Orthogonal deblocking of nucleotides

Abstract

Orthogonal deblocking of nucleotides is provided. A method comprising the following steps: (a) providing an array of sites, wherein each site comprises a mixture of different nucleic acid templates; (b) extending primers hybridized to the different nucleic acid templates at each site with different nucleotide analogs containing different reversible blocking moieties, respectively, thereby generating different primer extension products at each site; (c) detecting the different primer extension products to distinguish the different nucleotide analogs at each site; and (d) at each site, removing the different reversible blocking moieties from the primer extension products using a first treatment selective for a first different reversible blocking moiety and a second treatment selective for a second different reversible blocking moiety.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of July 8, 2016, application number: 201680036416.6, and invention name: "Orthogonal Deblocking of Nucleotides". Background Art

[0002] The present disclosure relates generally to nucleic acid analysis, and more particularly to nucleic acid sequencing.

[0003] Commercial platforms currently available for DNA sequencing are relatively expensive. These platforms use so-called "synthesis sequencing" methods because DNA polymers are synthesized while detecting each monomer (i.e., nucleotide) of the polymer structure added to the growth. Since the template DNA chain strictly guides the synthesis of new DNA polymers, the sequence of the DNA template can be inferred from a series of nucleotide monomers added to the growing chain during synthesis. The ability to detect monomer addition is promoted by special engineered variants of biochemical components that usually perform DNA synthesis in biological systems. These engineered components are relatively expensive to manufacture and are relatively consumed in large quantities during synthesis sequencing. In addition, monitoring reactions use relatively expensive hardware, such as lasers, detection optics (optics) and complex fluid delivery systems. Even before synthesis sequencing can be started, the most successful commercial platforms so far also require expensive reagents and hardware to amplify DNA templates. The complexity and cost of these platforms have hindered their use in clinical and research environments where technology has a clear demand.

[0004] Therefore, there is a need to improve the test-by-synthesis platform to make it more cost-effective, fast and convenient. The present disclosure addresses this need and also provides other advantages. Summary of the invention

[0005] The present disclosure provides a method for identifying a nucleic acid template. The method may include the following steps: (a) providing a site array, wherein each site contains a mixture of at least two different nucleic acid templates; (b) extending primers hybridized with the different nucleic acid templates at each site with different nucleotide analogs containing different reversible blocking parts, respectively, thereby generating different primer extension products at each site; (c) detecting the different primer extension products to distinguish the different nucleotide analogs at each site; and (d) at each site, using a first treatment selective for the first different reversible blocking part and a second treatment selective for the second different reversible blocking part to remove the different reversible blocking parts from the primer extension products. Optionally, the method also includes (e) repeating (b) to (d) to determine the sequence of the different nucleotide analogs added to each of the different extension products at each site.

[0006] Also provided is a method for sequencing a nucleic acid template, which can include the following steps: (a) providing an array of sites, wherein each site comprises a first nucleic acid template and a second nucleic acid template, wherein the first nucleic acid template has a sequence different from the sequence of the second nucleic acid template, wherein a first primer is bound to the first nucleic acid template, and wherein a second primer is bound to the second nucleic acid template, and a reversible blocking portion is attached to the second primer; (b) extending the first primer by adding a first nucleotide analog attached to the reversible blocking portion, wherein the reversible blocking portion attached to the first nucleotide is attached to the reversible blocking portion attached to the second primer; (c) selectively removing the reversible blocking portion attached to the second primer while retaining the reversible blocking portion attached to the nucleotide analog added to the first primer; (d) extending the second primer by adding a second nucleotide analog attached to the reversible blocking portion, wherein the reversible blocking portion attached to the first nucleotide analog is different from the reversible blocking portion attached to the second nucleotide analog; and (e) detecting the nucleotide analog added to the first primer and the nucleotide analog added to the second primer at each site, thereby determining the different sequences of the first template and the second template at each site. Optionally, the method may further include the step of (f) selectively removing the reversible blocking portion attached to the first nucleotide analog added to the first primer, while retaining the reversible blocking portion attached to the second nucleotide analog added to the second primer; (g) after (f), extending the first primer by adding a third nucleotide analog attached to the reversible blocking portion; (h) selectively removing the reversible blocking portion attached to the second nucleotide analog added to the second primer, while retaining the reversible blocking portion attached to the third nucleotide analog added to the first primer; (i) after (h), extending the second primer by adding a fourth nucleotide analog attached to the reversible blocking portion, wherein the reversible blocking portion attached to the third nucleotide analog is different from the reversible blocking portion attached to the fourth nucleotide analog; and (j) at each site, detecting the nucleotide analog added to the first primer in (g) and the nucleotide analog added to the second primer in (i), thereby determining the different sequences of the first template and the second template at each site. Optionally, steps (f) to (j) may be repeated.

[0007] Also provided is a method for sequencing a nucleic acid template, which can include the following steps: (a) providing an array of sites, wherein each site comprises a first nucleic acid template and a second nucleic acid template, wherein the first nucleic acid template has a sequence different from the sequence of the second nucleic acid template, wherein a first primer is bound to the first nucleic acid template, and a reversible blocking portion is attached to the first primer, wherein a second primer is bound to the second nucleic acid template, and a reversible blocking portion is attached to the second primer, and wherein the reversible blocking portion attached to the first primer is different from the reversible blocking portion attached to the second primer; (b) selectively removing the reversible blocking portion attached to the first primer, while retaining the reversible blocking portion attached to the second primer; (c) selectively removing the reversible blocking portion attached to the first primer, and (d) selectively removing the reversible blocking portion attached to the second primer; and (e) selectively removing the reversible blocking portion attached to the second primer by adding (d) selectively removing the reversible blocking portion attached to the second primer while retaining the reversible blocking portion attached to the nucleotide analog added to the first primer; (e) extending the second primer by adding a second nucleotide analog attached to the reversible blocking portion, wherein the reversible blocking portion attached to the first nucleotide analog is different from the reversible blocking portion attached to the second nucleotide analog; and (f) detecting the nucleotide analog added to the first primer and the nucleotide analog added to the second primer at each of the sites, thereby determining the different sequences of the first template and the second template at each of the sites.

[0008] The present disclosure also provides a nucleic acid array comprising a plurality of sites on a solid support, wherein each site comprises a first nucleic acid template and a second nucleic acid template, wherein the first nucleic acid template has a sequence different from the sequence of the second nucleic acid template, wherein a first primer binds to the first nucleic acid template and a first reversible blocking moiety is attached to the first primer, wherein a second primer binds to the second nucleic acid template and a second reversible blocking moiety is attached to the second primer, and wherein the first reversible blocking moiety is different from the second reversible blocking moiety. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Shown is a cycle diagram of simultaneous sequencing-by-synthesis of a mixture of two templates at a site on an array, where subsets of the label and deblocked moieties present on the same extension product are cleaved simultaneously.

[0010] Figure 2 A cycle diagram of simultaneous sequencing-by-synthesis of a mixture of two templates at a site on the array is shown, wherein label moieties present on different extension products (ie, produced in both the R1 and R2 reactions) are cleaved simultaneously. DETAILED DESCRIPTION

[0011] The present disclosure provides a method for high-density detection of nucleic acids. Specific embodiments of the disclosed methods utilize known techniques to operate and detect nucleic acids. However, the improvements listed below provide orthogonal processing to increase the information density obtained using these techniques.

[0012] The example of detection technology based on primer extension illustrates the information density of the increase that can be obtained. Specifically, the target sequence of nucleic acid can be hybridized with primers, and the primers can be extended by DNA polymerase to add the nucleotide analogs of the mark. An array format with multiple sites can be used, and each site has a single target sequence different from the target sequence existing at other sites. Optionally, several different nucleotide analog types can also be used, each with distinguishable markers. Primer extension causes the nucleotide analogs of the mark to be recruited to the nucleic acid with the target sequence. In the array format using the nucleotide analogs of different marks, the markers recruited to each site can be distinguished, and the target nucleic acid at the site can be identified using this information. The information density obtained from this array format is a target sequence identified at each site.

[0013] In the orthogonal form of the present disclosure, each site of the array can contain a mixture of two or more different target sequences that are processed (e.g., with a chemical reagent or physical operation) and observed (e.g., with a detector whose resolution is too low to spatially distinguish the nucleic acids in the mixture). Nevertheless, the orthogonal processing described herein produces different effects that allow different target sequences to be distinguished from each other. In this case, the information derived from the array can be at least doubled. Two different primers can be delivered to the array, and due to differential complementation, they will hybridize with two different template sequences at their respective individual sites. The first primer may have a first reversible blocking portion that prevents it from extending until the first deblocking treatment is applied, and the second primer may have a second reversible blocking portion that prevents it from extending until the second deblocking treatment is applied. In this example, the first deblocking treatment is selective for the first reversible blocking portion compared to the second reversible blocking portion, and the second deblocking treatment is selective for the second reversible blocking portion compared to the first reversible blocking portion. This selective deblocking capability provides orthogonality such that the primers (despite exposure to each deblocking treatment) can be individually modified for extension and detection. Thus, even if the molecules themselves cannot be separated sufficiently to allow spatial distinction using the detection system used, the two primers, and more importantly the templates that direct their extension, can be distinguished by this chemical switch.

[0014] The concept of orthogonality as exemplified above for primer extension based detection techniques can be readily applied to sequencing by synthesis (SBS) techniques. Figure 1 In the exemplary cycle diagram of the orthogonal SBS for two templates (R1 and R2) at the site is shown. The first column in the figure represents the treatment to which the array is exposed (that is, both templates are exposed to the treatment). The second and third columns respectively illustrate the influence of the treatment on the first template and the second template. In the first step of the first cycle, the mixture of primers is contacted with the array, which results in hybridization with the respective primer binding sites. After step 1, the R1 primer hybridized to the R1 template is blocked by blocker 1, and the R2 primer lacks a blocking group (preferably, the primer R2 can be blocked by an orthogonal blocking portion (blocker 2)). In this way, the two primers can be extended separately. For example, in the second step of the first cycle, nucleotides with blocker 2 and marker 2 are delivered to the array, which results in the selective extension of the unblocked R2 primer (that is, not extending the R1 primer). Then, in the third step of the first cycle, the array can be exposed to the treatment, for example, by cutting blocker 1 (that is, not cutting blocker 2), which selectively blocks the R1 primer. In the fourth step of the first cycle, nucleotides with Blocker 1 and Label 1 are delivered to the array, which results in the selective extension of the unblocked R1 primer (i.e., without extending the R2 primer). The array can then be viewed using a detection device so that the two labels can be distinguished at each site. Figure 1 As shown, the cycle of delivering blocked and labeled nucleotides, cleaving the blocking group and label, and detecting can be repeated cyclically.

[0015] As above and Figure 1 As shown, the nucleotide analogs used to extend the primers can include a reversible blocking portion that is selectively cleavable to provide an orthogonal control through multiple cycles of the synthesis sequencing process. Thus, the nucleotide analogs can be provided in two groups: a first group having a first reversible blocking portion (e.g., the same as the reversible blocking portion on the first primer, Blocker 1) and a second group having a second reversible blocking portion (i.e., Blocker 2). In some embodiments, the nucleotide analogs in the first group can have a label that is distinguishable from the label on the nucleotide analogs in the second group (e.g., Figure 1 In the array, the first group is marked at marker 1 and the second group is marked at marker 2). The resulting orthogonality in biochemical reactivity and marker management allows two primer extension events to be distinguished from each other at each site of the array. Thus, two target sequences can be distinguishably detected.

[0016] It should be understood that other methods may also benefit from orthogonal manipulation and detection as described in more detail below.Thus, the compositions, devices and methods described herein need not be limited to sequencing applications.

[0017] Orthogonality can be used to increase information density acquisition by 2 or more times. For example, more than a 2-fold increase in information density can be obtained by using more than two orthogonal reagent sets. As an example, three reagent sets can be used, which contain three different deblocking treatments, each of which is selective for the primers and / or nucleotide analogs in one of the reagent sets.

[0018] As shown above and as will be further elaborated below, the present disclosure provides the advantages of super-resolution imaging of arrays, wherein the number of target sequences that can be resolved simultaneously at a given position is greater than 1. Super-resolution imaging can provide the benefit of simultaneously distinguishing a number of different target nucleic acids that is greater than the number of sites on the array. Similarly, super-resolution is provided in that two different target sequences can be distinguished on a solid support using a detector having a resolution lower than the spatial resolution that would otherwise be required to distinguish two target sequences on a substrate.

[0019] In a specific embodiment, the present disclosure provides reagents and hardware configurations for efficient nucleic acid detection. An exemplary configuration uses a marker with fewer numbers of nucleotide analog species to be distinguished in a primer extension step. For example, based on the detection of a single marker species, four nucleotide analogs can be distinguished. As further described in detail below, this can be achieved by using a first group of nucleotide analogs comprising the following four species: (1) a species with a first marker, (2) a species with a ligand, (3) a species with a cleavable connection to the first marker, and (4) a species lacking any marker or ligand for subsequent steps, wherein all four species have a blocking portion selectively deblocked by the first treatment. An orthogonal group of nucleotide analogs can include the following four species (5) a species with a first marker, (6) a species with a mixture of the first and second markers, (7) a species with a cleavable connection to the second marker, and (8) a species lacking any marker or ligand for subsequent steps, wherein all four species have a blocking portion selectively deblocked by the second treatment. In particular, the first treatment does not result in substantial deblocking of a first set of nucleotide analogs, and the second treatment does not result in substantial deblocking of an orthogonal set of nucleotide analogs.

[0020] The species within each of the above groups can be distinguished from each other based on appropriate counts of which markers appear or disappear after a particular fluid step, and two orthogonal groups of nucleotide analogs can be distinguished based on two different markers. More specifically, species (1) and (5) can be distinguished from each other based on different markers and from other species due to their appearance after an initial labeling step and their resistance to various cleavage agents; species (2) can be distinguished based on the appearance of the marker after incubation with a labeled receptor; species (3) and (7) can be distinguished from each other based on different markers and from all other species based on the initial appearance of the markers and then disappearance after treatment with the respective cleavage agents; species can be distinguished from all other species based on the intensity of both markers at half the intensity of the fully labeled species; and species (4) and (8) can be distinguished based on the inference of the absence of any other species in the groups that have been detected. Many other configurations can vary the number of markers, the number of fluid operations during the detection phase, and / or the complexity of the detection equipment that distinguishes a particular number of markers. Thus, the configuration can be adjusted to suit a particular method or application.

[0021] Unless otherwise specified, the terms used herein shall be understood to have their ordinary meanings. Several examples of terms and their definitions used herein are described below.

[0022] As used herein, the term "amplicon" when referring to nucleic acid use, refers to the product of replicated nucleic acid, wherein the product has a nucleotide sequence identical or complementary to at least a portion of the nucleotide sequence of nucleic acid.Amplicon can be produced by any of the multiple amplification methods using nucleic acid or its amplicon as template, and the amplification method includes, for example, polymerase extension, polymerase chain reaction (PCR), rolling circle amplification (RCA), connection extension or connection chain reaction.Amplicon can be a single copy (for example, PCR product) with a specific nucleotide sequence, or a nucleic acid molecule with multiple copies (for example, concatemer products of RCA) of a nucleotide sequence. The first amplicon of a target nucleic acid is typically a complementary copy. After generating the first amplicon, subsequent amplicon is a copy created from the target nucleic acid or from another amplicon.Subsequent amplicon can have a sequence that is substantially complementary to the target nucleic acid, or substantially identical to the target nucleic acid.

[0023] As used herein, the term "array" refers to a group of sites that can be distinguished from each other according to relative positions. Different molecules at different sites of an array can be distinguished from each other according to the position of the site in the array. The individual sites of an array may include one or more molecules of a specific type. For example, a site may include a single target nucleic acid molecule with a specific sequence, or a site may include several nucleic acid molecules with the same sequence (and / or its complementary sequence). Alternatively, a site may, for example, include a mixture of target nucleic acid sequences so that individual molecules each contain two or more different molecules, or so that two or more molecules each contain a single target sequence of a mixture. The array sites may be different features located on the same substrate. Exemplary features include, but are not limited to, holes in substrates, beads (or other particles) in or on substrates, projections from substrates, ridges on substrates, or channels in substrates. The array sites may be various individual substrates carrying different molecules. Different molecules attached to individual substrates may be identified according to the position of the substrate on the surface associated with the substrate, or according to the position of the substrate in a liquid or gel. Exemplary arrays in which individual substrates are located on the surface include, but are not limited to, those having beads in holes.

[0024] As used herein, the term "attachment" refers to a state in which two things are joined, fastened, adhered, connected or combined. For example, an analyte, such as a nucleic acid, can be attached to a material, such as a gel or a solid support, by covalent or non-covalent bonds. A covalent bond is characterized by sharing electron pairs between atoms. A non-covalent bond is a chemical bond that does not include a shared electron pair and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions. Nucleic acids can be attached to a solid support by a gel coating on a solid support.

[0025] As used herein, when used to refer to nucleotide analogs, the term "blocking portion" means a portion of a nucleotide analog that inhibits or prevents a nucleotide analog from forming a covalent bond with a second nucleotide analog. For example, in the case of a nucleotide analog with a pentose moiety, the blocking portion prevents the formation of a phosphodiester bond between the 3' oxygen of the nucleotide analog and the 5' phosphate of the second nucleotide analog. The blocking portion can be a portion of a nucleotide analog (which is a monomer subunit present in a nucleic acid polymer), or the blocking portion can be a portion of a free nucleotide analog (such as a nucleoside triphosphate). The blocking portion as a portion of a nucleotide analog can be reversible, so that the blocking portion can be removed or modified to provide a nucleotide analog that can form a covalent bond with a second nucleotide analog. Particularly useful reversible blocking portions are phosphates, phosphate esters, alkyl azides, acetals, esters, ethers, etc. Other examples of reversible blocking portions that can be used are listed below and in the references set forth below, and the references are incorporated herein by reference. In specific embodiments, a blocking moiety (eg, a reversible blocking moiety) can be attached to the 3' position or the 2' position of the pentose moiety of the nucleotide analog.

[0026] As used herein, when referring to the use of nucleic acids, the term "cluster" refers to a nucleic acid population attached to a solid support to form a feature or site. Nucleic acids are typically of a single species, thereby forming a homogeneous cluster. However, in some embodiments, nucleic acids can be heterogeneous, so that individual molecules with different sequences exist at a site or feature. Nucleic acids are typically covalently attached to a solid support, for example via their 5' ends, but in some cases, other attachment methods are possible. The nucleic acids in the cluster can be single-stranded or double-stranded. In some but not all embodiments, clusters are prepared by a solid phase amplification method called bridge amplification. Exemplary configurations of clusters and methods for making them are shown, for example, in U.S. Pat. No. 5,641,658, U.S. Pat. Publication No. 2002 / 0055100; U.S. Pat. No. 7,115,400; U.S. Pat. Publication No. 2004 / 0096853; U.S. Pat. Publication No. 2004 / 0002090; U.S. Pat. Publication No. 2007 / 0128624; and U.S. Pat. Publication No. 2008 / 0009420, each of which is incorporated herein by reference.

[0027] As used herein, the term "deblocking agent" means a catalyst, enzyme, reagent or other substance that can modify or remove a blocking portion. In a specific embodiment, the deblocking agent can be specific for a specific blocking portion. Thus, the deblocking agent can selectively remove a specific blocking portion from a nucleotide analog compared to another blocking portion. Exemplary deblocking agents include, but are not limited to, enzymes such as phosphatases, esterases, alkyltransferases or methyltransferases; or chemical reagents such as phosphine, protons, or chemical catalysts such as palladium catalysts. Further examples of deblocking agents are described in detail below.

[0028] As used herein, the term "different", when used in reference to nucleic acids, means that the 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 a substantial portion of their length. For example, two or more nucleic acids may have target nucleotide sequence portions that are different from each other while also having a common sequence region that is the same for both. Typically, when two species are referred to as "different" herein, one species will have structural properties that are different from the structural properties of the second species. For example, two different polymer species (such as two proteins) may have different monomer subunit sequences (such as different amino acid sequences of two different proteins).

[0029] As used herein, the term "each", when used in reference to a collection of items, is meant to identify a single item in the collection, but not necessarily every single item in the collection. Exceptions may be made if explicitly disclosed or the context clearly dictates otherwise.

[0030] As used herein, the term "nucleic acid" is intended to be consistent with its use in the art, and includes naturally occurring nucleic acids or functional analogs thereof. Particularly useful functional analogs can hybridize to nucleic acids in a sequence-specific manner, or can be used as templates for replicating specific nucleotide sequences. Naturally occurring nucleic acids generally have a backbone containing a phosphodiester bond. The analog structure can have an alternative backbone connection, including any of a variety of connections known in the art. Naturally occurring nucleic acids generally have deoxyribose (e.g., found in deoxyribonucleic acid (DNA)) or ribose (e.g., found in ribonucleic acid (RNA)). Nucleic acids can contain any of a variety of analogs of these sugar moieties known in the art. Nucleic acids can include natural or non-natural bases. In this regard, natural deoxyribonucleic acids can have one or more bases selected from the group consisting of adenine, thymine, cytosine or guanine, and ribonucleic acids can have one or more bases selected from the group consisting of uracil, adenine, cytosine or guanine. Useful non-natural bases that can be included in nucleic acids are known in the art. The term "target" when used in reference to a nucleic acid is intended as a semantic identifier for the nucleic acid within the context of a method or composition described herein, and does not necessarily limit the structure or function of the nucleic acid beyond that expressly indicated in other contexts.

[0031] As used herein, the term "nucleic acid template" refers to a nucleic acid or portion thereof that can be used as a guide to perform polymerase-catalyzed replication. A nucleic acid molecule can include multiple templates along its length, or alternatively, only a single template per molecule is used in specific embodiments herein. Nucleic acid templates can also function as a guide to perform ligase-catalyzed primer extension.

[0032] As used herein, the term "nucleotide" or "nucleotide analog" is intended to include natural nucleotides, non-natural nucleotides, ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, and other molecules called nucleotides. For example, the term is used herein to generally refer to a nucleoside portion (whether ribose, deoxyribose, or its analogs) that includes a base portion and is optionally attached to one or more phosphate portions. The term can be used to refer to a monomer unit present in a polymer, for example, to identify a subunit present in a DNA or RNA chain. The term can also be used to refer to a monomer molecule that is not necessarily present in a polymer, for example, a molecule that can be incorporated into a polynucleotide in a template-dependent manner by a polymerase.

[0033] Exemplary nucleotide analogs include, but are not limited to, ribonucleotide monophosphates (sometimes referred to as ribonucleoside monophosphates), ribonucleotide diphosphates (sometimes referred to as ribonucleoside diphosphates), ribonucleotide triphosphates (sometimes referred to as ribonucleoside triphosphates), deoxynucleotide monophosphates (sometimes referred to as deoxynucleoside monophosphates), deoxynucleotide diphosphates (sometimes referred to as deoxynucleoside diphosphates), and deoxynucleotide triphosphates (sometimes referred to as deoxynucleoside triphosphates). For clarity, when it is desired to distinguish RNA components from DNA components, the term "ribonucleotide" may be used to specify RNA nucleotides, such as ribouridine triphosphate, riboguanosine triphosphate, ribocytidine triphosphate, or riboadenosine triphosphate; and the term "deoxynucleotide" may be used to specify DNA nucleotides, such as deoxythymidine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, or deoxyadenosine triphosphate. In specific embodiments, the nucleotide is "extendable", for example, lacking an extension blocking portion at the 3' hydroxyl or any other position on the nucleotide. In other embodiments, the nucleotide is "blocked," having a moiety that prevents the 3' position from participating in extension by the addition of another nucleotide or oligonucleotide.

[0034] As used herein, the term "pitch" refers to the center-to-center distance of two sites in an array. The pattern of sites can be conventional, so that the coefficient of variation around the average spacing is small, or the pattern can be unconventional, in which case the coefficient of variation can be relatively large. In either case, the average spacing can be, for example, at least 10nm, 0.1μm, 0.5μm, 1μm, 5μm, 10μm, 100μm or more. Alternatively / in addition, the average spacing can be, for example, at most 100μm, 10μm, 5μm, 1μm, 0.5μm, 0.1μm or less. Of course, the average spacing of a particular pattern of sites can be between one of the lower limits and one of the upper limits selected from the above range.

[0035] As used herein, the term "primer" means a nucleic acid having a sequence that binds to a primer binding site at or near a template sequence. Typically, a primer is bound to a structure that allows, for example, replication of the template via polymerase extension of the primer. A primer can be a first portion of a nucleic acid molecule that binds to a second portion of a nucleic acid molecule, the first portion being a primer sequence and the second portion being a primer binding sequence (e.g., a hairpin primer). Alternatively, a primer can be a first nucleic acid molecule that binds to a second nucleic acid molecule having a template sequence. A primer can be composed of DNA, RNA, or its analogs.

[0036] As used herein, the term "primer extension product" means a primer that has been modified by adding at least one nucleotide analog. For example, a primer can be modified by adding one or more nucleotide analogs (e.g., via polymerase catalysis) to its 3' end, thereby forming a primer extension product. Alternatively, a primer extension product can be prepared by connecting an oligonucleotide to the 3' or 5' end of a primer. In this case, the primer extension product extends a length equal to the length of the oligonucleotide. The primer extension product can be at least 1, 2, 5, 10, 500, 1000 or more nucleotides longer than the primer. Alternatively or in addition, the primer extension product can be no more than 1, 2, 5, 10, 500 or 1000 nucleotides longer than the primer. For example, an extension product that is at least 1 nucleotide longer than a primer and no more than 1 nucleotide longer than a primer is provided using a sealed nucleotide analog.

[0037] As used herein, reference to "selectively" manipulating a first thing as compared to a second thing is intended to mean that the manipulation has a greater effect on the first thing than on the second thing. The manipulation need not have any effect on the second thing. The manipulation may have an effect on the first thing that is at least 1%, 5%, 10%, 25%, 50%, 75%, 90%, 95%, or 99% greater than the effect on the second thing. The manipulation may have an effect on the first thing that is at least 2 times, 3 times, 4 times, 5 times, 10 times, 100 times, 1x10 3 times, 1x10 4 times or 1x10 6 The manipulation may include, for example, modification, contacting, treating, altering, cleavage (e.g., cleavage of a chemical bond), photochemical cleavage (e.g., cleavage of a chemical bond), formation (e.g., formation of a chemical bond), photochemical formation (e.g., photochemical formation of a chemical bond), covalent modification, non-covalent modification, destruction, photo-ablation, removal, synthesis, polymerization, photopolymerization, amplification (e.g., amplification of a nucleic acid), replication (e.g., replication of a nucleic acid), extension (e.g., extension of a nucleic acid), ligation (e.g., ligation of a nucleic acid), or other manipulations described herein or otherwise known in the art.

[0038] As used herein, the term "sequence" when used in reference to a nucleic acid refers to the order of nucleotides (or bases) in a nucleic acid. In the case where different species of nucleotides are present in a nucleic acid, the sequence comprises the identification of the species of nucleotides (or bases) at various positions in the nucleic acid. A sequence is a property of a whole or part of a nucleic acid molecule. The term can be similarly used to describe the order and positional identity of monomeric units in other polymers, such as amino acid monomeric units of protein polymers.

[0039] As used herein, the term "site" means a location in an array where at least one analyte molecule is present. A site may contain only a single analyte molecule or it may contain a population of several analyte molecules of the same species. In some embodiments, a site may contain a plurality of different analyte molecule species, each species being present in one or more copies. Array sites are typically discrete. Discrete sites may be continuous or they may have intervals between each other.

[0040] As used herein, the term "kind" or "type" is used to identify molecules that share the same chemical structure. For example, a mixture of nucleotide analogs may include several dCTP molecules. The dCTP molecules will be understood to be the same kind or type as each other, but different kinds or types compared to dATP, dGTP, dTTP, etc. Similarly, individual DNA molecules with the same nucleotide sequence are of the same kind or type, while DNA molecules with different sequences are of different kinds or types. As another example, DNA polymerases are different polymerase kinds or types compared to RNA polymerases (even if the two polymerases are derived from the same organism).

[0041] As used herein, the term "universal sequence" refers to a sequence common to two or more nucleic acid molecules, even regions in which the molecules also have sequences that are different from each other. Universal sequences present in different members of a set of molecules can allow a population of universal capture nucleic acids complementary to the universal sequence to capture multiple different nucleic acids. Similarly, universal sequences present in different members of a set of molecules can allow a population of universal primers complementary to the universal sequence to replicate or amplify multiple different nucleic acids. Therefore, universal capture nucleic acids or universal primers include sequences that can specifically hybridize to universal sequences. Target nucleic acid molecules can be modified to attach universal adapters, such as at one or both ends of different target sequences.

[0042]

[0066] With the above definitions in mind, the embodiments set forth below and recited in the claims can be understood.

[0043] The present disclosure provides a method for sequencing a nucleic acid template. The method may include the following steps: (a) providing a site array, wherein each site comprises a mixture of at least two nucleic acid templates; (b) extending primers hybridized to the different nucleic acid templates at each site with different nucleotide analogs containing different reversible blocking parts, respectively, thereby generating different primer extension products at each site; (c) detecting the different primer extension products to distinguish the different nucleotide analogs at each site; (d) at each site, using a first treatment selective for the first different reversible blocking part and a second treatment selective for the second different reversible blocking part to remove the different reversible blocking part from the primer extension product; and (e) repeating (b) to (d) to determine the sequence of the different nucleotide analogs added to each different extension product at each site.

[0044] As described above, the method of the present disclosure may include the step of providing the first and second nucleic acid templates, wherein the sequences of the two templates are different. The two template sequences may be parts of a single nucleic acid molecule, or alternatively, the two template sequences may be located on separate molecules. As further described in detail elsewhere herein, the two template sequences may be adjacent, which are too close to be resolved spatially with the detection system used. However, the orthogonal detection method of the present disclosure allows these template sequences to be distinguished. The orthogonal detection scheme is taken as an example of two template sequences, but can be used together with two or more template sequences. Therefore, the system or method described herein may include at least 2, 3, 4, 5, 10 or more template sequences, which are extremely close on a single nucleic acid molecule, for example, at a single site of an array, or are adjacent in other cases, which are too close to be resolved spatially with the detection system used.

[0045] The target nucleic acid used herein can be composed of DNA, RNA or its analog. The source of the target nucleic acid can be genomic DNA, messenger RNA or other nucleic acid from natural sources. In some cases, the target nucleic acid derived from such source can be amplified and then used in the method or composition herein.

[0046] Exemplary biological samples from which target nucleic acids can be derived include, for example, those from mammals, such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cattle, cats, dogs, primates, humans or non-human primates; plants, such as Arabidopsis, corn, sorghum, oats, wheat, rice, canola or soybeans; algae, such as Chlamydomonas reinhardtii; nematodes, such as Caenorhabditis elegans; insects, such as Drosophila melanogaster, mosquitoes, fruit flies, bees or spiders; fish, such as zebrafish; reptiles; amphibians, such as frogs or African clawed frogs; dictyostelium discoideum; fungi, such as pneumocystis carinii, Takifugu rubripes, yeast, Saccharomyces cerevisiae and Schizosaccharomyces pombe; or Plasmodium falciparum. The target nucleic acid may also be derived from a prokaryotic organism such as bacteria, E. coli, Staphylococcus, or Mycoplasma pneumoniae; an archaea; a virus such as hepatitis C virus or human immunodeficiency virus; or a viroid. The target nucleic acid may be derived from a homogenous culture or population of the above organisms, or alternatively from a collection of several different organisms, such as in a community or ecosystem. The nucleic acid may be isolated using methods known in the art, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual , 3rd edition, Cold Spring Harbor Laboratory, New York (2001) or in Ausubel et al., Current Protocols in Molecular Biology , John Wiley and Sons, Baltimore, Md. (1998), each of which is incorporated herein by reference.

[0047] In a specific embodiment, nucleic acid samples can be modified or prepared for use in one or more methods described herein. In some cases, it is desirable to add one or more primer binding sites to nucleic acid. Known molecular biology techniques can be used to introduce primer binding sites located upstream of each template sequence, such as by inserting an adapter with a primer binding site, creating a mutation in a primer binding site, connecting an adapter with a primer binding site, etc. Sambrook et al., the same and Ausubel et al., the same describe useful methods. U.S. Patent Application Publication No. 2015 / 0031560A1 (which is incorporated herein by reference) provides an illustration of a technology based on tag fragmentation (tagmentation) for introducing a primer binding site. Tag fragmentation is particularly useful for introducing one or more primer binding sites, and for example, can be implemented using U.S. Patent Nos. 6,294,385 and 8,383,345, and PCT Publication No. WO 2012 / 106546 (each incorporated herein by reference). It should be understood that in some cases in the methods set forth herein, naturally occurring sequence regions present in the upstream of the respective template sequences can be utilized as primer binding sites. Other desired sequence elements, such as promoter elements or tag sequences for extension based on RNA polymerase, can be introduced using methods similar to those exemplified above for primer binding sites.

[0048] Universal priming site is particularly useful for the multiple applications of the method set forth herein. Universal priming site provides a sequence region common to two or more nucleic acid molecules, wherein the molecule also has a template or target region containing different sequences. Universal priming sequences present in different members of a molecular set can allow the use of a single universal primer species complementary to the universal priming sequence to replicate, increase or detect a plurality of different sequences. Therefore, universal primers include sequences that can hybridize with universal priming sequence specificity. Examples of methods for attaching universal sequences to a set of target nucleic acids can be found in U.S. Patent Publication No. 2007 / 0128624A1 (which is incorporated herein by reference).

[0049] In some embodiments, target nucleic acid can be obtained as a fragment of one or more larger nucleic acids. Fragmentation can be implemented using any of a variety of techniques known in the art, such as including atomization, ultrasonic treatment, chemical cutting, enzymatic cutting, or physical shearing. Fragmentation can also be produced by using a specific amplification technique, which produces an amplicon by only copying a part of a larger nucleic acid. For example, PCR amplification produces a fragment with a size limited by the fragment length between the flanking primers for amplification.

[0050] The colony of target nucleic acid or its amplicon can have the specific application expectation or suitable average chain length for the method or composition set forth herein.For example, average chain length can be lower than about 100,000 nucleotides, 50,000 nucleotides, 10,000 nucleotides, 5,000 nucleotides, 1,000 nucleotides, 500 nucleotides, 100 nucleotides, or 50 nucleotides.Or / in addition, average chain length can be higher than about 10 nucleotides, 50 nucleotides, 100 nucleotides, 500 nucleotides, 1,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, 50,000 nucleotides, or 100,000 nucleotides.The average chain length of the colony of target nucleic acid colony or its amplicon can be in the scope between the maximum value and the minimum value listed herein.It should be understood that the amplicon generated at the amplification site (or prepared or used herein in other cases) can have the scope between the upper limit and the lower limit selected from the numerical value of the above example.

[0051] In some cases, it is possible to generate or configure the target nucleic acid colony in other cases to have the maximum length of its members.For example, the maximum length of the member prepared or used as described herein can be less than about 100,000 nucleotides, 50,000 nucleotides, 10,000 nucleotides, 5,000 nucleotides, 1,000 nucleotides, 500 nucleotides, 100 nucleotides, or 50 nucleotides.Or / in addition, it is possible to generate or configure the colony of the target nucleic acid or its amplicon under the conditions to have the minimum length of its members in other cases.For example, the minimum length of the member used in one or more steps of the method described herein or the minimum length of the member present in the specific composition can be higher than about 10 nucleotides, 50 nucleotides, 100 nucleotides, 500 nucleotides, 1,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, 50,000 nucleotides, or 100,000 nucleotides.The maximum and minimum chain length of the target nucleic acid in the colony can be in the range between the maximum and minimum values ​​described above. It will be appreciated that amplicons generated at an array site (or otherwise prepared or used herein) may have a maximum and / or minimum chain length ranging between the upper and lower limits exemplified above.

[0052] Any of a variety of known amplification techniques can be used to increase the amount of template sequences present for the methods set forth herein. Exemplary techniques include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA) or random primer amplification (RPA) of nucleic acid molecules with template sequences. It should be understood that the amplification of target nucleic acids before use in the methods or compositions set forth herein is optional. Thus, target nucleic acids will not be used before use in some embodiments of the methods and compositions set forth herein. Target nucleic acids may optionally be derived from synthetic libraries. Synthetic nucleic acids may have natural DNA or RNA compositions or may be analogs thereof. Solid phase amplification methods may also be used, including, for example, cluster amplification in the context of array-based methods, bridge amplification or other methods set forth below.

[0053] The nucleic acid used in the method described herein can be a solution phase or a solid phase. When in a solution phase, the nucleic acid is generally soluble, but it can also be in a suspended form that can be precipitated, as is the case with some large nucleic acid species such as chromosomes or nucleic acid nanospheres (see, for example, U.S. Patent Publication No. 2007 / 0099208A1, which is incorporated herein by reference). The nucleic acid of the solid phase may be present in or on a solid support. Exemplary solid supports include those made of glass, nitrocellulose, silica, metal, plastic, and other materials described in other parts of this article, such as array forms and flow cells. Similarly, nucleic acids may be present in or on semi-solid supports such as gels. Useful exemplary gels include, but are not limited to, those having a colloidal structure, such as agarose; a polymer mesh structure, such as gelatin; or a cross-linked polymer structure, such as polyacrylamide. Hydrogels are particularly useful, such as those shown in U.S. Patent Publication No. 2011 / 0059865A1 and U.S. Patent No. 9,012,022, each of which is incorporated herein by reference.

[0054] Attaching nucleic acid to a support (whether rigid or semi-rigid) can occur by covalent or non-covalent attachment. Exemplary attachments are described in U.S. Patent Nos. 6,737,236, 7,259,258, 7,375,234, and 7,427,678; and U.S. Patent Application Publication No. 2011 / 0059865A1, each of which is incorporated herein by reference. In some embodiments, nucleic acid or other reaction components can be attached to a gel or other semi-solid support, which in turn is attached or adhered to a solid support. In such embodiments, nucleic acid or other reaction components will be understood as a solid phase.

[0055] Multiple reactions can use a solid support (also known as a solid support). A solid support can be used to separate individual reactions so that each reaction can be separated or individually interrogated. For example, several different nucleic acids in a mixture can be attached to a solid support. Nucleic acids can be attached to a solid support in an array format.

[0056] In some embodiments, an array of sites is provided, wherein each site comprises a first nucleic acid template and a second nucleic acid template, and wherein the first nucleic acid template has a sequence different from the sequence of the second nucleic acid template. In some embodiments, each site can have more than 2 different templates. Exemplary array materials and manufacturing methods that can be modified for use herein include, but are not limited to, Inc. (San Diego, CA) or arrays such as those described in U.S. Pat. Nos. 6,266,459, 6,355,431, 6,770,441, 6,859,570 or 7,622,294 or PCT Publication No. WO 00 / 63437, each of which is incorporated herein by reference. Further examples of commercial arrays that can be used include, for example VLSIPS TM Other arrays synthesized using the Very Large Scale Immobilized Polymer Synthesis (Very Large Scale Immobilized Polymer Synthesis) technique. According to some embodiments, dotted arrays may also be used. An exemplary dotted array is available from Amersham Biosciences at CodeLink TM Another useful array is one made using an inkjet printing method, such as SurePrint available from Agilent Technologies. TM technology.

[0057] Other useful arrays that can be used (e.g., by modifying sites to include a variety of different template nucleic acid sequences) include those used in nucleic acid sequencing applications. For example, arrays with amplicons of genomic fragments (commonly referred to as clusters) are particularly useful, such as in Bentley et al., Nature 456:53-59 (2008), PCT Publication Nos. WO 04 / 018497, WO 91 / 06678, or WO 07 / 123744; U.S. Patent Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019, or 7,405,281; or those described in U.S. Patent Application Publication No. 2008 / 0108082A1, each of which is incorporated herein by reference.

[0058] Nucleic acid clusters can be produced by solid phase amplification methods. For example, nucleic acids with one or more template sequences to be detected can be attached to a surface and amplified using bridge amplification. Useful bridge amplification methods are described in, for example, U.S. Patent Nos. 5,641,658 or 7,115,400; or U.S. Patent Application Publication Nos. 2002 / 0055100A1, 2004 / 0096853A1, 2004 / 0002090A1, 2007 / 0128624A1, or 2008 / 0009420A1, each of which is incorporated herein by reference. Another useful method for amplifying nucleic acids on surfaces is rolling circle amplification (RCA), for example, as described in Lizardi et al., Nat.Genet.19:225-232 (1998) and U.S. Patent Application Publication No. 2007 / 0099208A1, each of which is incorporated herein by reference. Another useful array type is an array of particles generated from an emulsion PCR amplification technique. Examples are described in Dressman et al., Proc. Natl. Acad. Sci. USA 100: 8817-8822 (2003), PCT Application Publication No. WO05 / 010145, or U.S. Patent Application Publication No. 2005 / 0130173A1 or 2005 / 0064460A1, each of which is incorporated herein by reference. Although the above arrays have been described in the context of sequencing applications, it should be understood that the arrays can be used in other embodiments, including, for example, embodiments using non-cyclic primer extension techniques.

[0059] Detection can be carried out on the array at the overall or single molecule level. Overall level detection is a detection that occurs in the following manner, so that several copies of a single template sequence (such as multiple amplicons of a template) are detected at each individual site and the individual copies at the site are not distinguished from each other. Therefore, overall detection provides an average signal from multiple copies of a specific template sequence at the site. For example, the site may include at least 10, 100, 1000 or more copies of a specific template sequence. Of course, the site may include a plurality of different template sequences, each of which exists as a whole. Alternatively, detection at the single molecule level includes detection that occurs in the following manner, so that individual template sequences (each at different sites) are resolved separately on the array. Therefore, single molecule detection provides a signal from an individual molecule, which is distinguished from one or more signals of a molecular population that can be derived from the individual molecule. Of course, even in a single molecule array, a site may include several different template sequences (e.g., two or more template sequence regions located along a single nucleic acid molecule).

[0060] The array of sites can be presented as a grid of points or sheets. The sites can be positioned in a repeated pattern or in an irregular non-repeating pattern. Particularly useful patterns are hexagonal patterns, straight line patterns, grid patterns, patterns with reflection symmetry, patterns with rotational symmetry, etc. Asymmetric patterns can also be useful.

[0061] The size of the sites in the array and / or the spacing between the sites may be different to achieve high density, medium density or lower density. High density arrays are characterized as sites with spacings less than about 15 μm. Medium density arrays have sites with spacings of about 15 to 30 μm, while low density arrays have sites with spacings greater than 30 μm. In some embodiments, useful arrays may have sites with spacings less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm. The embodiments of the methods described herein may be used to image the array with a resolution sufficient to distinguish sites with the above density or density range. However, the detection step will generally use a detector having a spatial resolution that is too low to distinguish points at a distance equal to the spacing between the first template (or the first primer extension product hybridized therewith) and the second template (or the second primer extension product hybridized therewith) at individual sites. In a specific embodiment, the array sites may each have a spacing greater than about 100 nm. 2 , 250nm 2 , 500nm 2 , 1μm 2 , 2.5μm 2 , 5μm 2 , 10μm 2 , 100μm 2 , or 500μm 2 Alternatively, or in addition, the array sites may each have an area of ​​less than about 1 mm 2 , 500μm 2 , 100μm 2 , 25μm 2 , 10μm 2 , 5μm 2 , 1μm 2 , 500nm 2 , or 100nm 2 In fact, the site may have a size within a range between the upper and lower limits selected from those exemplified above.

[0062] The methods described herein may use arrays having sites at any of a variety of densities, including, for example, at least about 10 sites / cm 2 , 100 sites / cm 2 , 500 sites / cm 2 , 1,000 sites / cm2 , 5,000 sites / cm 2 , 10,000 sites / cm 2 , 50,000 sites / cm 2 , 100,000 sites / cm 2 , 1,000,000 sites / cm 2 , 5,000,000 sites / cm 2 or higher.

[0063] Usually, array will have the site that contains different nucleic acid sequence content.Therefore, each site in the array can contain the unique nucleic acid sequence compared with the nucleic acid sequence in other sites in the array.Yet, in some cases, array can have redundancy so that two or more sites have the same nucleic acid content.

[0064] It should be understood that the steps of the methods described herein can be implemented in a certain manner to expose the entire site or multiple sites of the array with a treatment. For example, a primer extension reagent can be delivered to the array so that multiple nucleic acids (such as different nucleic acids in a mixture) at each of one or more sites of the array are contacted with the primer extension reagent, thereby performing steps involving primer extension. Similarly, the array can be exposed to a deblocking treatment so that multiple nucleic acids (such as different nucleic acids in a mixture) at each of one or more sites of the array are contacted with the treatment, so that a step of deblocking the blocked primer extension products can be performed.

[0065] In synthetic sequencing, or any given point of other primer extension reactions, the nucleotide species present in the first template at the position complementary to the primer extension site can be identical with the nucleotide species present in the second template at the position complementary to the primer extension site. In other words, the first nucleic acid template at the specific site of the array can include at least one base portion, which is the same species as the base portion at the specific site in the second nucleic acid template, and complementary nucleotide analogs can be added to each primer at the position in the template where those base portions are located. Whether the template sequence of primer hybridization is identical or different, this can be the case. The technology described in further detail below can be used to distinguish two templates, for example using nucleotide analogs of different groups with mutually distinguishable markers.

[0066] Any of a variety of polymerases can be used in the method or composition set forth herein, for example, separated from an enzyme based on protein and its functional variants of a biological system. It will be appreciated that reference to specific polymerases such as those of the following examples includes functional variants thereof, unless otherwise indicated. The particularly useful function of a polymerase is to use existing nucleic acids as the polymerization of template catalyzed nucleic acid chains. Other useful functions are described in other parts of this paper. Examples of useful polymerases include DNA polymerases, reverse transcriptases, and RNA polymerases.

[0067] Polymerases with inherent 3' to 5' proofreading exonuclease activity may be useful for some embodiments. Polymerases substantially lacking 3' to 5' proofreading exonuclease activity are also useful in some embodiments, such as in most sequencing embodiments. The lack of exonuclease activity may be a wild-type characteristic or a characteristic conferred by a variant or engineered polymerase structure. For example, the exo minus Klenow fragment is a mutant form of the Klenow fragment that lacks 3' to 5' proofreading exonuclease activity.

[0068] Depending on the embodiment to be used, the polymerase can be thermophilic or heat-inactivatable. Thermophilic polymerases are generally useful for high temperature conditions or in thermal cycling conditions such as those employed by polymerase chain reaction (PCR) techniques. Examples of thermophilic polymerases include, but are not limited to, 9°N DNA polymerase, Taq DNA polymerase, DNA polymerase, Pfu DNA polymerase, RB69 DNA polymerase, KOD DNA polymerase and DNA polymerase. Most polymerases isolated from non-thermophilic organisms are heat inactivatable. Examples are DNA polymerases from bacteriophages. It should be understood that polymerases from any of a variety of sources can be modified to increase or decrease their tolerance to high temperature conditions. Particularly useful polymerases for incorporating nucleotide analogs with markers and / or reversible termination moieties are described in US2006 / 0281109A1, which is incorporated herein by reference.

[0069] In the specific embodiments of the methods and compositions set forth herein, only a single type of polymerase is used. In such examples, each site of the array interacts with only one polymerase at a given time, even though multiple polymerases may be present at the site and bind to multiple initiated templates at each site. For example, all sites of the array may interact with a particular type of DNA polymerase, and other polymerases (such as RNA polymerases) will not be in the array.

[0070] Other extension techniques that can be modified and used for the method or composition set forth herein are based on a ligase system, which is selective for incorporating oligonucleotides rather than monomeric nucleotides incorporated by the polymerase extension system described above. The DNA ligase system using the first group of oligonucleotides is orthogonal to the reagent system using the second group of oligonucleotides, wherein the first group of oligonucleotides has a reversibly sealed portion selectively removed by the first removal of the seal, and the second group of oligonucleotides is selectively removed by the second removal of the seal. The removal of the primer extended with the reversibly sealed oligonucleotide can be carried out in an orthogonal form, which is very similar to the mode exemplified herein for the removal of the primer extended with the nucleotide analog of the reversible seal. Extension (Extension byligation) can be carried out in the sequencing application of a population of partially random probe oligonucleotides using one or two base coding schemes. Ligation-based extension technology in the context of sequencing that can be modified for use herein is described in McKernan et al., Genome Research 19(9): 1527-41 (2009); Shendure et al., Science 309: 1728-1732 (2005); or U.S. Pat. Nos. 5,599,675 or 5,750,341, each of which is incorporated herein by reference.

[0071] The nucleic acid extension reaction or other cyclic reactions carried out using the method set forth herein can carry out one or more cycles. In a specific embodiment, multi-cycle reactions may include at least 2 cycles, 5 cycles, 10 cycles, 50 cycles, 100 cycles, 500 cycles, 1,000 cycles, 5,000 cycles, 10,000 cycles or more. Alternatively or additionally, the reaction may have an upper limit, wherein no more than 1 cycle, 2 cycles, 5 cycles, 10 cycles, 50 cycles, 100 cycles, 500 cycles, 1,000 cycles, 5,000 cycles or 10,000 cycles occur. In some embodiments, each cycle will result in the incorporation of a single nucleotide analog to the primer extended. In this case, the minimum cycle number or the maximum cycle number of the above examples can be understood as the minimum number or the maximum number of nucleotides incorporated into the extension product in the reaction catalyzed by the exemplary polymerase.

[0072] Some embodiments may use non-cyclic extension reactions, such as single base extension (SBE) reactions or allele-specific primer extension (ASPE) reactions. Reversible terminators may be used in part to achieve orthogonal extension of two different primers in a non-cyclic extension format. Since the deblocking step is not necessary for the continuation of these non-cyclic reactions (i.e., once the primer has been extended), instead, the nucleotide analogs used in the extension step may be irreversibly terminated. For example, dideoxynucleotides may be used. Exemplary reagents and related techniques for SBE, ASPE, and other useful non-cyclic extension techniques are described in, for example, U.S. Patent No. 7,670,810 or U.S. Patent Application Publication Nos. 2003 / 0108867, 2003 / 0108900, 2003 / 0170684, 2003 / 0207295, or 2005 / 0181394, each of which is incorporated herein by reference. Examples of commercially available products that use non-cyclic extension technology and can be modified to increase information content through the orthogonal detection methods described herein are available from Illumina, Inc. (San Diego, CA). Genotyping Products.

[0073] Cyclic and non-cyclic reactions can also include steps to separate the reaction components from each other or remove them from the reaction environment. One or more reaction components can be separated, for example, by separating the solid phase component from the liquid phase component. A washing step can be optionally included to more completely remove unwanted one or more liquid phase components from one or more solid phase components. A reaction vessel particularly useful for such separations is a flow cell, such as those commonly used in cycle sequencing methods. Exemplary flow cells, their methods of manufacture, and methods of use thereof are described in U.S. Patent Application Publication Nos. 2010 / 0111768A1 or 2012 / 0270305A1; or PCR Application Publication No. WO05 / 065814, each of which is incorporated herein by reference. Whether or not a solid phase separation method is used, the reaction components can be removed by any of a variety of other techniques known in the art, including liquid-liquid extraction, solid phase extraction, chromatography, filtration, centrifugation, etc.

[0074] The reversible terminator moiety provides a convenient way to control the extension reaction to add only a single nucleotide to the primer until a subsequent deblocking step is performed. As described herein, the use of an orthogonal blocking moiety and deblocking process provides super-resolution detection, in which the template complexity that can be monitored and detected is greater than that otherwise allowed using non-orthogonal techniques. Examples of reversible blocking moieties and their deblocking conditions include, but are not limited to, moieties that can be removed from the 3' position by photocleavage, such as o-nitrobenzyl ethers and alkyl o-nitrobenzyl carbonates / esters; ester moieties that can be removed by alkaline hydrolysis; allyl moieties that can be removed with NaI, trimethylsilyl chloride and Na2S2O3 or Hg(II) in acetone / water; available phosphines, such as tris(2-carboxyethyl)phosphine (T CEP) or tris (hydroxypropyl) phosphine (THP) cleavage - azidomethyl (-CH2-N3); acetal, such as tert-butyloxy-ethoxy cleavage in an acidic environment; MOM (-CH2OCH3) moiety cleavageable with LiBF4 and CH3CN / H2O; 2,4-dinitrobenzenesulfinyl cleavageable with nucleophiles such as thiophenol and thiosulfate; tetrahydrofuran ether cleavageable with Ag (I) or Hg (II); and 3' phosphate cleavageable with phosphatases (such as polynucleotide kinases). Other useful reversible moieties include esters, -F, -NH2, -OCH3, -N3, -NHCOCH3, and 2-nitrophenyl carbonate. Useful deblocking treatments include irradiation with light (such as to induce photocleavage), heating, exposure to chemical reagents, exposure to catalysts, exposure to electric current (such as to induce electrolysis), etc.

[0075] The specific embodiments of the method herein can, for example, utilize reversibly blocked primers and reversibly blocked nucleotide analogs in multiple cycles (such as sequencing by synthesis). As previously illustrated herein, specific reversibly blocked primers and reversibly blocked nucleotide analogs of a specific group can be susceptible to the same deblocking conditions. This may be because of the reversibly blocked portions of the same type present on the primers and on the nucleotide analogs in the group. However, different blocking portions may also be used, but the blocking portions are still susceptible to the same deblocking treatment.

[0076] The reversible blocking portion can be attached to the 3' nucleotide of the primer. Typically, the reversible blocking portion is attached to the 3' position of the ribose moiety. However, instead, the blocking portion can be attached to an alternative position, such as included in the 2' position of the ribose or on the base portion. The blocking portion can also be attached to the 5' end of the primer, such as at the 5' position of the ribose of the terminal nucleotide, or at the 5' phosphate moiety. The 5' end-blocked primer can be used for embodiments using ligation techniques.

[0077] The 3' or 5' end of the primer may be attached to a marker moiety, such as an optical marker. The marker may be present whether or not a reversible blocking moiety is also present on the primer. In some cases, a particular moiety may function both as a marker and as a reversible blocker for primer extension.

[0078] The same attachment points for labels and / or reversible blocking moieties exemplified above for primers may be useful for individual nucleotides.

[0079] Further exemplary guidance regarding blocking moieties and deblocking processes is described, for example, in Bentley et al., Nature 456:53-59 (2008), PCT Application Publication Nos. WO 04 / 018497, WO 91 / 06678, or WO 07 / 123744; U.S. Pat. Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019, 8,088,575, or 7,405,281; or U.S. Patent Application Publication No. 2008 / 0108082A1, each of which is incorporated herein by reference.

[0080] Orthogonal manipulation and detection according to the present disclosure do not require that the two template sequences differ at every position along their length. Specifically, identical base portions may be present at positions detected on the first template and the second template, respectively. The two positions may be distinguished based on the distinguishable characteristics of the markers present in the orthogonal reagent system and the specificity of the reagent system used to extend the appropriately deblocked primers. This information can then be used to distinguishably detect two different template sequences, even if the two positions are detected simultaneously using a detector having a resolution that is too low to resolve points at a distance equivalent to the separation of the two template sequences.

[0081] Any of a variety of labels can be used. When used to detect nucleotide analogs, particularly useful label moieties can be any portion of a nucleotide analog that provides distinguishable features when compared to other molecules present in its environment. Distinguishing features can be, for example, optical signals such as absorption of radiation, fluorescence emission, luminescent emission, fluorescence lifetime, fluorescence polarization, etc.; binding affinity to a ligand or receptor; magnetic properties; electrical properties; charge; mass; radioactivity, etc. Exemplary label moieties include, but are not limited to, fluorophores, luminophores, chromophores, radioisotopes, mass labels, charge labels, spin labels, receptors, ligands, etc. The label moiety can be a portion of a nucleotide of a monomeric unit present in a nucleic acid polymer, or the label moiety can be a portion of a free nucleotide analog (e.g., nucleoside triphosphates).

[0082] Fluorophores are particularly useful and include, for example, fluorescent nanocrystals; quantum dots, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, Cy3, Cy5, stilbene, Lucifer Yellow, Cascade Blue, Texas Red, Alexa dyes, SETA dyes, Atto dyes, phycoerythrin, bodipy, and the like. Useful optical probes are described in Lakowicz, Principles of Fluorescence Spectroscopy , 3rd edition Springer (2006); Haugland, Handbook of FluorescentProbesandResearchProducts 9th Edition, Molecular Probes, Inc, (2002); Shapiro, Practical Flow Cytometry , 4th edition, John Wiley & Sons (2003); PCT Patent Application Publication No. WO98 / 59066 or WO91 / 06678; or U.S. Patent Application Publication No. 2010 / 0092957A1, each of which is incorporated herein by reference.

[0083] Other labels, some of which are non-optical labels, can be used in various embodiments of the methods and compositions described herein. Examples include, but are not limited to, isotopic labels such as naturally non-abundant radioactive or heavy isotopes; magnetic materials; electron-rich materials such as metals; electrochemiluminescent labels such as Ru(bpy); 32+ ; or a portion that can be detected based on nuclear magnetic, paramagnetic, electrical, charge-to-mass ratio or thermal characteristics. The marker may also include magnetic particles or optically encoded nanoparticles. Such markers can be detected using appropriate methods known to those skilled in the art. For example, charged markers can be detected using electrical detectors, such as those used in commercially available sequencing systems from Ion Torrent (Guilford, CT, a subsidiary of Life Technologies) or in detection systems described in U.S. Patent Application Publication Nos. 2009 / 0026082A1; 2009 / 0127589A1; 2010 / 0137143A1; or 2010 / 0282617A1, each of which is incorporated herein by reference. It should be understood that for some embodiments, nucleotide analogs may lack one or more markers set forth herein.

[0084] The label moiety can be attached to the nucleotide in a variety of ways. Exemplary attachment and label compositions useful for nucleotide analogs are described in Bentley et al., Nature 456:53-59 (2008), PCT Application Publication Nos. WO 04 / 018497, WO 91 / 06678, or WO 07 / 123744; U.S. Patent Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019, 8,088,575, or 7,405,281; or U.S. Patent Application Publication No. 2008 / 0108082A1, each of which is incorporated herein by reference.

[0085] The detection step of the method described herein can be carried out using a device suitable for the specific marker used in the method of the present disclosure. For example, an optical detector such as a fluorescence detector, an absorbance detector, a luminescence detector, etc. can be used to detect appropriate optical markers. Systems designed for array-based detection are particularly useful. For example, an optical system used with the method described herein can be constructed to include various elements and accessories, such as in U.S. Patent Nos. 8,241,573, 7,329,860, or 8,039,817; or described in U.S. Patent Application Nos. 2009 / 0272914A1 or 2012 / 0270305A1, each of which is incorporated herein by reference.

[0086] Orthogonal detection system, such as the system for sequencing by synthesis, can use different markers to distinguish the different nucleotides added to every kind of primer.In one embodiment, each nucleotide species will have the unique optical marker that produces unique signal to distinguish the nucleotide species.An example is 8 dye method.In this example, the first group of 4 kinds of different nucleotide analogs each have different fluorescent dyes that distinguish each of 4 kinds of different nucleotide analogs from each other, wherein the 4 kinds of different nucleotides in the first group have the blocking part that can be processed by the first selectivity to block.The second group of 4 kinds of different nucleotide analogs each have different fluorescent dyes that distinguish each of 4 kinds of different nucleotide analogs from each other, wherein the 4 kinds of different nucleotides in the second group have the blocking part that can be processed by the second selectivity to block.In this case, the first processing is selective for the blocking part in the first group of nucleotides, and the second processing is selective for the blocking part in the second group of nucleotides.These two groups of dyes are unique, so that these 8 kinds of dyes produce 8 kinds of distinguishable signals respectively.

[0087] In the embodiment where all nucleotide analogs are distinguishably labeled, such as the above-mentioned 8 dye method, a pair of template sequences can be contacted with all nucleotide analogs, and then can be detected thereafter. Here, the ability of unique optical markers to distinguish all nucleotide analogs provides the benefit of relatively simple fluid operation, wherein all nucleotide analogs can be delivered to the template sequence, so that they exist simultaneously. In a relatively simple and preferred embodiment, all 8 kinds of nucleotide analogs are delivered simultaneously; however, if necessary, one or more subsets can be delivered sequentially. Detection can occur during or after the nucleotide analog delivery process. This relatively simple fluid process is provided by a relatively complex detection device with the ability to distinguish all different signals. For example, a fluorescence detection system that can distinguish 8 different fluorescent signals can be used for the 8 dye method. Those skilled in the art will know or be able to determine the appropriate fluorescence detection device for realizing such signal distinction. For example, the excitation and emission properties of fluorescent markers can be appropriately matched with the combination of the excitation wavelength produced by the fluorometer and the emission wavelength detected. Exemplary guidance for optics and labels that can be used for multi-wavelength fluorescence detection is provided in Lakowicz, Principles of Fluorescence Spectroscopy, 3rd Edition Springer (2006); Haugland, Handbook of Fluorescent Probes and Research Products 9th Edition, Molecular Probes, Inc, (2002); and Shapiro, Practical Flow Cytometry, 4th Edition, John Wiley & Sons (2003), each of which is incorporated herein by reference.

[0088] The principle of the system exemplified above for all nucleotides being distinguishably labeled can be easily extended to array format.When treated with a primer extension reaction system, it is expected that the array of different template sequences with sufficient number and type will incorporate all labeled nucleotides.More specifically, in an array-based method with extremely multiple nucleic acids between array sites and with two different templates / sites, it is expected that after all 8 kinds of nucleotides are delivered to the primer extension cycle of the array, the dye combination of all possible 2-dye appears on the array.Site can be spatially distinguished using optical devices known in the art, such as in U.S. Patent Nos. 8,241,573, 7,329,860 or 8,039,817; or those described in U.S. Patent Application Publication Nos. 2009 / 0272914A1 or 2012 / 0270305A1, each of which is incorporated herein by reference.Such detection systems can be easily modified to adapt to 8 color fluorescence detections as described above. A detection system modified in this manner would be capable of multiplexed orthogonal detection, thus enabling discrimination between two different templates (eg, by sequencing) at multiple sites, each of which has a different sequence composition.

[0089] In some embodiments, the number of different signals distinguished in a particular method is less than the number of different nucleotide analogs used in the method. For example, a subset of a plurality of different nucleotide analogs may have the same marker and / or nucleotide species may be unlabeled. The example of a configuration using the same marker for a plurality of different nucleotide species is the case of an orthogonal primer extension method, wherein 4 different nucleotide analogs of the first group share a common first marker and are susceptible to the first selective deblocking process, while 4 different nucleotide analogs of the second group share a common second marker and are susceptible to the second selective deblocking process. In this example, the first marker is optically distinguishable from the second marker, and the first process is selective to the first group of nucleotide analogs and the second process is selective to the second group of nucleotide analogs. In this configuration, 4 different nucleotides in the first group can be distinguished from each other by the sequential circulation of delivery and detection (i.e., the separate circulation of each different nucleotide analog). As long as the first and second labels in this example are distinguishable, members of the two groups of nucleotides can be delivered in pairs (1 single nucleotide species each from the first group and 1 single nucleotide species each from the second group) in 4 cycles of delivery and detection. Thus, members of the first group of nucleotide analogs used in the primer extension reaction can include only one type of optical label to be detected, and a second group of nucleotide analogs orthogonal to the first group can also include only one type of optical label to be detected, wherein the label used in the first group is optically distinguishable from the label used in the second group.

[0090] Grayscale (greyscaling) allows the use of a variety of different nucleotide analogs with the same marker. Here, different nucleotide analog types can be distinguished based on the intensity of the detected marker signal. For example, the nucleotide analogs of each type can be delivered as a unique proportional mixture of this nucleotide type of marked and unlabeled form. For each type of marked: the change in the ratio of unlabeled nucleotide analogs will result in the unique grayscale signal output of each mixture. By more specific examples, the first nucleotide analog can be fully labeled (mixture of unlabeled and unlabeled first nucleotide analogs), the second nucleotide analog can be 75% labeled (mixture of 75% labeled and 25% unlabeled second nucleotide analogs), the third nucleotide analog can be 50% labeled (mixture of 50% labeled and 50% unlabeled third nucleotide analogs), and the fourth nucleotide analog can be 25% labeled (mixture of 25% labeled and 75% unlabeled fourth nucleotide analogs). These four nucleotide analog species can be distinguished based on the resulting differences in signal intensity, where (e.g., at an array site) an appropriate deblocking primer population will produce a full signal due to incorporation of the first nucleotide analog; a 75% signal due to incorporation of the second nucleotide analog; a 50% signal due to incorporation of the third nucleotide analog; and a 25% signal due to incorporation of the fourth nucleotide analog.

[0091] In specific embodiments, at least one of the nucleotide analogs can be completely unlabeled. Therefore, in the case where an optical marker is present on other nucleotide analogs in the nucleotide analog group, "dark" nucleotide analogs may also be present. Primer extension can be determined by inferring based on the lack of a marker to mix dark nucleotide analogs or unlabeled nucleotide analogs in other cases, and the lack of the marker will be expected when other nucleotide analogs in the group have been mixed by extension reaction. Therefore, in some embodiments, only the subset of the nucleotide analogs used in the primer extension reaction set forth herein needs to have a marker.

[0092] The use of completely unlabeled nucleotide analog species can be combined with grayscale. For example, three of four different nucleotide analog species in a group (i.e., a group that can be deblocked by conventional treatment) can have a distinguishable non-zero amount of specific markers (e.g., the ratio of labeled and unlabeled nucleotide analogs in a mixture) and the fourth nucleotide analog species can lack the marker. Alternatively or in addition, grayscale can be combined with the use of several optically distinguishable markers. For example, some nucleotide analog species can appear as a mixture in an extension reaction, and the mixture is a mixture of nucleotides of the same type but with different markers. Such configurations are exemplified in U.S. Patent Application Publication No. 2013 / 0079232A1 or 2015 / 0031560A1, which are incorporated herein by reference.

[0093] As a plurality of different markers, gray scale, and / or the alternative of the use of unlabeled species or in addition, the embodiment set forth herein can use nucleotide analogs, and this nucleotide analog has part, can cut joint or provides the acquisition of marker or other parts of loss due to limiting treatment.In U.S. Patent Application Publication No. 2013 / 0079232A1 or 2015 / 0031560A1, described this type of reagent system, some of these nucleotide analog species have part, make can be based on the initial lack of detectable signal, then the signal after the receptor treatment with suitable mark appears to distinguish them from other nucleotide analogs.These references also illustrate the purposes of nucleotide analogs, and described nucleotide analogs can be based on the initial detectable signal, thereafter owing to losing or at least reducing to distinguish with the reagent treatment of modified marker (such as by the chemical cutting of the joint between marker and nucleotide part).In this case, other nucleotide analog species in group are not susceptible to modification (such as lacking can cut joint) and distinguish based on the persistence of signal generation after processing.

[0094] As exemplified above, in some embodiments, the label can be attached to the nucleotide analog via a cleavable linker. In a specific embodiment, a light-cleavable linker can be used instead of the chemically cleavable linker exemplified above. In some embodiments, the linker is selected from an acid-labile linker (including a double alkoxybenzyl linker, a Sieber linker, an indole linker, a tert-butyl Sieber linker), an electrophilic cleavable linker, a nucleophilic cleavable linker, a light-cleavable linker, a linker cleaved under reducing conditions or oxidizing conditions, a safety plug linker, and a linker cleaved by an elimination mechanism. In some such embodiments, the linker is selected from a disulfide linker (-SS-), an ester, nitrobenzene, an imine, an enzymatic or chemically cleavable peptide and a polynucleotide, such as DNA.

[0095] In some embodiments, members of a first set of nucleotide analogs used in a primer extension reaction (e.g., nucleotide analogs selectively deblocked by a first conventional treatment) will include only one type of optical label to be detected, and a second set of nucleotide analogs orthogonal to the first set (e.g., nucleotide analogs selectively deblocked by a second conventional treatment) will also include only one type of optical label to be detected, wherein the labels used in the first set are optically distinguishable from the labels used in the second set. In this embodiment, the one type of optical label can be attached to substantially all nucleotide analogs of a first species in the first set, one type of optical label can be attached to a subset of nucleotide analogs of a second species in the first set, substantially all nucleotide analogs of a third species in the first set can be attached to a ligand, and substantially all nucleotide analogs of a fourth species in the first set are not attached to one type of optical label or a ligand.

[0096] In another embodiment, the members of the first group of nucleotide analogs used in the primer extension reaction (e.g., nucleotide analogs selectively deblocked by the first conventional treatment) will include only two types of optical labels to be detected, and the second group of nucleotide analogs orthogonal to the first group (e.g., nucleotide analogs selectively deblocked by the second conventional treatment) will also include only two types of optical labels to be detected. In this embodiment, the first of the two types of optical labels can be attached to substantially all nucleotide analogs of the first type in the first group, the second of the two types of optical labels can be attached to substantially all nucleotide analogs of the second type in the first group, the first of the two types of optical labels and the second of the two types of optical labels can be attached to nucleotide analogs of the third type in the first group, and substantially all nucleotide analogs of the fourth type in the first group are not attached to one of the two types of optical labels or the second of the two types of optical labels.

[0097] From above example, it will be understood that reducing the number of different markers in the orthogonal detection system can provide the advantage of reducing the complexity of the detection device needed for different nucleotides that distinguish the primer that combines to template.But, in many embodiments, this is achieved by: increasing the complexity of the fluid step, so that the number of fluid operations used in the detection step process is increased compared with the fluid step used when each nucleotide species has a unique marker.The general advantage of this method is that those skilled in the art can select the appropriate combination of marker, fluid step and detection device to adapt to specific application or environment.

[0098] As the embodiment illustrated above, in some cases, two or more primers that hybridize with two or more different nucleic acid templates at the site respectively can exist simultaneously during the primer extension step. Alternatively, the first of the two primers that hybridize with two or more different templates at the site can be removed from the site, and then the second of the two or more primers that hybridize with two or more different templates at the site can be extended.

[0099] In addition, the two or more different primer extension products produced by the above steps can be present at the site simultaneously during the detection step. Alternatively, the first of the two or more different primer extension products can be removed from the site, and then the second of the two or more different primer extension products at the site can be detected.

[0100] In addition, during the blocking step, two or more primer extension products with different reversible blocking parts can exist simultaneously. The blocking step can be configured to selectively remove (or otherwise modify) one or only a subset of different reversible blocking parts, so that at least one other reversible blocking part is retained (or not modified) after processing. Or, for example, a combination of blocking treatment or a universal blocking treatment can be used to remove all different reversible blocking parts that exist simultaneously. As another alternative, the first of two or more different primer extension products can be removed from the site, and the second of two or more primer extension products can be blocked afterwards.

[0101] The present disclosure provides reaction mixtures (also referred to herein as reagent systems) comprising various combinations of components. In several cases, several combinations of reaction components and components are described in the context of the exemplary methods as described in the previous paragraphs. It should be understood that reaction mixtures and components thereof do not need to be limited to use in the methods illustrated herein. Other uses may also be considered. Therefore, components may be assembled in various useful combinations, such as creating test kits. Test kits may be used to describe the storage, transportation or commercial transactions of components herein. Test kits may optionally include instructions for implementing one or more methods described herein.

[0102] In a specific embodiment, the present disclosure provides a method for sequencing a nucleic acid template, which may include the following steps: (a) providing an array of sites, wherein each site comprises a first nucleic acid template and a second nucleic acid template, wherein the first nucleic acid template has a sequence different from the sequence of the second nucleic acid template, wherein a first primer is bound to the first nucleic acid template, and a reversible blocking portion is attached to the first primer, wherein a second primer is bound to the second nucleic acid template, and a reversible blocking portion is attached to the second primer, and wherein the reversible blocking portion attached to the first primer is different from the reversible blocking portion attached to the second primer; (b) selectively removing the reversible blocking portion attached to the first primer, while retaining the reversible blocking portion attached to the second primer; (c) extending the first primer by adding a first nucleotide analog attached to the reversible blocking portion; (d) selectively removing the reversible blocking portion attached to the second primer, while retaining the reversible blocking portion attached to the nucleotide analog added to the first primer; (e) extending the second primer by adding a second nucleotide analog attached to the reversible blocking portion, wherein the reversible blocking portion attached to the first nucleotide analog is different from the reversible blocking portion attached to the second nucleotide analog; and (f) detecting the nucleotide analog added to the first primer and the nucleotide analog added to the second primer at each of the sites, thereby determining the different sequences of the first template and the second template at each of the sites.

[0103] In some embodiments, the above method may further include the following steps: (g) selectively removing the reversible blocking portion attached to the first nucleotide analog added to the first primer, while retaining the reversible blocking portion attached to the second nucleotide analog added to the second primer; (h) after (g), extending the first primer by adding a third nucleotide analog attached to the reversible blocking portion; (i) selectively removing the reversible blocking portion attached to the second nucleotide analog added to the second primer, while retaining the reversible blocking portion attached to the first nucleotide analog added to the first primer; (j) after (i), extending the second primer by adding a fourth nucleotide analog attached to the reversible blocking portion, wherein the reversible blocking portion attached to the third nucleotide analog is different from the reversible blocking portion attached to the fourth nucleotide analog; and (k) at each of the sites, detecting the nucleotide analog added to the first primer in (h) and the nucleotide analog added to the second primer in (j), thereby determining different sequences of the first template and the second template at each of the sites. Optionally, steps (g) to (k) may be repeated.

[0104] It should be understood that the steps described in the above embodiments and other embodiments disclosed in the present invention do not need to be in the order of the examples. Taking the embodiments in the preceding paragraphs as an example, step (f) (which describes the detection activity) does not need to occur after step (e). On the contrary, before extending the second primer in step (d), the first primer extended in step (c) can be detected. Generally, the detection step can occur before or after removing one or more different reversible blocking parts.

[0105] The order of other steps may be varied to suit a particular application of the method. Figure 1 and Figure 2 The cycle diagram in is used to illustrate an example of two methods that can use similar steps but in different orders. Figure 1 In the cycle diagram of , the marker on the primer extension product is detected before both the deblocking and cleavage of the marker. The deblocking and marker cleavage of nucleotides in each group are shown to occur simultaneously, which may be convenient, for example, when both are susceptible to the same treatment or due to compatibility treatments. However, deblocking and marker cleavage may occur separately. Figure 2 In the example of a cycle diagram of , label cleavage and deblocking occur separately. In this example, different labels present in two different reactions (i.e., R1 and R2) are cleaved simultaneously. This is convenient, for example, when two types of labels can be cleaved using the same treatment or with compatible treatments. Figure 1 and Figure 2 Comparison of the cycle graphs in also illustrates other differences. For example, Figure 1 In the case of R1 and R2, the deblocking of the extension products of the two reactions (R1 and R2) occurs after the detection step of the previous cycle, whereas in Figure 2 In the method described herein, the R2 extension product is deblocked before the detection step of the previous cycle and the R1 extension product is deblocked after the detection step of the previous cycle. Various arrangements and orders of steps can be used in the methods described herein. Based on the teachings described herein and the known reaction characteristics of the reagents used, those skilled in the art will be able to easily determine the desired arrangement and order.

[0106] In addition, as described above, the steps of the methods described herein may be performed sequentially or simultaneously. For example, selective removal of different reversible blocking moieties (e.g., steps (b) and (d) in the preceding paragraph) may be performed simultaneously or sequentially. Similarly, extension of different primers (e.g., steps (c) and (e) in the preceding paragraph) that have been deblocked using respective different deblocking treatments may occur simultaneously or sequentially.

[0107] Universal priming sites are particularly useful for the multiple applications of the method set forth herein. Universal priming sites provide a sequence region common to two or more nucleic acid molecules, in which molecules also have different sequences in their template region. Universal priming sequences present in different members of a molecular set can allow the use of a single universal primer species complementary to the universal sequence to replicate, amplify or detect a plurality of different sequences. Examples of methods for attaching universal sequences to a set of target nucleic acids are found in U.S. Patent Application Publication No. 2007 / 0128624A1, which is incorporated herein by reference.

[0108] In embodiments of the present disclosure, the first primer can have a first universal primer sequence that is complementary to the first universal priming site of the first template at a site. The same universal priming site can be present for a plurality of different first templates at different sites of the array. Therefore, a single species of the first universal primer can be used to amplify or extend different first templates at a site. Continuing this example, the second primer can have a second universal primer sequence that is complementary to the second universal priming site of the second template at a site also located at the first template. The same second universal priming site can be present for a plurality of different second templates at different sites of the array. Therefore, a single species of the second universal primer can be used to amplify or extend different second templates at a site.

[0109] The orthogonal sequencing methods described herein can be used for paired end sequencing methods. Typically, paired end sequencing involves determining the sequences at both ends of a template sequence region, wherein the length of the template sequence region is known. Methods for fragmenting a target nucleic acid sample (e.g., a genomic DNA sample), attaching primers to accommodate paired end reads, and reading sequences from the ends of the fragments are known and can be performed as described, for example, in U.S. Patent Nos. 7,754,429, 8,017,335, or 8,192,930, each of which is incorporated herein by reference.

[0110] In the case of a synthetic sequencing embodiment, a nucleic acid fragment can be constructed to have two template sequences, and paired reads can be obtained from each of the two templates to obtain 4 reads from a single fragment. An exemplary construct for obtaining 4 reads from a single fragment and a method for preparing the construct are described in U.S. Patent Publication No. 2015 / 0031560A1, which is incorporated herein by reference.

[0111] The present disclosure also provides a nucleic acid array, which is included in a plurality of sites on a solid support, wherein each array comprises a first nucleic acid template and a second nucleic acid template, wherein the first nucleic acid template has a sequence different from the sequence of the second nucleic acid template, wherein a first primer is bound to the first nucleic acid template, a first reversible blocking portion is attached to the first primer, wherein a second primer is bound to the second nucleic acid template, a second reversible blocking portion is attached to the second primer, and wherein the first reversible blocking portion is different from the second reversible blocking portion. The nucleic acid array can also include one or more components described in the context of the method of the present disclosure. In some embodiments, it is intended that the product inherently derived from the method described herein is a component of a nucleic acid.

[0112] In specific embodiments, nucleic acid array will be included in the first polymerase combined with the first primer and the first nucleic acid template at the site. In addition, the second polymerase can be combined with the second primer and the second nucleic acid template at the site. In some cases, the first polymerase and the second polymerase are polymerases of the same type. However, in some embodiments, it is also useful that the first and second polymerases are different types (such as archaeal dna polymerase and RNA polymerase).

[0113] The nucleic acid array of the present disclosure can be present in a detection device such as a nucleic acid sequencing device. Exemplary detection devices are described herein and in the references incorporated herein by reference. In general, the detection device can include a nucleic acid array and a detector positioned to detect one or more sites in the array. Typically, the detector will have a spatial resolution that is too low to resolve a point at a distance equivalent to the interval between the first primer and the second primer at each of the sites. However, using orthogonal primers to seal and extend allows two primers to be resolved. The detector can be configured to observe any of the multiple signals illustrated herein. For example, in some embodiments, the detector is an optical detector. The array site can have an optical marker that can be detected by an optical detector. Different primers at each site can be extended to incorporate different optical markers, and an optical detector can be configured to optically distinguish different markers (such as due to light absorption wavelength, luminescent excitation wavelength, or luminescent emission wavelength difference). In some configurations, the pixel of the detector is configured to simultaneously obtain signals from both the first primer and the second primer.

[0114] Throughout this application, various publications, patents, and patent applications are cited. The disclosures of these publications are hereby incorporated into this application in their entirety by reference.

[0115] The term "comprising" is intended herein to be open ended and include not only the stated elements but also any additional elements.

[0116] A number of embodiments have been described. However, it should be understood that various modifications may be made. Therefore, other embodiments are within the scope of the following claims.

[0117] 1. A method for sequencing a nucleic acid template, comprising:

[0118] (a) providing an array of sites, wherein each site comprises a first nucleic acid template and a second nucleic acid template,

[0119] wherein the first nucleic acid template has a sequence different from the sequence of the second nucleic acid template,

[0120] wherein a first primer is bound to the first nucleic acid template, a reversible blocking moiety is attached to the first primer,

[0121] wherein a second primer is bound to the second nucleic acid template, a reversible blocking moiety is attached to the second primer, and

[0122] wherein the reversible blocking moiety attached to the first primer is different from the reversible blocking moiety attached to the second primer;

[0123] (b) selectively removing the reversible blocking portion attached to the first primer while retaining the reversible blocking portion attached to the second primer;

[0124] (c) extending the first primer by adding a first nucleotide analog attached to the reversible blocking moiety;

[0125] (d) selectively removing the reversible blocking portion attached to the second primer while retaining the reversible blocking portion attached to the nucleotide analog added to the first primer;

[0126] (e) extending the second primer by adding a second nucleotide analog attached to a reversible blocking moiety, wherein the reversible blocking moiety attached to the first nucleotide analog is different from the reversible blocking moiety attached to the second nucleotide analog; and

[0127] (f) detecting the nucleotide analog added to the first primer and the nucleotide analog added to the second primer at each of the sites, thereby determining the different sequences of the first template and the second template at each of the sites.

[0128] 2. The method of claim 1, further comprising:

[0129] (g) selectively removing the reversible blocking portion attached to the first nucleotide analog added to the first primer, while retaining the reversible blocking portion attached to the second nucleotide analog added to the second primer;

[0130] (h) following (g), extending the first primer by adding a third nucleotide analog attached to a reversible blocking moiety;

[0131] (i) selectively removing the reversible blocking portion attached to the second nucleotide analog added to the second primer, while retaining the reversible blocking portion attached to the first nucleotide analog added to the first primer;

[0132] (j) after (i), extending the second primer by adding a fourth nucleotide analog attached to a reversible blocking moiety, wherein the reversible blocking moiety attached to the third nucleotide analog is different from the reversible blocking moiety attached to the fourth nucleotide analog; and

[0133] (k) detecting the nucleotide analog added to the first primer in (h) and the nucleotide analog added to the second primer in (j) at each of the sites, thereby determining different sequences of the first template and the second template at each of the sites.

[0134] 3. The method of item 2, further comprising repeating steps (g) to (k).

[0135] 4. The method of item 1 or 2, wherein the detection uses a detector having a spatial resolution that is too low to resolve points at a distance equivalent to the spacing between the first primer and the second primer at each of the sites.

[0136] 5. The method of any one of items 1-4, wherein the detector is an optical detector.

[0137] 6. The method of any one of items 1-5, wherein the nucleotide analogue comprises an optical label.

[0138] 7. The method of claim 6, wherein the first nucleotide analogue is from a first group of nucleotide analogues, wherein the second nucleotide analogue is from a second group of nucleotide analogues, and wherein the optical marker of the first group of nucleotide analogues is different from the optical marker of the second group of nucleotide analogues.

[0139] 8. The method of any one of items 1-7, wherein the first nucleotide analogue is from a first group of nucleotide analogues, wherein the second nucleotide analogue is from a second group of nucleotide analogues, and wherein a subset of the nucleotide analogues in the first group of nucleotide analogues comprises an optical label.

[0140] 9. The method of claim 8, wherein a subset of the nucleotide analogs in the second set of nucleotide analogs comprises an optical marker.

[0141] 10. The method of claim 9, wherein a subset of nucleotide analogs in the first group of nucleotide analogs comprises an optical label, which is different from the optical label of the subset of nucleotides in the second group of nucleotide analogs.

[0142] 11. The method of any one of items 1-7, wherein the first nucleotide analogue is from a first group of nucleotide analogues, wherein the second nucleotide analogue is from a second group of nucleotide analogues, wherein the first group of nucleotide analogues contains only one type of optical marker detected in step (f), and the second group of nucleotide analogues contains only one type of optical marker detected in step (f).

[0143] 12. The method of claim 11, wherein said one type of optical label is attached to substantially all of the nucleotide analogs of the first type in said first group,

[0144] attaching said one type of optical label to a subset of said nucleotide analogs of a second type in said first group,

[0145] attaching substantially all of the nucleotide analogs of the third species in the first group to a ligand, and

[0146] Substantially all of the nucleotide analogues of the fourth species in the first group are not attached to the one species of optical label or the ligand.

[0147] 13. The method of any one of items 1-7, wherein the first nucleotide analogue is from a first group of nucleotide analogues, wherein the second nucleotide analogue is from a second group of nucleotide analogues, wherein the first group of nucleotide analogues only contains two types of optical markers detected in step (f), and the second group of nucleotide analogues only contains two types of optical markers detected in step (f).

[0148] 14. The method of claim 13, wherein only one of the two types of optical labels is attached to substantially all nucleotide analogs of the first type in the first group,

[0149] attaching only the second of the two types of optical labels to substantially all nucleotide analogs of the second type in the first group,

[0150] attaching one of the two types of optical labels and a second of the two types of optical labels to a third type of nucleotide analog in the first group, and

[0151] Substantially all of the nucleotide analogs of the fourth species in the first group are not attached to one of the two types of optical labels or to the second of the two types of optical labels.

[0152] 15. The method of any one of items 1-14, wherein the pixels of the detector simultaneously acquire signals from both the first primer and the second primer.

[0153] 16. The method of any one of items 1 to 15, wherein the first nucleic acid template comprises at least one base portion that is the same type as the base portion in the second nucleic acid template.

[0154] 17. The method of any one of items 1 to 16, wherein steps (b) and (d) are performed simultaneously.

[0155] 18. The method of any one of items 1-16, wherein steps (b) and (d) are performed sequentially.

[0156] 19. The method of any one of items 1 to 18, wherein steps (c) and (e) are performed simultaneously.

[0157] 20. The method of any one of items 1-18, wherein steps (c) and (e) are performed sequentially.

[0158] 21. The method of any one of items 1 to 20, wherein a single nucleic acid molecule contains the first nucleic acid template and the second nucleic acid template.

[0159] 22. The method of any one of items 1-20, wherein the first nucleic acid template and the second nucleic acid template are on different nucleic acid molecules.

[0160] 23. The method of any one of items 1 to 22, wherein the site has a diameter of 100 μm 2 or less area.

[0161] 24. The method of any one of items 1-23, wherein the sites comprise multiple amplicons of the first nucleic acid template and multiple amplicons of the second nucleic acid template.

[0162] 25. The method of claim 24, wherein the plurality of amplicons comprise nucleic acid clusters.

[0163] 26. The method of any one of items 1-25, wherein the plurality of sites have a pitch of 10 μm or less.

[0164] 27. The method of any one of items 1 to 26, wherein the plurality of sites comprises at least 1 x 10 6 A site.

[0165] 28. The method of any one of items 1-27, wherein each of said sites comprises a nucleic acid sequence that is unique compared to said nucleic acid sequences at other sites in said plurality.

[0166] 29. The method of any one of items 1-28, wherein the first primer comprises a first universal primer sequence and the first nucleic acid template at each of the plurality of sites comprises a first universal primer binding sequence complementary to the first universal primer sequence.

[0167] 30. The method of claim 29, wherein the second primer comprises a second universal primer sequence and the second nucleic acid template at each of the plurality of sites comprises a second universal primer binding sequence complementary to the second universal primer sequence, wherein the first universal primer binding sequence is different from the second universal primer binding sequence.

[0168] 31. The method of any one of items 1-30, wherein steps (c) and (e) are performed by the same polymerase or the same type of polymerase.

[0169] 32. The method of any one of items 1 to 31, wherein azidomethyl and tert-butoxy-ethoxy are used as reversible blocking moieties.

[0170] 33. The method of claim 32, wherein the azidomethyl group is selectively removed by treatment with phosphine and the tert-butoxy-ethoxy group is selectively removed by treatment with an acid.

[0171] 34. The method of any one of items 1-31, wherein the selective removal of the reversibly blocking portion comprises chemical treatment, thermal treatment, irradiation or electrical treatment.

[0172] 35. The method of any one of items 1-34, wherein the reversible blocking moiety attached to the first primer is of the same kind as the reversible blocking moiety attached to the first nucleotide analogue.

[0173] 36. The method of claim 35, wherein the reversible blocking portion attached to the second primer is of the same type as the reversible blocking portion attached to the second nucleotide analog.

[0174] 37. A method for sequencing a nucleic acid template, comprising:

[0175] (a) providing an array of sites, wherein each site comprises a mixture of at least two different nucleic acid templates;

[0176] (b) extending primers hybridized to the different nucleic acid templates at each of the sites with different nucleotide analogs comprising different reversible blocking moieties, respectively, thereby generating different primer extension products at each site;

[0177] (c) detecting the different primer extension products to distinguish the different nucleotide analogs at each site;

[0178] (d) removing the different reversible blocking moieties from the primer extension products at each of the sites using a first treatment selective for a first one of the different reversible blocking moieties and a second treatment selective for a second one of the different reversible blocking moieties; and

[0179] (e) repeating (b) to (d) to determine the sequence of the different nucleotide analogs added to each of said different extension products at each of said sites.

[0180] 38. The method of claim 37, wherein the primers hybridized to the different nucleic acid templates are present simultaneously during the extension of (b).

[0181] 39. The method of any one of items 37 to 38, wherein the different primer extension products are present simultaneously during the detection period of (c).

[0182] 40. The method of any one of items 37 to 39, wherein the first of the different reversible blocking portions and the second of the different reversible blocking portions are simultaneously present on the primer extension product during the removal of (d).

[0183] 41. The method of claim 37, wherein a first primer that hybridizes to a first of the different nucleic acid templates is removed from the site array, and then a second primer that hybridizes to a second of the different nucleic acid templates is extended.

[0184] 42. The method of claim 37, wherein the first of the different primer extension products is removed from the site array and then the second of the different primer extension products is detected.

[0185] 43. The method of claim 37, wherein the first of the different primer extension products is removed from the site array prior to the second processing.

[0186] 44. The method of any one of items 37-43, wherein (c) is performed after (d).

[0187] 45. The method of any one of items 37-43, wherein the detection uses a detector having a spatial resolution that is too low to resolve points at distances equivalent to the spacing between the different primer extension products at each of the sites.

[0188] 46. ​​The method of any one of items 37-45, wherein the detector is an optical detector.

[0189] 47. The method of any one of items 37-46, wherein the nucleotide analog comprises an optical label.

[0190] 48. The method of any one of items 37-47, wherein a first group of nucleotide analogs is added to a first of the different primer extension products, and a second group of nucleotide analogs is added to a second of the different primer extension products, wherein the first group of nucleotide analogs comprises an optical label that is different from the optical label of the second group of nucleotide analogs.

[0191] 49. The method of any one of items 37-48, wherein pixels of the detector simultaneously acquire signals from both the first primer extension product and the second primer extension product.

[0192] 50. The method of any one of items 37-49, wherein the first nucleic acid template comprises at least one base portion that is the same type as the base portion in the second nucleic acid template.

[0193] 51. The method of any one of items 37-50, wherein a single nucleic acid molecule contains at least two different nucleic acid templates.

[0194] 52. The method of any one of items 37-50, wherein the first nucleic acid template and the second nucleic acid template of the at least two different nucleic acid templates are on different nucleic acid molecules.

[0195] 53. The method of any one of items 37-52, wherein the site has a diameter of 100 μm 2 or less area.

[0196] 54. The method of items 37-53, wherein the site comprises multiple amplicons for each of the at least two different nucleic acid templates.

[0197] 55. The method of claim 54, wherein the plurality of amplicons comprise nucleic acid clusters.

[0198] 56. The method of any one of items 37-55, wherein the plurality of sites have a spacing of 10 μm or less.

[0199] 57. The method of any one of items 37-56, wherein the plurality of sites comprises at least 1 x 10 6 A site.

[0200] 58. The method of any one of items 37-57, wherein each of said sites comprises a nucleic acid sequence that is unique compared to said nucleic acid sequences at other sites in said plurality.

[0201] 59. The method of any one of items 37-58, wherein the same polymerase or the same type of polymerase performs extension of the primer hybridized to the different nucleic acid template at each of the sites.

[0202] 60. The method of any one of items 37-59, wherein the selective treatment comprises using phosphine treatment to selectively remove the reversible blocking portion comprising an azidomethyl group, and using acid treatment to selectively remove the reversible blocking portion comprising a tert-butoxy-ethoxy group.

[0203] 61. The method of any one of items 37-59, wherein the selective removal of the reversibly blocking portion comprises chemical treatment, thermal treatment, irradiation or electrical treatment.

[0204] 62. A nucleic acid array comprising:

[0205] a plurality of sites on a solid support, wherein each site comprises a first nucleic acid template and a second nucleic acid template,

[0206] wherein the first nucleic acid template has a sequence that is different from the sequence of the second nucleic acid template,

[0207] wherein a first primer binds to the first nucleic acid template, a first reversible blocking moiety is attached to the first primer,

[0208] wherein a second primer binds to the second nucleic acid template, a second reversible blocking moiety is attached to the second primer, and

[0209] Wherein the first reversible sealing portion is different from the second reversible sealing portion.

[0210] 63. The nucleic acid array of item 62, wherein each site on the solid support occupies 100 μm 2 or smaller area.

[0211] 64. The nucleic acid array of item 62 or 63, wherein the plurality of sites have a spacing of 10 μm or less.

[0212] 65. The nucleic acid array of any one of items 62-64, wherein the plurality of sites comprises at least 1 x 10 6 A site.

[0213] 66. The nucleic acid array of claim 65, wherein each of the sites comprises a nucleic acid sequence that is unique compared to the nucleic acid sequences at other sites in the plurality.

[0214] 67. The nucleic acid array of any one of items 62-65, wherein the first reversible blocking portion is attached to the 3' nucleotide of the first primer.

[0215] 68. The nucleic acid array of claim 67, wherein the 3' nucleotide of the first primer is attached to a first optical marker.

[0216] 69. The nucleic acid array of any one of items 67-68, wherein the second reversible blocking portion is attached to the 3' nucleotide of the second primer.

[0217] 70. The nucleic acid array of item 69, wherein the 3' nucleotide of the second primer is attached to a second optical marker, wherein the second optical marker is optically distinguishable from the first optical marker.

[0218] 71. The nucleic acid array of any one of items 67-70, wherein the first and second optical markers comprise fluorophores.

[0219] 72. The nucleic acid array of any one of items 62-71, wherein the first nucleic acid template and the second nucleic acid template comprise DNA.

[0220] 73. The nucleic acid array of any one of items 62-72, wherein a single nucleic acid molecule contains the first nucleic acid template and the second nucleic acid template.

[0221] 74. The nucleic acid array of any one of items 62-73, wherein the first nucleic acid template and the second nucleic acid template are located on different nucleic acid molecules.

[0222] 75. The nucleic acid array of any one of items 62-74, wherein the sites comprise multiple amplicons of the first nucleic acid template and multiple amplicons of the second nucleic acid template.

[0223] 76. The nucleic acid array of claim 75, wherein the plurality of amplicons comprise nucleic acid clusters.

[0224] 77. A nucleic acid array of any one of items 62-76, wherein the first primer comprises a first universal primer sequence and the first nucleic acid template at each of the multiple sites comprises a first universal primer binding sequence complementary to the first universal primer sequence.

[0225] 78. A nucleic acid array of item 77, wherein the second primer comprises a second universal primer sequence, and the second nucleic acid template at each of the multiple sites comprises a second universal primer binding sequence complementary to the second universal primer sequence, wherein the first universal primer binding sequence is different from the second universal primer binding sequence.

[0226] 79. The nucleic acid array of any one of items 62-68, wherein a first polymerase is combined with the first primer and the first nucleic acid template.

[0227] 80. The nucleic acid array of item 79, wherein a second polymerase is combined with the second primer and the second nucleic acid template, and wherein the first polymerase and the second polymerase are the same type of polymerase.

[0228] 81. A nucleic acid array of any one of items 62-80, wherein the first reversible blocking portion comprises an azidomethyl group and wherein the second reversible blocking portion comprises a tert-butoxy-ethoxy group.

[0229] 82. A detection device comprising

[0230] The nucleic acid array of any one of items 62-81, and

[0231] A detector positioned to detect multiple sites.

[0232] 83. A detection device according to claim 82, wherein said detector has a spatial resolution which is too low to resolve points at a distance equivalent to the spacing between said first primer and said second primer at each of said sites.

[0233] 84. The detection device of item 82 or 83, wherein the detector is an optical detector.

[0234] 85. The detector of any one of items 82-83, wherein pixels of the detector are configured to simultaneously acquire signals from both the first primer and the second primer.

Claims

1. A method for sequencing a nucleic acid template, comprising the following steps: (a) providing an array of sites, wherein each site comprises a first nucleic acid template and a second nucleic acid template, wherein the first nucleic acid template has a sequence different from the sequence of the second nucleic acid template, wherein a first primer is bound to the first nucleic acid template, and wherein a second primer is bound to the second nucleic acid template, and a reversible blocking moiety is attached to the second primer; (b) extending the first primer by adding a first nucleotide analog attached to a reversible blocking moiety, wherein the reversible blocking moiety attached to the first nucleotide analog is different from the reversible blocking moiety attached to the second primer; (c) selectively removing the reversible blocking moiety attached to the second primer while retaining the reversible blocking moiety attached to the nucleotide analog added to the first primer; (d) extending the second primer by adding a second nucleotide analog attached to a reversible blocking moiety, wherein the reversible blocking moiety attached to the first nucleotide analog is different from the reversible blocking moiety attached to the second nucleotide analog; as well as (e) detecting the nucleotide analog added to the first primer and the nucleotide analog added to the second primer at each site, thereby determining different sequences of the first template and the second template at each site in an orthogonal manner by using selective deblocking of nucleotides.

2. The method of claim 1, further comprising the steps of: (f) selectively removing the reversible blocking moiety attached to the first nucleotide analog added to the first primer, while retaining the reversible blocking moiety attached to the second nucleotide analog added to the second primer; (g) after (f), extending the first primer by adding a third nucleotide analog attached to a reversible blocking moiety; (h) selectively removing the reversible blocking portion attached to the second nucleotide analog added to the second primer, while retaining the reversible blocking portion attached to the third nucleotide analog added to the first primer; (i) after (h), extending the second primer by adding a fourth nucleotide analog attached to a reversible blocking moiety, wherein the reversible blocking moiety attached to the third nucleotide analog is different from the reversible blocking moiety attached to the fourth nucleotide analog; as well as (j) detecting the nucleotide analog added to the first primer in (g) and the nucleotide analog added to the second primer in (i) at each site, thereby determining the different sequences of the first template and the second template at each site.

3. The method of claim 2, wherein steps (f) to (j) are repeated.

4. The method of any one of claims 1-3, wherein the detection uses a detector having a spatial resolution that is too low to resolve points at a distance equivalent to the spacing between the first primer and the second primer at each of the sites.

5. The method of any one of claims 1 to 3, wherein the detector is an optical detector.

6. The method of any one of claims 1 to 3, wherein the nucleotide analogs comprise an optical label.

7. The method of claim 6, wherein the first nucleotide analog is from a first group of nucleotide analogs, wherein the second nucleotide analog is from a second group of nucleotide analogs, and wherein the optical label of the first group of nucleotide analogs is different from the optical label of the second group of nucleotide analogs.

8. The method of any one of claims 1-3, wherein the first nucleotide analog is from a first group of nucleotide analogs, wherein the second nucleotide analog is from a second group of nucleotide analogs, and wherein a subset of the nucleotide analogs in the first group of nucleotide analogs comprises an optical label.

9. The method of claim 8, wherein a subset of said nucleotide analogs in said second set of nucleotide analogs comprises an optical label.

10. The method of claim 9, wherein a subset of nucleotide analogs in said first set of nucleotide analogs comprises an optical label that is different from said optical label of said subset of nucleotides in said second set of nucleotide analogs.

11. The method of any one of claims 1-3, wherein the first nucleotide analog is from a first group of nucleotide analogs, wherein the second nucleotide analog is from a second group of nucleotide analogs, wherein the first group of nucleotide analogs comprises only one type of optical label detected in step (e), and the second group of nucleotide analogs comprises only one type of optical label detected in step (e).

12. The method of claim 11, wherein said one type of optical label is attached to all nucleotide analogs of the first species in said first group, attaching said one type of optical label to a subset of said nucleotide analogs of a second type in said first group, attaching all nucleotide analogs of the third species in the first group to a ligand, and Not all nucleotide analogues of the fourth species in the first group are attached to the one type of optical label or the ligand.

13. The method of any one of claims 1-3, wherein the first nucleotide analog is from a first group of nucleotide analogs, wherein the second nucleotide analog is from a second group of nucleotide analogs, wherein the first group of nucleotide analogs comprises only two types of optical labels detected in step (e), and the second group of nucleotide analogs comprises only two types of optical labels detected in step (e).

14. The method of claim 13, wherein only one of the two types of optical labels is attached to all nucleotide analogs of the first species in the first group, attaching only the second of the two types of optical labels to all nucleotide analogs of the second type in the first group, attaching one of the two types of optical labels and a second of the two types of optical labels to a third type of nucleotide analog in the first group, and All nucleotide analogs of the fourth species in the first group are not attached to one of the two types of optical labels or to the second of the two types of optical labels.

15. The method of any one of claims 1-3, wherein pixels of the detector simultaneously acquire signals from both the first primer and the second primer.

16. The method of any one of claims 1-3, wherein the first nucleic acid template comprises at least one base portion that is the same species as a base portion in the second nucleic acid template.

17. The method of any one of claims 1 to 3, wherein steps (b) and (d) are performed simultaneously.

18. The method of any one of claims 1 to 3, wherein steps (b) and (d) are performed sequentially.

19. The method of any one of claims 1-3, wherein a single nucleic acid molecule contains the first nucleic acid template and the second nucleic acid template.

20. The method of any one of claims 1-3, wherein the first nucleic acid template and the second nucleic acid template are on different nucleic acid molecules.

21. The method of any one of claims 1 to 3, wherein the site has a diameter of 100 μm 2 or less area.

22. The method of any one of claims 1-3, wherein the sites comprise a plurality of amplicons of the first nucleic acid template and a plurality of amplicons of the second nucleic acid template.

23. The method of claim 22, wherein the plurality of amplicons comprises nucleic acid clusters.

24. The method of any one of claims 1-3, wherein the plurality of sites have a pitch of 10 μm or less.

25. The method of any one of claims 1 to 3, wherein the plurality of sites comprises at least 1 x 10 6 A site.

26. The method of any one of claims 1-3, wherein each of said sites comprises a nucleic acid sequence that is unique compared to said nucleic acid sequences at other sites in said plurality.

27. The method of any one of claims 1-3, wherein the first primer comprises a first universal primer sequence and the first nucleic acid template at each site in the plurality of sites comprises a first universal primer binding sequence that is complementary to the first universal primer sequence.

28. The method of claim 27, wherein the second primer comprises a second universal primer sequence and the second nucleic acid template at each site in the plurality of sites comprises a second universal primer binding sequence that is complementary to the second universal primer sequence, wherein the first universal primer binding sequence is different from the second universal primer binding sequence.

29. The method of any one of claims 1 to 3, wherein steps (b) and (d) are performed by the same polymerase or the same class of polymerases.

30. The method of any one of claims 1 to 3, wherein azidomethyl and tert-butoxy-ethoxy are used as reversible blocking moieties.

31. The process of claim 30, wherein the azidomethyl group is selectively removed by treatment with phosphine and the tert-butoxy-ethoxy group is selectively removed by treatment with an acid.

32. The method of any one of claims 1-3, wherein said selective removal of said reversibly blocking moiety comprises chemical treatment, thermal treatment, irradiation, or electrical treatment.

33. The method of any one of claims 1-3, wherein the reversible blocking moiety attached to the second primer is of the same species as the reversible blocking moiety attached to the second nucleotide analog.

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