flow cell. Transposase was removed from the DNA using SDS, and gaps were repaired using a polymerase / ligase mixture. Fragments were allowed to be seeded onto the flow cell surface near the initial capture site. Bridge amplification was performed to amplify fragments in situ to form clonal clusters. The amplified DNA was sequenced. Sequencing data was analyzed to identify cluster proximity groups to indicate which fragments originated from the same DNA initiation molecule. This information was then used to determine the phase of SNPs in the sample and displayed. Figure 10 middle.

[0195] Human genomic DNA (Coriell sample NA12878) was prepared using the recommended method and sequenced on the Illumina HiSeq platform. Twelve indices were added to the sample preparation to allow for higher-density clustering and more precise identification of proximity groups. After alignment with a reference genome (HG19), data from two or three channels were combined and cluster proximity groups were analyzed. Information from the proximity groups was then used to determine the phase of heterogeneous SNPs in the sample. The phase determination data are shown in Table 1 below.

[0196] Table 1: Phase Fixing Data Sample: Human DNA, Coriell NA12878

[0197] Throughout this application, various publications, patents, or patent applications have been cited. The disclosures of these publications are incorporated herein by reference in their entirety in order to provide a more comprehensive description of the level of art relating to this invention.

[0198] The term "includes" is intended to be open-ended in this document, encompassing not only the listed elements but also any additional elements.

[0199] Although the present invention has been described with reference to the embodiments provided above, it should be understood that various modifications can be made without departing from the invention. Therefore, the invention is defined only by the claims.

Claims

1. A method for sequencing target nucleic acid polymers, comprising: (a) Modifying a target nucleic acid polymer to generate a modified nucleic acid polymer, wherein the modified nucleic acid polymer comprises a plurality of sequence regions derived from the target nucleic acid polymer; (b) Fragments of the modified nucleic acid polymer are generated in a container including a solid support surface, each fragment including one of the sequence regions; (c) The fragment is randomly captured at a location within a region on the surface of the solid support; (d) Determining the nucleotide sequence of the sequence region by detecting the fragment at the stated location; and (e) Generate a nucleotide sequence characterization of the target nucleic acid polymer based on the relative distance between the nucleotide sequence from the fragment and the position on the surface of the solid support.

2. A method for sequencing target nucleic acid polymers, comprising: (a) Modifying a target nucleic acid polymer to generate a modified nucleic acid polymer, wherein the modified nucleic acid polymer comprises a plurality of sequence regions derived from the target nucleic acid polymer; (b) Attaching the modified nucleic acid polymer to a region on the surface of a solid support; (c) Generate a fragment of the modified nucleic acid polymer attached to the surface of the solid support, wherein the fragment is attached to the location of the region on the surface of the solid support; (d) Determine the nucleotide sequence from the fragment by detecting the fragment at the stated position; and (e) Generate a nucleotide sequence characterization of the target nucleic acid polymer based on the relative distance between the nucleotide sequence from the fragment and the position on the surface of the solid support.

3. A method for sequencing target nucleic acid polymers, comprising: (a) Adding an insert to a target nucleic acid polymer to form a modified nucleic acid polymer comprising a plurality of internal inserts; (b) Fragments of the modified nucleic acid polymer are generated in a fluid in contact with the surface of a solid support, thereby releasing the fragments, each of the fragments comprising at least a portion of the insert having a primer site; (c) The fragments from the fluid are randomly captured at locations on the surface of a solid support; (d) Determine the nucleotide sequence from the fragment by detecting the fragment at the stated position; and (e) Generate a nucleotide sequence characterization of the target nucleic acid polymer based on the relative distance between the nucleotide sequence from the fragment and the position on the surface of the solid support.

4. A method for sequencing target nucleic acid polymers, comprising: (a) Adding an insert to a target nucleic acid polymer to form a modified nucleic acid polymer comprising a plurality of internal inserts, wherein the inserts have primer sites; (b) Fragments of the modified nucleic acid polymer are generated in a fluid in contact with the surface of a solid support, thereby releasing the fragments, each of the fragments comprising at least a portion of the insert having a primer site; (c) The fragments from the fluid are randomly captured at locations on the surface of a solid support; (d) Determine the nucleotide sequence from the fragment by extending the primer that has been hybridized to the primer site at the location; as well as (e) Generate a nucleotide sequence characterization of the target nucleic acid polymer based on the relative distance between the nucleotide sequence from the fragment and the position on the surface of the solid support.

5. A method for sequencing target nucleic acid polymers, comprising: (a) Adding an insert to a target nucleic acid polymer to form a modified nucleic acid polymer comprising a plurality of internal inserts; (b) Attaching the modified nucleic acid polymer to the surface of a solid support; (c) Generating fragments of the modified nucleic acid polymer attached to the surface of the solid support, wherein the fragments are attached to the surface of the solid support and wherein each fragment includes at least a portion of the insert; (d) Determine the nucleotide sequence from the fragment by detecting the fragment at the stated position; and (e) Generate a nucleotide sequence characterization of the target nucleic acid polymer based on the relative distance between the nucleotide sequence from the fragment and the position on the surface of the solid support.

6. A method for determining the origin of individual sequences in a mixture of sequences from different sources, comprising: (a) Providing a mixture of target nucleic acid polymers from multiple different sources; (b) Modifying the mixture of the target nucleic acid polymers to generate a mixture of modified nucleic acid polymers, wherein the mixture of modified nucleic acid polymers comprises a plurality of sequence regions from the different sources; (c) Fragments of the modified nucleic acid polymer are generated in a container having a solid support surface, each fragment comprising a sequence region from one of the different sources; (d) Given that fragments from a common target nucleic acid polymer are preferentially located at adjacent positions on the surface of the solid support, the fragments are randomly captured at positions on the surface of the solid support; (e) Determine the nucleotide sequence of the fragment at the stated location; as well as (f) Identify nucleotide sequences originating from a common source among the plurality of different sources based on the relative distance between the nucleotide sequence from the fragment and the position on the solid support.

7. A method for determining the origin of individual sequences in a mixture of sequences from different sources, comprising: (a) Providing a mixture of target nucleic acid polymers from multiple different sources; (b) Modifying the mixture of the target nucleic acid polymers to generate a mixture of modified nucleic acid polymers, wherein the mixture of modified nucleic acid polymers comprises a plurality of sequence regions from the different sources; (c) Attaching the modified nucleic acid polymer to the surface of a solid support; (d) Generate fragments of the modified nucleic acid polymer attached to the surface of the solid support, wherein fragments from a common source among the plurality of sources are attached to adjacent sites on the surface of the solid support; (e) Determine the nucleotide sequence of the fragment at the stated location; as well as (f) Identify nucleotide sequences originating from a common source among the plurality of different sources based on the relative distance between the nucleotide sequence from the fragment and the position on the solid support.

8. A method for determining the origin of individual sequences in a mixture of sequences from different sources, comprising: (a) Providing a mixture of target nucleic acid polymers from multiple different sources; (b) Adding inserts to the target nucleic acid polymer in the mixture to form a mixture of modified nucleic acid polymers, each polymer including a plurality of internal inserts; (c) Generating fragments of the modified nucleic acid polymer in a fluid in contact with the surface of a solid support, thereby releasing the fragments, each of the fragments comprising at least a portion of each of the inserts; (d) The fragments from the fluid are randomly captured at locations on the surface of a solid support; (e) Determine the nucleotide sequence from the fragment by detecting the fragment at the stated position; as well as (f) Identify nucleotide sequences originating from a common source among the plurality of different sources based on the relative distance between the nucleotide sequence from the fragment and the position on the solid support.

9. A method for determining the origin of individual sequences in a mixture of sequences from different sources, comprising: (a) Providing a mixture of target nucleic acid polymers from multiple different sources; (b) Adding inserts to the target nucleic acid polymer in the mixture to form a mixture of modified nucleic acid polymers, each polymer including a plurality of internal inserts; (c) Attaching the modified nucleic acid polymer to the surface of a solid support; (d) Generate fragments of the modified nucleic acid polymer attached to the surface of the solid support, wherein fragments from a common source among the plurality of sources are attached to adjacent sites on the surface of the solid support; (e) Determine the nucleotide sequence from the fragment by detecting the fragment at the stated position; as well as (f) Identify nucleotide sequences originating from a common source among the plurality of different sources based on the relative distance between the nucleotide sequence from the fragment and the position on the solid support.

Images