On surface nucleic acid target detection and next generation sequencing (NGS) library preparation

Abstract

A method and microarray for on surface nucleic acid target detection and next generation sequencing (NGS) library preparation.

Description

PATENTON SURFACE NUCLEIC ACID TARGET DETECTION AND NEXT GENERATION SEQUENCING (NGS) LIBRARY PREPARATIONRELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application No. 63 / 680,879, filed August 8, 2024, the subject matter of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Next generation sequencing (NGS) technology using DNA detecting microarrays relies on the highly parallel sequencing of single target polynucleotides immobilized on a surface, or the sequencing of clonal populations of target nucleotides, that were produced from the single target polynucleotides, e.g., by bridge amplification.Sequencing clonal populations of target polynucleotides yields much higher signal-to-noise ratios (SNRs) than sequencing single target polynucleotides, improves the sensitivity and accuracy of sequencing reactions, and allows for the use of low-cost optics in sequencing instrumentation.

[0003] In traditional methods to fabricate DNA detecting microarrays, clusters of DNA oligos are chemically synthesized on a microfluidic flowchip surface, or on the surface of beads and then loaded onto the flowchip.

[0004] In targeted next generation sequencing (NGS), library preparation process consists of multiple steps including fragmentation of genomic DNA, binding of targeted DNA fragments onto beads modified with DNA probes, separating the beads with targets and other DNA fragments, elution of the target DNA from the beads, and adapter conjugation.SUMMARY

[0005] We disclose methods of fabricating microarrays of nucleic acid molecules (DNA molecules) on microfluidic devices at a significantly lower cost than traditional methods. Compared to traditional bead deposition methods and in situ chemical synthesis methods, we use nucleic acid (e.g., DNA) strands with desired sequences to hybridize with primers tethered to the surface of a flowchip. The primers are extended so that they are complementary to the nucleic acid strands and form nucleic acid capture probes that are tethered to the surface. Strands of the tethered nucleic acid capture probes are amplified onthe surface of the flowchip using, for example, bridge PCR, to form a microarray(s) or an array (s) of amplicons or clusters of capture probes. The microarray can be used for capturing and detecting target nucleic acid strands, such as DNA strands, in a sample.

[0006] In some embodiments, the disclosed method does not pre-identify the nucleic acid capture probes on the surface prior to capture of the target nucleic acids. After the target nucleic acids are hybridized to or captured by the clusters of capture probes on the surface, barcode-tagged DNA strands with specific nucleic sequences complementary to the targets are sent in the flowchip to bind and indicate which target nucleic acids are present.

[0007] We also disclose a method of targeted NGS library preparation on the surface of microfluidic devices, of which the microarray described herein, as well as other devices of the same type, can be used. Our method of NGS library preparation with our microarray, and the multiple steps can be realized automatically on the microfluidic system.

[0008] Some embodiments therefore relate to a method of fabricating a nucleic acid detecting microarray. The method can include providing a plurality of primer pairs on a surface of a solid support of the microarray. A plurality of single stranded nucleic acid probe templates can be hybridized to at least a portion of the plurality of primers on the surface. Nucleic acid sequences of the primers hybridized to the single stranded capture nucleic acid probe templates can be extended to form a plurality of complementary capture probes hybridized to the plurality of single stranded capture nucleic acid probe templates. The probe templates can then be removed from the surface of the microarray and the capture probes can be amplified to form probe clusters on the surface of the microarray.

[0009] In some embodiments, the plurality of single stranded nucleic acid probe templates can include a first end with a first binding domain to one of the surface primers of the pair, a second opposite end with a second binding domain to the other of the surface primers of the pair, and a capture domain between the first end and second end that can bind to target nucleic acid of interest.

[0010] In some embodiments, the probe clusters are spatially and / or randomly dispersed on the surface of the microarray.

[0011] In some embodiments, the microarray can include a flowchip.

[0012] In some embodiments, the nucleic acid sequences are extended by adding nucleotide triphosphates (NTPs) and polymerase to the surface of the microarray.

[0013] In some embodiments, the probe templates are removed by denaturing.

[0014] In some embodiments, the capture probes are amplified by bridge PCR.

[0015] In some embodiments, the amplified capture probes are linearized.

[0016] In some embodiments, different probe clusters can bind to different target nucleic acids.

[0017] In some embodiments, the probe clusters can bind to target DNA.

[0018] In some embodiments, the method further includes characterizing and / or identifying the probe clusters to determine target nucleic acids to which the probes of respective clusters bind.

[0019] In some embodiments, the probe clusters are characterized and / or identified by hybridizing complementary nucleic strands with fluorescent labels to the respective probes and detecting the fluorescent properties of each probe cluster to determine the nucleic sequence of the capture probes of the respective cluster.

[0020] In some embodiments, the detected fluorescent properties include at least one of fluorescent intensity or wavelength.

[0021] In some embodiments, the hybridized complementary nucleic strands with fluorescent labels are removed after determining the capture probes of the respective cluster.

[0022] In some embodiments, target nucleic acids are detected by adding target nucleic acids to the microarray surface such that the target nucleic acids are captured by a respective capture probe having a complementary sequence. The respective capture probes hybridized to target nucleic acids can be extended with a labeled NTP and the label of labeled NTP hybridized during extension can be detected to detect the presence of the target nucleic acid.

[0023] In other embodiments, a nucleic acid detecting microarray formed by the methods described can be used to detect target nucleic acids by adding target nucleic acids to the microarray surface such that the target nucleic acids are captured by a respective capture probe having a complementary sequence. Complementary nucleic strands with fluorescent barcodes that hybridize to the captured target nucleic acids can be added to the microarray surface, and the fluorescent properties of each probe cluster can be detected to determine the identity of captured target nucleic acids.

[0024] Still other embodiments described herein relate to a method of forming a next generation sequencing (NGS) library using a nucleic acid detecting microarray, such as a nucleic acid detecting microarray formed by the methods described herein. The method can include providing on a surface of a microarray a plurality of nucleic strands that each includea surface primer domain (Surface primer 1), a sample barcode (or index) domain, and a probe domain complementary to NGS target nucleic acids (Probe 1). The nucleic acid strands can be amplified to increase the binding affinity of NGS target nucleic acids to the probe. NGS target nucleic acid strands in a sample can be captured with the probes. The probes with the captured NGS target can be extended. The captured NGS nucleic acid strands can be removed from the microarray. Second surface primer (Surface primer 2) strands can be then hybridized to the extended end of the nucleic strands provided on the surface, wherein the Surface primer 2 strands have a probe domain on the 3 ’ end that hybridizes to the end of the targeted region on the strands on the surface (Probe 2) and an optional index domain. The Surface primer 2 strands can be extended to form strands containing domains of, from 5 ’ end to 3’ end, Surface primer2, Index 2, Probe 2, target, Index 1, Surface primer 1. The formed strands can be denatured and collected to form a targeted library for NGS.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figs. l(A-B) illustrate the process of the fabrication of DNA microarrays by seeding capture probes and amplifying the capture probes on a support surface. A) The scheme of the steps. The flowchip surface is tethered with single stranded DNA primers. These are universal primer pairs. The probes with sequences that can capture target DNA of interest are loaded on the flowchip. These probes are designed to have surface primer binding domains on the two ends, so they would be able to hybridize to the surface primers. The probes are then copied by extending the binding surface primer, and the original probe strands are removed by denaturing. The copies of the probes are then amplified with bridges PCR to form arrays of probe clusters. B) The top view of the microarray after the bridge PCR.

[0026] Fig. 2 illustrates the DNA microarray fabricated with the method of Fig. 1 can be characterized by hybridizing strands with fluorescent barcodes. Once the identity of each cluster of probes is determined, the DNA microarray can be used to detect DNA targets. The probes with targets captured and hybridized can be extended with a fluorescent dye labeled nucleotide to indicate which targets were present in the sample.

[0027] Fig. 3 illustrates a method to detect the presence and species of target nucleic acids simultaneously on the DNA microarray of Fig. 1. The sample containing DNA targets to be detected is sent onto the flowchip. The probes on the flowchip capture their designatedtarget nucleic acids of interest. Then, a solution containing DNA strands with fluorescent labels is sent to the flowchip. The sequences of the barcoded DNA strands are designed so that each sequence would specifically hybridize with one DNA target of interest, and each possible target can be detected by one of the fluorescent barcodes. By detecting the presence of the barcodes, the presence of the respective DNA target can be determined.

[0028] Fig. 4 illustrates automated targeted NGS library preparation with DNA microarray.DETAILED DESCRIPTION

[0029] Terms used herein will be understood to take on their ordinary meaning in the relevant art unless specified otherwise. Several terms used herein and their meanings are set forth below.

[0030] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a biomarker” includes a mixture of two or more biomarkers, and the like.

[0031] The term “about,” particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.

[0032] The terms “includes,” “including,” “includes,” “including,” “contains,” “containing,” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition of matter that includes, includes, or contains an element or list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, product- by-process, or composition of matter.

[0033] The term “amplicon,” when used in reference to a nucleic acid, means the product of copying the nucleic acid, wherein the product has a nucleotide sequence that is the same as or complementary to at least a portion of the nucleotide sequence of the nucleic acid. An amplicon can be produced by any of a variety of amplification methods that use the nucleic acid, or an amplicon thereof, as a template including, for example, polymerase extension, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), ligation extension, or ligation chain reaction. An amplicon can be a nucleic acid molecule having a single copy of a particular nucleotidesequence (e.g., a PCR product) or multiple copies of the nucleotide sequence. A first amplicon of a target nucleic acid is typically a complementary copy. Subsequent amplicons are copies that are created, after generation of the first amplicon, from the target nucleic acid or from the first amplicon. A subsequent amplicon can have a sequence that is substantially complementary to the target nucleic acid or substantially identical to the target nucleic acid.

[0034] The term “array” refers to a population of features or sites that can be differentiated from each other according to relative location. Different molecules that are at different sites of an array can be differentiated from each other according to the locations of the sites in the array. An individual site of an array can include one or more molecules of a particular type. For example, a site can include a single target nucleic acid molecule having a particular sequence or a site can include several nucleic acid molecules having the same sequence (and / or complementary sequence, thereof). The sites of an array can be different features located on the same substrate. Exemplary features include without limitation, wells in a substrate, beads (or other particles) in or on a substrate, projections from a substrate, ridges on a substrate or channels in a substrate. The sites of an array can be separate substrates each bearing a different molecule. Different molecules attached to separate substrates can be identified according to the locations of the substrates on a surface to which the substrates are associated or according to the locations of the substrates in a liquid or gel.

[0035] The term “attached” refers to the state of two things being joined, fastened, adhered, connected or bound to each other. For example, an analyte, such as a nucleic acid, can be attached to a material, such as a solid support, by a covalent or non-covalent bond. A covalent bond is characterized by the sharing of pairs of electrons between atoms. A non- covalent bond is a chemical bond that does not involve the sharing of pairs of electrons and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions and hydrophobic interactions.

[0036] The term “index”, “index sequence”, or “barcode sequence” are used interchangeably and refer to a series of nucleotides in a nucleic acid that can be used to identify the nucleic acid, a characteristic of the nucleic acid, or a manipulation that has been carried out on the nucleic acid. The index or barcode sequence can be a naturally occurring sequence or a sequence that does not occur naturally in the organism from which the barcoded nucleic acid was obtained. An index or barcode sequence can be unique to a single nucleic acid species in a population or a barcode sequence can be shared by several differentnucleic acid species in a population. For example, each nucleic acid probe in a population can include different index or barcode sequences from all other nucleic acid probes in the population. Alternatively, each nucleic acid probe in a population can include different index or barcode sequences from some or most other nucleic acid probes in a population. For example, each probe in a population can have an index or barcode that is present for several different probes in the population even though the probes with the common barcode differ from each other at other sequence regions along their length. In particular embodiments, one or more barcode sequences that are used with a biological sample are not present in the genome, transcriptome or other nucleic acids of the biological sample. For example, index or barcode sequences can have less than 80%, 70%, 60%, 50% or 40% sequence identity to the nucleic acid sequences in a particular biological sample.

[0037] The term “biological sample” refers one or more cell, tissue, organism or portion thereof. A biological sample can be obtained from any of a variety of organisms. Exemplary organisms include, but are not limited to, a mammal such as a rodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate (z.e., human or non-human primate); a plant; an algae; a nematode; an insect, such as mosquito, fruit fly, honey bee or spider; a fish such as zebrafish; a reptile; an amphibian such as a frog; a fungi, or yeast. Target nucleic acids can also be derived from a prokaryote such as a bacterium; an archaea; a virus, such as Hepatitis C virus or human immunodeficiency virus; or a viroid. Samples can be derived from a homogeneous culture or population of the above organisms or alternatively from a collection of several different organisms, for example, in a community or ecosystem.

[0038] The term “clonal population” refers to a population of nucleic acids that is homogeneous with respect to a particular nucleotide sequence. The homogenous sequence can be at least 10 nucleotides long, or longer, for example, at least 50, 100, 250, 500 or 1000 nucleotides long. A clonal population can be derived from a single target nucleic acid or template nucleic acid. Essentially all of the nucleic acids in a clonal population have the same nucleotide sequence. It will be understood that a small number of mutations (e.g., due to amplification artifacts) can occur in a clonal population without departing from clonality.

[0039] The term “cluster,” when used in reference to nucleic acids, refers to a population of the nucleic acids that is attached to a solid support to form a feature or site.The nucleic acids are generally members of a single species, thereby forming a monoclonalcluster. A “monoclonal population” of nucleic acids is a population that is homogeneous with respect to a particular nucleotide sequence. Clusters need not be monoclonal. Rather, for some applications, a cluster can be predominantly populated with amplicons from a first nucleic acid and can also have a low level of contaminating amplicons from a second nucleic acid. For example, when an array of clusters is to be used in a detection application, an acceptable level of contamination would be a level that does not impact signal to noise or resolution of the detection technique in an unacceptable way. Accordingly, apparent clonality will generally be relevant to a particular use or application of an array made by the methods set forth herein. Exemplary levels of contamination that can be acceptable at an individual cluster include, but are not limited to, at most 0.1%, 0.5%, 1%, 5%, 10%, 5 25%, or 35% contaminating amplicons. The nucleic acids in a cluster are generally covalently attached to a solid support, for example, via their 5' ends, but in some cases other attachment means are possible. The nucleic acids in a cluster can be single stranded or double stranded. In some but not all embodiments, clusters are made by a solid-phase amplification method known as bridge amplification. Exemplary configurations for clusters and methods for their production are set forth, for example, in U.S. Pat. No. 5,641,658; U.S. Patent Publ. No. 2002 / 0055100; U.S. Pat. No. 7,115,400; U.S. Patent Publ. No. 2004 / 0096853; U.S. Patent Publ. No. 2004 / 0002090; U.S. Patent Publ. No. 2007 / 0128624; and U.S. Patent Publ. No. 2008 / 0009420, each of which is incorporated herein by reference.

[0040] The term “different”, when used in reference to nucleic acids, means that the nucleic acids have nucleotide sequences that are not the same as each other. Two or more nucleic acids can have nucleotide sequences that are different along their entire length. Alternatively, two or more nucleic acids can have nucleotide sequences that are different along a substantial portion of their length. For example, two or more nucleic acids can have target nucleotide sequence portions that are different for the two or more molecules while also having a universal sequence portion that is the same on the two or more molecules. Two beads can be different from each other by virtue of being attached to different nucleic acids.

[0041] The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.

[0042] The term “extend,” when used in reference to a nucleic acid, is intended to mean addition of at least one nucleotide or oligonucleotide to the nucleic acid. In particularembodiments one or more nucleotides can be added to the 3' end of a nucleic acid, for example, via polymerase catalysis (e.g., DNA polymerase, RNA polymerase or reverse transcriptase). Chemical or enzymatic methods can be used to add one or more nucleotide to the 3' or 5' end of a nucleic acid. One or more oligonucleotides can be added to the 3' or 5' end of a nucleic acid, for example, via chemical or enzymatic (e.g., ligase catalysis) methods. A nucleic acid can be extended in a template directed manner, whereby the product of extension is complementary to a template nucleic acid that is hybridized to the nucleic acid that is extended.

[0043] The term “flowchip” is intended to mean a vessel having a chamber where a reaction can be carried out, an inlet for delivering reagents to the chamber, and an outlet for removing reagents from the chamber. In some embodiments, the chamber is configured for the detection of the reaction that occurs in the chamber. For example, the chamber can include one or more transparent surfaces, allowing optical detection of biological samples, optically labeled molecules, or the like in the chamber. Examples of flowchips include, but are not limited to those used in a nucleic acid sequencing apparatus, such as flowchips for the Genome Analyzer, MiSeq, NextSeq or HiSeq platforms commercialized by Illumina, Inc. (San Diego, Calif.); or for the SOLiD or Ion Torrent sequencing platform commercialized by Life Technologies (Carlsbad, Calif.). Exemplary flowchips and methods for their manufacture and use are also described, for example, in WO 2014 / 142841 Al; U.S. Pat. App. Pub. No. 2010 / 0111768 Al and U.S. Pat. No. 8,951,781, each of which is incorporated herein by reference.

[0044] The terms “nucleic acid” and “nucleotide” are intended to be consistent with their use in the art and to include naturally occurring species or functional analogs thereof. Particularly useful functional analogs of nucleic acids are capable of hybridizing to a nucleic acid in a sequence specific fashion or capable of being used as a template for replication of a particular nucleotide sequence. Naturally occurring nucleic acids generally have a backbone containing phosphodiester bonds. An analog structure can have an alternate backbone linkage including any of a variety of those known in the art. Naturally occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)). A nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are known in the art. A nucleic acid can include native or non-native nucleotides. In this regard, a nativedeoxyribonucleic acid can have one or more bases selected from the group consisting of adenine, thymine, cytosine or guanine and a ribonucleic acid can have one or more bases selected from the group consisting of uracil, adenine, cytosine or guanine. Useful non-native bases that can be included in a nucleic acid or nucleotide are known in the art. The terms “probe” or “target,” when used in reference to a nucleic acid or sequence of a nucleic acid, are intended as semantic identifiers for the nucleic acid or sequence in the context of a method or composition set forth herein and does not necessarily limit the structure or function of the nucleic acid or sequence beyond what is otherwise explicitly indicated. The terms “probe” and “target” can be similarly applied to other analytes such as proteins, small molecules, cells or the like.

[0045] The term “random” can be used to refer to the spatial arrangement or composition of locations on a surface. For example, there are at least two types of order for an array described herein, the first relating to the spacing and relative location of features (also called “sites”) and the second relating to identity or predetermined knowledge of the particular species of molecule that is present at a particular feature. Accordingly, features of an array can be randomly spaced such that nearest neighbor features have variable spacing between each other. Alternatively, the spacing between features can be ordered, for example, forming a regular pattern such as a rectilinear grid or hexagonal grid. In another respect, features of an array can be random with respect to the identity or predetermined knowledge of the species of analyte (e.g., nucleic acid of a particular sequence) that occupies each feature independent of whether spacing produces a random pattern or ordered pattern. An array set forth herein can be ordered in one respect and random in another. For example, in some embodiments set forth herein a surface is contacted with a population of nucleic acids under conditions where the nucleic acids attach at sites that are ordered with respect to their relative locations but ‘randomly located’ with respect to knowledge of the sequence for the nucleic acid species present at any particular site. Reference to “randomly distributing” nucleic acids at locations on a surface is intended to refer to the absence of knowledge or absence of predetermination regarding which nucleic acid will be captured at which location (regardless of whether the locations are arranged in an ordered pattern or not).

[0046] The term “substrate” is intended to mean a solid support. The term includes any material that can serve as a solid or semi-solid foundation for creation of features, such as wells for the deposition of biopolymers, including nucleic acids, polypeptide and / or otherpolymers. A substrate of the invention is modified, for example, or can be modified to accommodate attachment of biopolymers by a variety of methods well known to those skilled in the art. Exemplary types of substrate materials include glass, modified glass, functionalized glass, inorganic glasses, microspheres, including inert and / or magnetic particles, plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, an optical fiber or optical fiber bundles, a variety of polymers other than those exemplified above and multiwell plates. Specific types of exemplary plastics include acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes and Teflon™. Particularly useful solid supports for some embodiments are located within a flowchip apparatus. Exemplary flowchips are set forth in further detail herein.

[0047] The term “universal sequence” refers to a series of nucleotides that is common to two or more nucleic acid molecules even if the molecules also have regions of sequence that differ from each other. A universal sequence that is present in different members of a collection of molecules can allow capture of multiple different nucleic acids using a population of universal capture nucleic acids that are complementary to the universal sequence. Similarly, a universal sequence present in different members of a collection of molecules can allow the replication or amplification of multiple different nucleic acids using a population of universal primers that are complementary to the universal sequence. Thus, a universal capture nucleic acid or a universal primer includes a sequence that can hybridize specifically to a universal sequence. Target nucleic acid molecules may be modified to attach universal adapters, for example, at one or both ends of the different target sequences.

[0048] Embodiments described herein relate to methods of fabricating microarrays of nucleic acid molecules (DNA molecules) on microfluidic devices or flowchips at a significantly lower cost than those of traditional methods. Fig. 1 illustrates a method of fabricating a DNA microarray on a solid support of a flowchip. The method includes providing a plurality of oligonucleotide primer pairs on a solid support surface of a microarray of a microfluidic device, such as a flowchip. A plurality of single-stranded nucleic acid probe templates are hybridized to at least a portion of the plurality of primers on the surface. The nucleic acid sequences of the primers hybridized to the single stranded capture nucleic acid probe templates are extended to form a plurality of complementary capture probes hybridized to the plurality of single-stranded capture nucleic acid probe templates.The probe templates are then removed from the microarray surface. The capture probes are amplified to form probe clusters on the surface of the microarray.

[0049] In some embodiments, the plurality of oligonucleotide primer pairs can be immobilized to the solid support surface. The plurality of oligonucleotide primer pairs can include different species of oligonucleotide molecules, each having a different sequence. For example, a plurality of oligonucleotide primer pairs can include at least two different species of oligonucleotides, at least three different species, at least four different species, or more, wherein a first species has a different sequence than the other species in the plurality. It will be understood that different species of oligonucleotides can share a common sequence so long as there is a sequence difference between at least a portion of the different species.

[0050] The term “immobilized” as used herein is intended to mean direct or indirect attachment to a solid support via covalent or non-covalent bond(s). In some embodiments, covalent attachment can be used, but all that is required is that the oligonucleotide primer pairs remain stationary or attached to a support under conditions in which it is intended to use the support, for example, in applications requiring nucleic acid amplification and / or sequencing. Oligonucleotides to be used as capture and / or amplification primer pairs can be immobilized such that a 3 '-end is available for enzymatic extension and at least a portion of the sequence is capable of hybridizing to a complementary sequence.

[0051] Any of a variety of solid supports can be used in the method described herein. Particularly useful solid supports are those used for nucleic acid arrays. Examples include glass, modified glass, functionalized glass, inorganic glasses, plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, an optical fiber or optical fiber bundles, polymers and multiwell (e.g., microtiter) plates. Examples of plastics include acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes and Teflon. Examples of silica- based materials include silicon and various forms of modified silicon.

[0052] In particular embodiments, a solid support can be within or part of a vessel such as a well, tube, channel, cuvette, Petri plate, bottle or the like. A particularly useful vessel is a flow-cell, for example, as described in WO 2014 / 142841 Al; U.S. Pat. App. Pub. No. 2010 / 0111768 Al and U.S. Pat. No. 8,951,781 or Bentley et al., Nature 456:53-59 (2008), each of which is incorporated herein by reference. Examples of flow-cells are those that are commercially available from Illumina, Inc. (San Diego, Calif.) for use with a sequencingplatform such as a Genome Analyzer®, MiSeq®, NextSeq® or HiSeq® platform. Another particularly useful vessel is a well in a multiwell plate or microtiter plate.

[0053] Optionally, a solid support can include a gel coating. Attachment of nucleic acids to a solid support via a gel is exemplified by flowchips available commercially from Illumina Inc. (San Diego, Calif.) or described in US Pat. App. Pub. Nos. 2011 / 0059865 Al, 2014 / 0079923 Al, or 2015 / 0005447 Al; or PCT Publ. No. WO 2008 / 093098, each of which is incorporated herein by reference. Exemplary gels that can be used in the methods and apparatus set forth herein include, but are not limited to, those having a colloidal structure, such as agarose; polymer mesh structure, such as gelatin; or cross-linked polymer structure, such as polyacrylamide, SFA (see, for example, US Pat. App. Pub. No. 2011 / 0059865 Al, which is incorporated herein by reference) or PAZAM (see, for example, US Pat. App. Publ. Nos. 2014 / 0079923 Al, or 2015 / 0005447 Al, each of which is incorporated herein by reference).

[0054] In some embodiments, a solid support can be configured as an array of features to which nucleic acids can be attached. The features can be present in any of a variety of desired formats. For example, the features can be wells, pits, channels, ridges, raised regions, pegs, posts or the like. In some embodiments, the features can contain beads. However, in particular embodiments the features need not contain a bead or particle. Exemplary features include wells that are present in substrates used for commercial sequencing platforms sold by 454 LifeSciences (a subsidiary of Roche, Basel Switzerland) or Ion Torrent (a subsidiary of Life Technologies, Carlsbad Calif.). Other substrates having wells include, for example, etched fiber optics and other substrates described in U.S. Pat Nos. 6,266,459; 6,355,431; 6,770,441; 6,859,570; 6,210,891; 6,258,568; 6,274,320; US Pat app. Publ. Nos. 2009 / 0026082 Al; 2009 / 0127589 Al; 2010 / 0137143 Al; 2010 / 0282617 Al or PCT Publication No. WO 00 / 63437, each of which is incorporated herein by reference. In some embodiments, wells of a substrate can include gel material (with or without beads) as set forth in US Pat. App. Publ. No. 2014 / 0243224 Al, which is incorporated herein by reference.

[0055] The features on a solid support can be metal features on a non-metallic surface such as glass, plastic or other materials exemplified above. A metal layer can be deposited on a surface using methods known in the art such as wet plasma etching, dry plasma etching, atomic layer deposition, ion beam etching, chemical vapor deposition, vacuum sputtering or the like. Any of a variety of commercial instruments can be used as appropriate including,for example, the FlexAl®, OpAL®, lonfab 300plus®, or Optofab 30000® systems (Oxford Instruments, UK). A metal layer can also be deposited by e-beam evaporation or sputtering as set forth in Thornton, Ann. Rev. Mater. Sei. 7:239-60 (1977), which is incorporated herein by reference. Metal layer deposition techniques, such as those exemplified above, can be combined with photolithography techniques to create metal regions or patches on a surface. Exemplary methods for combining metal layer deposition techniques and photolithography techniques are provided in U.S. Pat. No. 8,895,249 or US Pat App. Pub. No. 2014 / 0243224 Al, each of which is incorporated herein by reference.

[0056] Features can appear on a solid support as a grid of spots or patches. The features can be located in a repeating pattern or in an irregular, non-repeating pattern. Particularly useful repeating patterns are hexagonal patterns, rectilinear patterns, grid patterns, patterns having reflective symmetry, patterns having rotational symmetry, or the like. Asymmetric patterns can also be useful. The pitch can be the same between different pairs of nearest neighbor features or the pitch can vary between different pairs of nearest neighbor features.

[0057] In particular embodiments, features on a solid support can each have an area that is larger than about 100 nm2, 250 nm2, 500 nm2, 1 pm2, 2.5 pm2, 5 pm2, 10 pm2, 100 pm2, or 500 pm2. Alternatively or additionally, features can each have an area that is smaller than about 1 mm2, 500 pm2, 100 pm2, 25 pm2, 10 pm2, 5 pm2, 1 pm2, 500 nm2, or 100 nm2.

[0058] The oligonucleotide primer pairs may include nucleic acid sequences, which are specific for primer binding domains on the 5’ end or 3’ end of template nucleic acid strands in a nucleic acid sample. Thus, only a subset of template nucleic acid strands in the sample may, in these and related embodiments, be selected by the oligonucleotide primer pairs to become single stranded nucleic acid probe templates. The primer binding sequences will generally be of known sequence and will therefore be complementary to primer binding domains of single stranded nucleic acid probe templates.

[0059] The length of primer oligonucleotides can be, for example, 16-50 nucleotides, more particularly 16-40 nucleotides, and yet more particularly 20-30 nucleotides in length. The desired length of the primer oligonucleotides will depend upon a number of factors. However, the primers are typically long (complex) enough so that the likelihood of annealing to sequences other than a primer binding domain of the single stranded nucleic acid probetemplates is very low. Accordingly, known sequences that flank a template sequence can include a primer binding portion.

[0060] The oligonucleotide primer pairs are capable of annealing or hybridizing specifically to primer binding domains of single stranded nucleic acid probe templates under conditions encountered in a primer annealing step. In some embodiments, a first primer of a primer pair can include a first universal capture region, and a second capture primer of the primer pair can include a second universal capture region. For example, the first primer of a primer pair can include an Illumina® P5 primer nucleotide sequence, and the second primer of the primer pair can include an Illumina® P7 primer nucleotide sequence. It will be appreciated that other know universal primer pairs can be used.

[0061] Primer pairs can include a single primer pair or a plurality of primer pairs. For example, the first primer pair can be a plurality of first primer pairs. In some embodiments, the first primer pair is at least one primer pair. In another example, the second primer pair can be a plurality of second capture primer pairs. In some embodiments, the second primer pair is at least one primer pair.

[0062] Primers may additionally comprise non-nucleotide chemical modifications, for example, to facilitate covalent attachment of the primer to a solid support. Certain chemical modifications may themselves improve the function of the molecule as a primer or may provide some other useful functionality, such as providing a cleavage site that enables the primer (or an extended polynucleotide strand derived therefrom) to be cleaved from a solid support. Useful chemical modifications can also provide reversible modifications that prevent hybridization or extension of the primer until the modification is removed or reversed. Similarly, other molecules attached to a surface in accordance can include cleavable linker moieties and or reversible modifications that alter a particular chemical activity of function of the molecule.

[0063] In certain embodiments, primer pairs are immobilized by covalent attachment to the solid support at or near the 5' end of the primer, such that a portion of the primer is free to anneal to its single stranded nucleic acid probe template and the 3' hydroxyl group is free to function in primer extension. In certain embodiments, a subset of modified primers can be provided that are prevented from hybridization and / or extension until the modification is removed, reversed or altered. In particular embodiments, the primer oligonucleotides will be incapable of hybridization to the initial single stranded nucleic acid probe templates.

[0064] In some embodiments, hybridization of the single stranded nucleic acid probe templates will typically be specific for the primer oligonucleotides such that the amount of primer oligonucleotides on the surface determines the amount of single stranded nucleic acid probe templates captured and thus the density of the resulting amplified clusters.

[0065] The chosen attachment chemistry for attaching the primer pairs to solid support surface can depend on the nature of the solid support and any functionalization or derivatization applied to it. In the case of nucleic acid embodiments, the primer itself may include a moiety which may be a non-nucleotide chemical modification to facilitate attachment. For example, the primer may include a sulfur containing nucleophile, such as a phosphorothioate or thiophosphate at the 5' end. In the case of solid supported polyacrylamide hydrogels, this nucleophile may bind to a bromoacetamide group present in the hydrogel. In one embodiment, the means of attaching primers to the solid support is via 5' phosphorothioate attachment to a hydrogel comprised of polymerized acrylamide and N- (5-bromoacetamidylpentyl) acrylamide (BRAPA).

[0066] A uniform, homogeneously distributed ‘lawn’ of immobilized oligonucleotide primer oligonucleotide primers may be formed by coupling (grafting) a solution of oligonucleotide primer pairs onto the solid support. The solution can contain a homogeneous population of primer pairs. Each surface that is exposed to the solution therefore reacts with the solution to create a uniform density of immobilized sequences over the whole of the exposed solid support. A suitable density of oligonucleotide primer pairs is at least about 1 fmol / mm2(6xlO10per cm2), or more optimally, at least about 10 fmol / mm2(6x10’1per cm2). The density of the oligonucleotide primer pairs can be controlled to give an optimum cluster density.

[0067] In some embodiments, the plurality of single stranded nucleic acid probe templates that are hybridized to at least one of the primer pairs on the surface of the solid support of the flowchip can include a first end with a first primer binding domain that is complementary or hybridizes to one of the surface primers of the pair, a second opposite end with second primer binding domain that is complementary or hybridizes to the other of surface primers of the pair, a capture domain between the first end and second end having a nucleic acid sequence that can bind, is complementary, and / or hybridizes to target nucleic acids of interest, and optionally a barcode or index domain or sequence between the first end and second.

[0068] In some embodiments, the nucleic acid sequence of the single stranded nucleic acid probe templates can include flanking universal primer binding domains at its 3'-end and / or 5 '-end, a target- specific capture domain, and optionally one or more restriction sites and an index domain or sequence. The single stranded nucleic acid probe templates can hybridize with an immobilized primer via one or more of the template's flanking universal capture regions and the primer's 3 '-terminal universal capture region.

[0069] The nucleic acid sequence of the target- specific capture domain is generally such that a complementary capture probe can be formed that has nucleic acid sequence that is complementary to a portion of a target nucleic acid sequence. The nucleic acid sequence of the target-specific capture domain can be any of a variety of lengths including, for example, lengths of at least 2, 4, 6, 8, 10, 12, 15, 20 or more nucleotides in length. Alternatively or additionally, the length of the barcode sequence can be at most 20, 15, 12, 10, 8, 6, 4 or fewer nucleotides.

[0070] In multiplex embodiments, a plurality of different capture probes can include different target capture sequences that hybridize to different target nucleic acid sequences from a biological specimen. Different target capture sequences can be used to selectively bind to one or more desired target nucleic acids from a biological specimen. In some cases, the different nucleic acid capture probes can include a target capture sequence that is common to all or a subset of the probes on a solid support.

[0071] The optional index or barcode sequence can be of any of a variety of lengths. Longer sequences can generally accommodate a larger number and variety of barcodes for a population. A barcode sequence can be at least 2, 4, 6, 8, 10, 12, 15, 20 or more nucleotides in length. Alternatively or additionally, the length of the barcode sequence can be at most 20, 15, 12, 10, 8, 6, 4 or fewer nucleotides. Examples of barcode sequences that can be used are set forth, for example in, US Pat. App. Publ. No. 2014 / 0342921 Al and U.S. Pat. No. 8,460,865, each of which is incorporated herein by reference.

[0072] In some embodiments, the plurality of single stranded nucleic acid probe templates can include a plurality of the same single stranded nucleic acid probe templates and / or a plurality of different single stranded nucleic acid probe templates. Different single stranded nucleic acid probe templates can be distinguished from each other, e.g., by having different target- specific capture regions or by having the same target-specific capture region in different locations.

[0073] In some embodiments, the single stranded nucleic acid probe templates can be hybridized to the primer pairs by providing a fluid that contains a mixture of different single stranded nucleic acid probe templates and contacting this fluidic mixture with the primer pairs immobilized on the solid support. The contact can result in the fluidic mixture being in contact with primer pairs to which many different single stranded nucleic acid probe templates from the fluidic mixture will attach. Thus, the single stranded nucleic acid probe templates have random access to primer pairs on the surface. Accordingly, the single stranded nucleic acid probe templates can be randomly located on the solid support.

[0074] The total number and variety of different single stranded nucleic acid probe templates that end up hybridized to primers on the surface can be selected for a particular application or use. For example, in embodiments where a fluidic mixture of different single stranded nucleic acid probe templates is contacted with primer pairs on the solid support for purposes of attaching the single stranded nucleic acid probe templates to the support, the number and variety of different single stranded nucleic acid probe templates can be less than the occupancy of the primers.

[0075] In some embodiments, attachment of a single stranded nucleic acid probe template is non-specific with regard to any sequence differences between the single stranded nucleic acid probe template and other single stranded nucleic acid probe templates that are or will be hybridized to the primers. For example, different single stranded nucleic acid probe templates can have a universal sequence that complements surface-attached primers. Alternatively, each of the different probes (or a subpopulation of different probes) can have a unique sequence that complements a unique primer on the solid support. In such cases, the unique primers, optionally, be attached at predefined locations in order to selectively capture particular single stranded nucleic acid probe templates, or particular types of single stranded nucleic acid probe templates, at the respective predefined locations.

[0076] When hybridizing template nucleic acids with immobilized primers on a patterned flowchip, hybridization conditions can be adjusted such that only a single stranded nucleic acid probe template hybridizes with an immobilized primer. Methods of hybridization for formation of stable duplexes between complementary sequences by way of Watson-Crick base-pairing are known in the art. Single stranded nucleic acid probe templates not hybridized to a primer can be separated and removed, for example by washing.

[0077] Primers hybridized with a single stranded nucleic acid probe template can be extended to form a plurality of complementary captures probes hybridized to the plurality of single stranded capture nucleic acid probe templates. An extension reaction may be carried out wherein the primer oligonucleotide is extended by sequential addition of nucleotides to generate a complementary copy of the single stranded nucleic acid probe template attached to the solid support. In some embodiments, the nucleic acid sequences are extended by adding nucleotide triphosphates (NTPs) and polymerase to the surface of the microarray.

[0078] Extension of primers can be carried out using methods exemplified herein or otherwise known in the art for amplification of nucleic acids or sequencing of nucleic acids. In particular embodiments, one or more nucleotides can be added to the 3' end of a nucleic acid, for example, via polymerase catalysis (e.g., DNA polymerase, RNA polymerase or reverse transcriptase). Chemical or enzymatic methods can be used to add one or more nucleotide to the 3' or 5' end of a nucleic acid. One or more oligonucleotides can be added to the 3' or 5' end of a nucleic acid, for example, via chemical or enzymatic (e.g., ligase catalysis) methods. A nucleic acid can be extended in a template directed manner, whereby the product of extension is complementary to a template nucleic acid that is hybridized to the nucleic acid that is extended. In some embodiments, a DNA primer is extended by a reverse transcriptase using an RNA template, thereby producing a cDNA. Thus, an extended complementary capture probe made in a method set forth herein can be a reverse transcribed DNA molecule. Exemplary methods for extending nucleic acids are set forth in US Pat. App. Publ. No. US 2005 / 0037393 Al or U.S. Pat. No. 8,288,103 or 8,486,625, each of which is incorporated herein by reference.

[0079] All or part of a single stranded nucleic acid probe template that is hybridized to a primer can be copied by extension. For example, an extended probe can include at least, 1, 2, 5,10, 25, 50, 100, 200, 500, 1000 or more nucleotides that are copied from a target nucleic acid. The length of the extension product can be controlled, for example, using reversibly terminated nucleotides in the extension reaction and running a limited number of extension cycles. The cycles can be run as exemplified for nucleic acid extension techniques.

[0080] The single stranded nucleic acid probe templates can be separated from the complementary immobilized capture probes and removed, for example, by washing. The terms ‘separate’ and ‘separating’ when used in reference to strands of a nucleic acid, refer to the physical dissociation of the DNA bases that interact within for example, a Watson-CrickDNA-duplex of the single stranded nucleic acid probe templates and its complement. After the extension reaction, the duplex is immobilized through a single 5' attachment, and hence strand separation can result in loss of one of the strands from the surface. In some embodiments, the single stranded nucleic acid probe templates are removed by denaturing the probe templates from the capture probes.

[0081] Following extension and denaturing to form the plurality single stranded capture probes, the plurality single stranded capture probes can be amplified by, for example, bridge amplification or PCR, to form amplicons or clusters of the single stranded capture probes attached to the surface of the substrate. Any of a variety of amplification techniques can be used to form the amplicons or clusters of the single stranded capture probes. Examples of amplification techniques polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), or random prime amplification (RPA). In some embodiments, the amplification can be carried out on solid phase. For example, the 3 '-end of the complementary capture probe can hybridize with a non-extended immobilized capture primer via its complementary 3 '-terminal universal capture region, thereby forming a bridge structure and amplified by bridge PCR or bridge amplification. Exemplary reagents and conditions that can be used for bridge amplification are described, for example, in U.S. Pat. Nos. 5,641,658, 7,115,400, or 8,895,249; or U.S. Pat. Publ. Nos. 2002 / 0055100 Al, 2004 / 0096853 Al, 2004 / 0002090 Al, 2007 / 0128624 Al or 2008 / 0009420 Al, each of which is incorporated herein by reference. In some embodiments, one or more rounds of bridge amplification are conducted to form a monoclonal clusters of different capture probes.

[0082] Solid-phase PCR amplification can also be carried out with one of the primers attached to the solid support and the second primer in solution. An exemplary format that uses a combination of a surface attached primer and soluble primer is the format used in emulsion PCR as described, for example, in Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003), WO 05 / 010145, or U.S. Pat. App. Publ. Nos. 2005 / 0130173 Al or 2005 / 0064460 Al, each of which is incorporated herein by reference.

[0083] Amplification sites or clusters in an array need not be entirely clonal in all embodiments. Rather, for some applications, an individual amplification site or cluster can be predominantly populated with amplicons from a first capture probe and can also have a low level of contaminating amplicons from a second capture probe. An array can have one or more amplification sites that have a low level of contaminating amplicons so long as the levelof contamination does not have an unacceptable impact on a subsequent use of the array. For example, when the array is to be used in a detection application, an acceptable level of contamination would be a level that does not impact signal to noise or resolution of the detection technique in an unacceptable way. Accordingly, apparent clonality will generally be relevant to a particular use or application of an array made by the methods set forth herein. Exemplary levels of contamination that can be acceptable at an individual amplification site for particular applications include, but are not limited to, at most 0.1%, 0.5%, 1%, 5%, 10% or 25% contaminating amplicons. An array can include one or more amplification sites having these exemplary levels of contaminating amplicons. For example, up to 5%, 10%, 25%, 50%, 75%, or even 100% of the amplification sites in an array can have some contaminating amplicons.

[0084] A composition for amplifying capture probes at amplification sites, referred to herein as an “amplification reagent,” is typically capable of rapidly making copies of capture probes at amplification sites. An amplification reagent used in a method of the present disclosure will generally include a polymerase and nucleotide triphosphates (NTPs). Any of a variety of polymerases known in the art can be used, but in some embodiments it may be preferable to use a polymerase that is exonuclease negative. Examples of nucleic acid polymerases suitable for use in embodiments of the present invention include, but are not limited to, DNA polymerase (such as Klenow fragment, T4 DNA polymerase, Bst (Bacillus stearothermophilus) polymerase), thermostable DNA polymerases (such as Taq, Vent, Deep Vent, Pfu, TH, and 9° N DNA polymerases) as well as their genetically modified derivatives (TaqGold, VENTexo, Pfu exo). In some embodiments, an amplification reagent can also include recombinase, accessory protein, and single-stranded DNA binding (SSB) protein for recombinase-facilitated amplification.

[0085] The NTPs can be deoxyribonucleotide triphosphates (dNTPs) for embodiments where DNA copies are made. Typically the four native species, dATP, dTTP, dGTP and dCTP, will be present in a DNA amplification reagent; however, analogs can be used if desired. The NTPs can be ribonucleotide triphosphates (rNTPs) for embodiments where RNA copies are made. Typically the four native species, rATP, rUTP, rGTP and rCTP, will be present in an RNA amplification reagent; however, analogs can be used if desired. NTPs can be modified with a fluorescent or radioactive group. A large variety of synthetically modified nucleic acids have been developed for chemical and biological methods in order toincrease the detectability and / or the functional diversity of nucleic acids. These functionalized / modified molecules (e.g., nucleotide analogs) can be fully compatible with natural polymerizing enzymes, maintaining the base pairing and replication properties of the natural counterparts.

[0086] The rate at which an amplification reaction occurs can be increased by increasing the concentration or amount of one or more of the active components of an amplification reaction. For example, the amount or concentration of polymerase, nucleotide triphosphates, or primers. In some cases, the one or more active components of an amplification reaction that are increased in amount or concentration (or otherwise manipulated in a method set forth herein) are non-nucleic acid components of the amplification reaction.

[0087] Amplification rate can also be increased in a method set forth herein by adjusting the temperature. For example, the rate of amplification at one or more amplification sites can be increased by increasing the temperature at the site(s) up to a maximum temperature where reaction rate declines due to denaturation or other adverse events. Optimal or desired temperatures can be determined from known properties of the amplification components in use or empirically for a given amplification reaction mixture. Such adjustments can be made based on a priori predictions of primer melting temperature (Tm) or empirically. In certain embodiments the temperature of an amplification reaction are at least 35°C. to no greater than 70° C. For instance, an amplification reaction can be at least 35°C. to no greater than 42°C., or at least 57°C. to no greater than 63°C.

[0088] The result of bridging amplification is a population of clonal “bridged” amplification products or capture probe clusters at the amplification sites. Both strands of the amplicon are immobilized on the surface of an amplification site at the 5' ends, where this attachment is derived from the original attachment of the oligonucleotide primer pairs. The amplicons within amplification sites will be clonal and derived from amplification of a single capture probe, or with acceptable levels of another amplicon as described herein.

[0089] To facilitate identification of the capture probe clusters, one of the strands of the double stranded bridged structure can be selectively removed from the surface to allow efficient hybridization of a complementary probe or target nucleic acid. The selective removal of a specific strand is referred to herein as “linearization.” Examples of suitablemethods for linearization are described herein and are described in more detail in application number WO 2007 / 010251 and U.S. Pat. Application Pub. 2012 / 0309634.

[0090] In one embodiment, linearization is achieved by cleaving one strand of the bridged double stranded amplicons and then subjecting the resulting structure to conditions that remove the strand that is no longer attached to the amplification site surface. Cleavage can be accomplished through the use of a primer that includes a cleavage site. The cleavage site is typically in a location that results in a substantial portion of one strand of the bridged structure to be free of the surface of the amplification site — no longer immobilized — and susceptible to loss after the removal step.

[0091] In one embodiment, a cleavage site is treated to remove a nucleotide and make an abasic site. An “abasic site” is a nucleotide position in a nucleic acid from which the base component has been removed. Abasic sites can be formed chemically under artificial conditions or by the action of enzymes. Once formed, abasic sites may be cleaved (e.g., by treatment with an endonuclease or other single-stranded cleaving enzyme, exposure to heat or alkali), providing a means for site-specific cleavage of a nucleic acid.

[0092] In one embodiment, an abasic site may be created at a pre-determined position on one strand of an immobilized amplicon. This can be achieved, for example, by incorporating a specific nucleotide at the pre-determined position.

[0093] In one embodiment, a deoxyuridine (U) is incorporated in one of the primers attached to the surface of an amplification site. The enzyme uracil DNA glycosylase (UDG) can then be used to remove the uracil base, generating an abasic site on one strand. The polynucleotide strand including the abasic site can then be cleaved at the abasic site by treatment with endonuclease (e.g., DNA glycosylase-lyase Endonuclease VIII), heat or alkali. In a particular embodiment, the USER reagent available from New Englad Biolabs (NEB # M5505S) is used for the creation of a single nucleotide gap at an uracil base in an immobilized. In one embodiment, the amplification sites are exposed to a mixture containing the appropriate glycosylase and one or more suitable endonucleases. Treatment with endonuclease enzymes gives rise to a 3 '-phosphate moiety at the cleavage site, which can be removed with a suitable phosphatase such as alkaline phosphatase.

[0094] Treatment with endonuclease enzymes gives rise to a 3'-phosphate moiety at the cleavage site, and the presence of 3' phosphate is known to inhibit the activity of exonuclease I (Lehman and Nussbaum, 1964, J. Biol. Chem., 239: 2628-2636). Both exonuclease andlinearization steps can occur at the same time by combining the enzymes. The reduction of these two steps into one results in faster sequencing runs as two steps are now preformed simultaneously. Moreover, combining both steps does not have a detrimental effect on primary metrics, read quality, dual indexing, or genome build metrics.

[0095] Abasic site generation and cleavage results in a free 5 '-end on the strand that is no longer immobilized to the surface. This strand can be completely removed from the surface by exposing the amplification site to suitable conditions. In one embodiment, removal is by denaturation. The denaturation can be performed thermally or isothermally, for example, using chemical denaturation. The chemical denaturant may be urea, hydroxide, or formamide or other similar reagents. In another embodiment, removal can be achieved by treatment with an exonuclease with 5 '-3' activity, such as lambda or T7 exonuclease.Removal of the unattached strand results in a remaining single strand that can act as a capture probe for a target nucleic acid.

[0096] Optionally, the 3' ends of the capture probes at the amplification sites are repaired. The exonuclease can remove some of the nucleotides at the 3' ends of the nucleic acids after the linearization. Without intending to be limiting, it is possible the 3’ ends are “breathing” slightly resulting in a small number of nucleotides becoming single stranded and available to the exonuclease for digestion. Repair can be achieved by exposing the nucleotides to a DNA polymerase, such as the DNA polymerase used for the bridging amplification.

[0097] After the DNA microarray is fabricated, the probe identities of each cluster are unknown. In some embodiments, the probe clusters can be characterized and / or identified to determine target nucleic acids to which the probes of respective clusters bind.

[0098] In some embodiments, capture probe identities can be determined or mapped on the solid support surface by hybridizing fluorescent barcodes to capture probes of respective clusters and detecting the fluorescent properties, such as fluorescent intensity or wavelength, of each capture probe cluster to determine or map the nucleic sequence of the capture probes of the respective cluster on the solid support surface. The fluorescent barcodes include nucleic strands complementary and specific to respective capture probes and a fluorescent label conjugated or attached to a respective complementary strand that has a fluorescent property that is unique to and / or different the fluorescent properties of other fluorescent labels such that the hybridizations of different fluorescent barcodes to respectivecomplementary capture probes can be distinguished by the unique or different fluorescent properties of the fluorescent label. The nucleic sequences of the complementary nucleic strands and the fluorescent properties of the fluorescent label can be cataloged or classified. Upon hybridization of a fluorescent barcode to a capture probe of a cluster on the substrate and detection of the fluorescent properties of the fluorescent barcode, the location and nucleic sequence of the capture probe can be determined or mapped on the solid support surface based on the cataloged or classified fluorescent properties of the respective fluorescent barcode.

[0099] In some embodiments, the fluorescent label may include one or more fluorophores. As such, a given specific nucleic strand, which is complementary and specific to a respective capture probe, may be labeled with fluorescent label that includes a single type of fluorophore. Alternatively, a given specific nucleic strand, which is complementary and specific to respective capture probe, may be labeled with a fluorescent label that includes two or more different types fluorophores, e.g., as found in tandem dyes, e.g., where a first fluorophore acts as a donor to a second fluorophore. Examples of fluorophores include, but are not limited to: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7- dichlorofluorescein; 5 -Carboxy fluorescein (5-FAM); 5-Carboxynapthofhiorescein; 5- Carboxytetramethyirhodamine (5-TAMRA); 5-FAM (5 -Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5- TAMRA (5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7- Amino-4-methylcoumarin; 7 -Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxvacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2- methoxy acridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acuiflavin Feulgen SITSA; Aequoin (Photoprotein); AFPs-AutoFluorescent Protein-(Quantum Biotechnologies); Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™, Alexa Fluor 633™, Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexion; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X;Aminoactinomycin D, Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; AiPC (Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 68; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™, FQ; Auramine; Aurophosphine G; Aurophosphine;BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; Bimane; Bisbenzamide, Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG; Blancophor SV: BOBO™-1; BOBO™-3; Bodipy 492 / 515, Bodipy 493 / 503; Bodipy 500 / 510; Bodipy 505 / 515, Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X, Bodipy 665 / 676; Bodipy FI; Bodipy FL ATP, Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate: Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; BTC; BTC- SN; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green- 1 Ca2+Dye; Calcium Green-2 Ca2+; Calcium Green-SN Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; Chlorophyll; Chromomycin A; Chromomycin A, CL-NERF; CMFDA; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy35™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; cyclic AMP Fluorosensor (FiCRhR), Dabcyl; -Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3' DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-DL16-ASP);Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer; DiD (DilC 18(5)); DIDS; Dihydrorhodamine 123 (DHR); Dil (DiIC18(3)); Dinitrophenol; DiO (DiOC18(3)), DiR; DiR (DilCl 8(7)); DM-NERF (high pH); DNP; Dopamine; DTAF: DY-630-NHS; DY- 635-NHS, ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight: Europium (III) chloride; EYFP, Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyde Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate: Fluoro-Emerald; FluoroGold (Hydroxystilbamidine); Fluor-Ruby; Fluor X; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™ / Fluo-3; Fura-2; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2), Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258, Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, high calcium; Indo-1, low calcium; Indodicarbocyanine (DiD), Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-IO-I; IO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS751 (RNA); Leucophor PAF; Leucophor SF; LeucophorWS; Lissamine Rhodamine;Lissamine Rhodamine B; Calcein / Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue- White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow / Blue; Mag Green, Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-indo- 1 ; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxiion Brilliant Flavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobronobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green 488-X; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5 5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA: Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma), PKH67; PMIA, Pontochrome Blue Black; POPO-I; POPO-3; PO-PRO-1; PO-PRO- 3; Primuline; Procion Yellow; Propidium lodid (PL); PyMPO; Pyrene; Pyronine, Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-TexasRed];Resoufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); S65A, S65C, S65L, S65T; SBF1, Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B: Sevron Orange: Sevron Yellow L; SITS; SITS (Primuline): SITS (Stilbene Isothiosulptionic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calccin; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen, SpectrumOrange; Spectrum Red; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13, SYTO 14; SYTO 15; SYTO 16;SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25;SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60;SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82, SYTO 83;SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline:Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate;Thiadicarbocyanine (DiSC3): Thiazine Red R; Thiazole Orange, Thioflavin 5: Thioflavin S, Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (CalcofluorWhite); TMR; TO- PRO-I; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; WW 781: X-Rhodamine, XRITC, Xylene Orange: Y66F; Y66H; Y66W; YO-PRO-1; YO- PRO-3, YOYO-1; YOYO-3, Sybr Green, Thiazole orange (interchelating dyes), or combinations thereof.

[0100] In some embodiments, the fluorescent label can include the Alexa Fluor dye series (from Molecular Probes / Invitrogen) which cover a broad spectrum and match the principal output wavelengths of common excitation sources such as Alexa Fluor 350, Alexa Fluor 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, and 750. Some embodiments of the present invention include the Cy Dye fluorophore series (GE Healthcare), also covering a wide spectrum such as Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7. Some embodiments of the present invention include the Oyster dye fluorophores (Denovo Biolabels) such as Oyster-500, -550, -556, 645, 650, 656. Some embodiments of the present invention include the DY-Labels series (Dyomics), for example, with maxima of absorption that range from 418 nm (DY-415) to 844 nm (DY-415) to 844 nm (DY-831) such as DIY- 415, -495, 505, -547, -548, -549, -550, -554, -555, -556, -560, -590, -610, -615, -630, -631, - 632, -633, -634, -635, -636, -647, -648, -649, -650, -651, -652, -675, -676, -677, -680, -681, - 682, -700, -701, -730, -731, -732, -734, -750, -751, -752, -776, -780, -781, -782, -831, - 480XL, -48 IXL, -485XL, -510XL, -520XL, -52 IXL. Some embodiments of the present invention include the ATTO fluorescent labels (ATTO-TEC GmbH) such as ATTO 390, 425, 465, 488, 495, 520, 532, 550, 565, 590, 594, 610, 61 IX, 620, 633, 635, 637, 647, 647N, 655, 680, 700, 725, 740. Some embodiments can include CAL Fluor and Quasar dyes (Biosearch Technologies) such as CAL Fluor Gold 540, CAL Fluor Orange 560, Quasar 570, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 635, Quasar 670 Some embodiments of the present invention include quantum dots such as the EviTags (Evident Technologies) or quantum dots of the Qdot series (Invitrogen) such as the Qdot 525, Qdot565, Qdot585, Qdot605, Qdot655, Qdot705, Qdot 800. Some embodiments of the present invention include fluorescein, rhodamine, and / or phycoerythrin.

[0101] In some embodiments, a specific nucleic strand, which is complementary and specific to a respective capture probe, is labeled with one or more polymeric dyes(e.g., fluorescent polymeric dyes). Fluorescent polymeric dyes that find use in the subject methods and systems are varied. In some embodiments of the method, the polymeric dye includes a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure that includes a backbone of alternating unsaturated bonds (e.g., double and / or triple bonds) and saturated (e.g., single bonds) bonds, where 71-electrons can move from one bond to the other. As such, the conjugated backbone may impart an extended linear structure on the polymeric dye, with limited bond angles between repeat units of the polymer. For example, proteins and nucleic acids, although also polymeric, in some cases do not form extended-rod structures but rather fold into higher-order three-dimensional shapes. In addition, CPs may form “rigid-rod” polymer backbones and experience a limited twist (e.g., torsion) angle between monomer repeat units along the polymer backbone chain. In some instances, the polymeric dye includes a CP that has a rigid rod structure. The structural characteristics of the polymeric dyes can have an effect on the fluorescence properties of the molecules.

[0102] Any convenient polymeric dye may be utilized in the subject devices and methods. In some embodiments, a polymeric dye is a multichromophore that has a structure capable of harvesting light to amplify the fluorescent output of a fluorophore. In some embodiments, the polymeric dye is capable of harvesting light and efficiently converting it to emitted light at a longer wavelength. In some embodiments, the polymeric dye has a lightharvesting multichromophore system that can efficiently transfer energy to nearby luminescent species (e.g., a “signaling chromophore”). Mechanisms for energy transfer include, for example, resonant energy transfer (e.g., Forster (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer), and the like. In some instances, these energy transfer mechanisms are relatively short range; that is, close proximity of the light harvesting multichromophore system to the signaling chromophore provides for efficient energy transfer. Under conditions for efficient energy transfer, amplification of the emission from the signaling chromophore occurs when the number of individual chromophores in the light harvesting multichromophore system is large; that is, the emission from the signaling chromophore is more intense when the incident light (the “excitation light”) is at a wavelength which is absorbed by the light harvestingmultichromophore system than when the signaling chromophore is directly excited by the pump light.

[0103] The multichromophore may be a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure and can be used as highly responsive optical reporters for chemical and biological targets. Because the effective conjugation length is substantially shorter than the length of the polymer chain, the backbone contains a large number of conjugated segments in close proximity. Thus, conjugated polymers are efficient for light harvesting and enable optical amplification via Forster energy transfer.

[0104] Polymeric dyes of interest include, but are not limited to, those dyes described in U.S. Pat. Nos. 7,270,956; 7,629,448; 8,158,444; 8,227,187; 8,455,613; 8,575,303; 8,802,450; 8,969,509; 9,139,869; 9,371,559; 9,547,008; 10,094,838; 10,302,648; 10,458,989;10,641,775 and 10,962,546 the disclosures of which are herein incorporated by reference in their entirety; and Gaylord et al., J. Am. Chem. Soc., 2001, 123 (26), pp 6417-6418; Feng et al., Chem. Soc. Rev., 2010,39, 2411-2419; and Traina et al., J. Am. Chem. Soc., 2011, 133 (32), pp 12600-12607, the disclosures of which are herein incorporated by reference in their entirety. Specific polymeric dyes that may be employed include, but are not limited to, BD Horizon Brilliant™ Dyes, such as BD Horizon Brilliant™ Violet Dyes (e.g., BV421, BV510, BV605, BV650, BV711, BV786); BD Horizon Brilliant™ Ultraviolet Dyes (e.g., BUV395, BUV496, BUV737, BUV8O5); and BD Horizon Brilliant™ Blue Dyes (e.g., BB515) (BD Biosciences, San Jose, CA). Any fluorochromes that are known to a skilled artisan-including, but not limited to, those described above — or are yet to be discovered may be employed in the subject methods.

[0105] In some embodiments, each of the plurality of distinguishably fluorescently labeled specific nucleic strands, which is complementary and specific to respective capture probe, that make up a given fluorescent barcode is excitable by common light source, such as a common laser. In such instances, each of the plurality of distinguishably fluorescently labeled specific nucleic strand, which is complementary and specific to respective capture probe, that make up a given barcode may have a common excitation maximum, but differ from each other in terms of emission maximum.

[0106] Any given two distinguishable fluorescent barcodes may be distinguishable from each other (and give rise to distinguishable fluorescent signatures) based on the types offluorophores making up the barcode and / or signal brightness provided thereby. As such, any two distinguishable fluorescent signatures or wavelengths obtained from different barcodes may be distinguishable based on fluorescent signals and / or intensity thereof, of the fluorescent signals collectively making up the fluorescent signature. For example, two distinguishable fluorescent barcodes may be distinguishable from each other because they are made up of combinations of different types fluorophores, e.g., where one includes fluorophores a, b and c and the other includes fluorophores b, c and d. Two distinguishable fluorescent barcodes may also be distinguishable from each other because they are made up of different amounts of fluorophores, e.g., where one is made up of fluorophores a, b and c present in a first amount on the various specific binding members and the other is made up of fluorophores present at a second amount that differs from the first amount at a value that can be detected, e.g., by a difference in brightness of signal. Different brightnesses may readily be provided by having differing amounts of fluorophores associated with the specific binding members. Combinations of type and amount of fluorophores may be employed to provide any desired number of unique fluorescent barcodes.

[0107] A fluorescent barcode may be associated with a given capture probe or cluster using any convenient protocol. For example, the surface of the substrate and clusters of capture probes formed thereon can be contacted with a barcode labeling composition that includes different fluorescently labeled specific nucleic strands, which are complementary and specific to respective capture probes. In other instances, the surface of substrate and clusters of capture probes formed thereon may be sequentially contacted with the different fluorescently labeled specific nucleic strands, which are complementary and specific to respective capture probes. For example, contact may be achieved under any suitable conditions that provide for specific binding of the fluorescently labeled specific nucleic strand of the fluorescent barcode to their complementary capture probes. The labeled specific nucleic strands may contact clusters of the microarray, e.g., by introducing the labeled specific nucleic strand onto the surface of the substrate, such as by manual or automated fluid dispensing.

[0108] In some embodiments, the fluorescence properties of the clusters hybridized with the fluorescent barcodes can be imaged through an optical system onto an image sensor. An analysis process is then used to analyze the images, find the positions or specified locations of the clusters of clonally amplified clusters of molecules on the solid supportsurface, based on the fluorescent properties of the fluorescent probes attached to the clusters, and correlate the position or specific location of a cluster with the nucleic sequences of the capture probes of a specific cluster.

[0109] Once the identity and location of each cluster of capture probes on the substrate is determined, the fluorescent barcodes can be separated from capture probes by, for example, denaturing and washing, and the microarray can be used to detect target nucleic acids or nucleic acid analytes. The term “nucleic acid analyte” or “target nucleic acid” or “target” or “analyte” refers to a nucleic acid (often derived from a biological sample and hence referred to also as a sample nucleic acid), to which the capture probe specifically hybridizes. It is recognized that the target nucleic acids can be derived from essentially any source of nucleic acids (e.g., including, but not limited to chemical syntheses, amplification reactions, forensic samples, etc.). It is either the presence or absence of one or more target nucleic acids that are to be detected, or the amount of one or more target nucleic acids that is to be quantified. The target nucleic acid(s) that are detected preferentially have nucleotide sequences that are complementary to the nucleic acid sequences of the corresponding capture probe(s) to which they specifically bind (hybridize). The term “target nucleic acid” may refer to the specific subsequence of a larger nucleic acid to which the probe specifically hybridizes, or to the overall sequence (e.g., gene or mRNA) whose abundance (concentration) and / or expression level it is desired to detect. The difference in usage will be apparent from context. In some cases, the term refers to amplicons of a nucleic acid.

[0110] In some embodiments, target nucleic acids can be detected by adding target nucleic acids to the microarray surface such that the target nucleic acids are captured by a respective capture probe having a complementary sequence. Respective capture probes hybridized to target nucleic acids can be extended with a labeled NTP. The label of the labeled NTP hybridized during extension can be detected to determine the location of the target nucleic acid on a substrate corresponding to the mapped cluster of capture probes. The location of the detected target nucleic acid on the substrate can be compared to the mapped location of the clusters to determine the presence of the cluster and corresponding identity of the target nucleic acid.

[0001] For example, Fig. 2 illustrates a method of characterizing the capture probes and detecting DNA in a sample. In the method, a solution containing fluorescent barcodes, i.e., nucleic strands complementary and specific to respective capture probes conjugated to afluorescent label, can be provided on a flowchip. Once the strands with the barcode hybridize to the surface capture probes, the fluorescence properties of each cluster are analyzed, and the identity of each probe cluster is determined. Then the fluorescent barcode tagged strands are denatured and removed. When the DNA microarray is in use, the sample containing DNA targets of interest is sent onto the flowchip, and the target can be captured by their designated capture probes. The probes with a target hybridized can then be extended with DNA polymerase and a nucleotide with fluorescence; thus allowing users to detect the presence of the target DNA.

[0002] In other embodiments, the target nucleic acids can be detected using the nucleic acid detecting microarray by adding target nucleic acids to the microarray surface such that the target nucleic acids are captured by respective capture probes having a complementary nucleic acid sequence. The target nucleic acids captured or hybridized to the respective capture probe can be detected using fluorescent barcodes that hybridize specifically to captured target nucleic acids and do not hybridize to capture probes, which do not include a hybridized or captured target nucleic acid. That is, the fluorescent barcodes include nucleic strands complementary and specific to respective target nucleic acids, not the capture probes. For example, the nucleic sequence of the nucleic strand of the fluorescent probe is sufficiently complementary to a portion of the captured target nucleic acid that is not hybridized to the capture probe. A fluorescent label is conjugated or attached to a respective complementary strand that has a fluorescent property unique to and / or different from the fluorescent properties of other fluorescent labels such that the hybridization of different fluorescent barcodes to respective captured target nucleic acids can be distinguished by the fluorescent properties of the fluorescent label. The nucleic sequence of the complementary nucleic strands and the fluorescent properties of the fluorescent label can be cataloged or classified. Upon hybridization of a fluorescent barcode to a target nucleic acid hybridized or captured by a capture probe of a cluster on the substrate and detection of the fluorescent properties of a cluster provided by the fluorescent barcode, the presence, location, and nucleic sequence of the target nucleic can be detected, determined or mapped on the solid support surface based on the cataloged or classified fluorescent properties of the respective fluorescent barcode.

[0003] In some embodiments, the solid support surface and clusters of capture probes formed thereon can be contacted with a plurality of target nucleic acids. Target nucleic acidsnot captured, hybridized, or annealed to a respective capture probe can be removed from the surface of the substrate by washing, and the solid support surface including clusters with captured target nucleic acids can be contacted with a barcode labeling composition that includes different fluorescently labeled specific nucleic strands, which are complementary and specific to respective captured target nucleic acids, that collectively make up the barcode for that composition.

[0004] In some embodiments, the fluorescence properties of the clusters including captured target nucleic acids hybridized with the fluorescent barcodes can be imaged through an optical system onto an image sensor. An analysis process is then used to analyze the images, detect the fluorescent properties of the clusters provided by the respective hybridized fluorescent barcodes to the capture target nucleic acids of the clusters, and detect or identify the target nucleic acids, based on the fluorescent properties of the fluorescent probes attached to the captured target nucleic acids of the clusters.

[0005] For example, Fig. 3 illustrates that after the DNA microarray is fabricated, a sample containing target nucleic acids of interest can be directly loaded onto a flowchip. The target nucleic acids can be captured by their designated capture probes. Then a solution containing fluorescent barcodes including single stranded DNA complementary to target nucleic acids captured by their capture probes and modified with fluorescent labels is sent to the flowchip. The sequences of the fluorescence barcoded DNA strands are designed to bind to specific regions of the DNA targets. This way, the presence and identity of the targets captured on the surface can be detected at the same time.

[0111] In some embodiments, the methods described herein can be used, for example, to determine the sequence identity of a nucleic acid analyte in solution by measuring the binding of the analyte with known probes. For example, the methods described herein can be used to measure nucleotide sequences in a variety of sample types including cDNA, genomic DNA, RNA, cells, or viruses.

[0112] In some embodiments, the methods described herein are useful for the determination of the presence and amount of multiple nucleotide sequences in a sample. Where the probe and analyte are nucleic acids, the present invention provides methods of expression monitoring and for measuring genetic information. The method allows for many nucleotide sequences relating to genes, e.g. 10, 100, 1,000, 10,000, 100,000, or more genes to be analyzed at once. The term expression monitoring is used to refer to the determination oflevels of expression of particular, typically preselected, genes. In some cases, the whole transcriptome can be measured comprising all or a substantial portion of the expression in a cell, or group of cells. In some embodiments the expression of only a few genes, such as 5 to 100 genes is measured, for example, to diagnose a specific condition. In some embodiments, the array has a high degree of probe redundancy (multiple probes per gene) the expression monitoring methods provide accurate measurement and do not require comparison to a reference nucleic acid.

[0113] In some embodiments, the methods and systems described herein may be used in a wide variety of circumstances, including the detection of disease, the identification of differential gene expression between two samples (e.g., a pathological as compared to a healthy sample), screening for compositions that upregulate or downregulate the expression of particular genes, and so forth. They can be used for the analysis of genetic DNA, including the determination of single-nucleotide polymorphisms (SNPs) for genotyping and allele discrimination assays and for the detection of DNA and RNA viruses. The methods and systems of the invention can also be used for the detection of genetically modified organisms (GMO).

[0114] The methods and systems described herein can be used for genetic testing and diagnostics. They can be used for example for newborn screening (e.g., For phenylketonuria or congenital hypothyroidism, diagnostic testing (such as to diagnose or rule out a specific genetic or chromosomal condition or to confirm a diagnosis when a particular condition is suspected based on physical mutations and symptoms); carrier testing: (e.g., to identify people who carry one copy of a gene mutation that, when present in two copies, predictive and presymptomatic testing (e.g., testing for BRCA1 in relation to the risk of breast cancer): forensic testing (e.g., to identify an individual for legal purposes, e.g., to identify crime or catastrophe victims, rule out or implicate a crime suspect, or establish biological relationships between people (e.g., paternity): or for research testing (e.g., finding unknown genes, learning how genes work and advancing our understanding of genetic conditions).

[0115] Still other embodiments relate to a method of forming a next generation sequencing (NGS) library using a nucleic acid microarray, such as described herein. Referring to Fig. 4, in the method, a nucleic acid detecting microarray is provided by modifying the surface of a flowchip with DNA strands containing surface primer domain (Surface primer 1), an optional sample barcode (also called index) domain, and a probedomain complementary to a NGS target nucleic acids (Probe 1). The surface of the microarray can be modified similar to the method of modifying a microarray described in Fig. 1. For example, a flowchip surface can be modified with pairs of universal primers. Single stranded DNA probe templates, which are designed to form complementary capture probes with domains complementary to a NGS target nucleic acids of interest, are synthesized with each end having a binding domain that is complementary or hybridizes to the surface primers. The probe templates are provided on the flowchip so that they hybridize with the surface primers. The surface primers hybridized to the single stranded probe templates are then extended with DNA polymerase and dNTPs to form capture probes (Probe 1) tethered to the surface of the flowchip. The templates are removed and strands of the capture probes (Probe 1) are then amplified with, for example, bridge PCR to form clusters or amplicons on the flowchip. The cluster or amplicons can be linearized to become single stranded DNA capture probes (Probe 1) that can readily hybridize with target DNA of interest.

[0116] Target DNA strands can be added to the flowchip and captured by single stranded DNA capture probes (Probe 1) having complementary capture nucleic acid sequences. After capturing a target containing a nucleic acid strand in a sample, the probe is extended with DNA polymerase and dNTPs with the target nucleic acids as the templates.

[0117] The probes can be extended beyond the targeted region. Then, the strands containing the DNA targets are removed by denaturing the double stranded DNA. A solution containing a surface primer (Surface primer 2) for the other end of the library strands is sent to the flowchip. The Surface primer 2 strands have a probe domain on the 3 ’ end that can hybridize to the end of the targeted region on the strands on the surface (Probe 2) and may also have an optional index domain.

[0118] These primers are then extended to form nucleic acid strand containing domains of, from 5’ end to 3’ end, Surface primer 2, Index 2, Probe 2, target, Index 1, Surface primer 1. These newly formed nucleic acid strands are then denatured and collected from the flowchip, forming a targeted library for NGS. This whole process can be operated on the microfluidic device, and automated by controlling with a computer program.

[0119] In some embodiments, a flowchip provides a convenient apparatus for use in a method set forth herein. For example, a flowchip is a convenient apparatus for housing a solid support that will be treated with multiple fluidic reagents, such as the repeated fluidicdeliveries used for some nucleic acid sequencing protocols or some nucleic acid hybridization protocols. In some embodiments, a biological specimen can be delivered to a solid support in a flowchip, for example, when a fluidic mixture of cells, subcellular components, viruses or viroids is delivered to the solid support. In some embodiments, it may be preferable to open a flowchip to expose a solid support inside or to remove the solid support from the flowchip in order to allow convenient delivery of a biological specimen to the solid support. For example, opening the flowchip or removing the solid support can allow a user or robotic device to lay a tissue section on the solid support. The opening of a flowchip or removal of a solid support from a flowchip can be temporary. Thus, the flowchip can subsequently be closed or the solid support returned to the flowchip to proceed with one or more subsequent steps of a method set forth herein.

[0120] In some embodiments, a flowchip can have a construction that allows it to be opened or taken apart. For example, the flowchip can be in a closed state while performing a sequencing reaction. Then the flowchip can be taken apart so that a sample can be placed on the flowchip surface. The flowchip can be held together by adhesive such that one or more surface can be removed to open it. For example, a flowchip can have a spacer with adhesive surfaces on the top or bottom (akin to single-sided or double-sided sticky tape) and this spacer can occur between two solid supports. One or both of the solid supports can be configured to attach nucleic acids and support a biological sample as set forth herein. The spacer can have open regions (e.g., created by laser cutting of the spacer material) that create fluidic channels bound by the two solid supports and the spacer. Thus, one or both of the solid supports can be non-permanently adhered to the spacer to allow one or both of them to be removed to allow access to the surface when placing a tissue or other specimen thereon.

[0121] From the above description of the invention, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes and modifications within the skill of the art are intended to be covered by the appended claims. All references, publications, and patents cited in the present application are herein incorporated by reference in their entirety.

Claims

Having described the invention, we claim:

1. A method of fabricating a nucleic acid detecting microarray, the method comprising: providing a plurality of primer pairs on a surface of microarray; hybridizing a plurality of single stranded nucleic acid probe templates to at least a portion of the plurality of primers on the surface; extending nucleic acid sequences of the primers hybridized to the single stranded capture nucleic acid probe templates to form a plurality of complementary captures probes hybridized to the plurality of single stranded capture nucleic acid probe templates; removing the probe templates; and amplifying the capture probes to form probe clusters on the surface of the microarray.

2. The method of claim 1 , wherein the plurality of single stranded nucleic acid probe templates include a first end with a first binding domain to one of the surface primers of the pair, a second opposite end with second binding domain to the other of the surface primers of the pair, and capture domain between the first end and second end that can bind to target nucleic acid of interest.

3. The method of claim 1 or 2, wherein the probe cluster are spatially dispersed on the surface of the microarray.

4. The method of any of claims 1 to 3, wherein the microarray comprises a flowchip.

5. The method of any of claims 1 to 4, wherein the nucleic acid sequences are extended by adding nucleotide triphosphates (NTPs) and polymerase to the surface of the microarray.

6. The method of any of claims 1 to 5, wherein the probe templates are removed by denaturing.

7. The method of any of claims 1 to 6, wherein the capture probes are amplified by bridge PCR.

8. The method of claim 7, wherein the amplified capture probes are linearized.

9. The method of any of claims 1 to 8, wherein different probe clusters bind to different target nucleic acids.

10. The method of any of claims 1 to 9, wherein the probe clusters bind to target DNA.

11. The method of any of claims 1 to 10, further comprising characterizing and / or identifying the probe clusters to determine target nucleic acids to which the probes of respective clusters bind.

12. The method of claim 11, wherein the probe clusters are characterized and / or identified by hybridizing fluorescent barcodes to the respective probes, the fluorescent barcodes including fluorescent labeled nucleic strands, which are complementary to the respective probes; and detecting the fluorescent properties of each probe cluster to determine the nucleic sequence of the capture probes of the respective cluster.

13. The method of claim 12, wherein the detected fluorescent properties include at least one of fluorescent intensity or wavelength.

14. The method of claims 12 or 13, removing the hybridized complementary nucleic strands with fluorescent barcodes after determining the capture probes of the respective cluster.

15. A nucleic acid detecting microarray formed by the methods of any of claims 116. A method of detecting nucleic acids in a sample, the method comprising: providing a nucleic acid microarray of claim 15; adding target nucleic acids to the microarray surface such that the target nucleic acids are captured by a respective capture probe having a complementary sequence; extending respective capture probes hybridized to target nucleic acids with a labeled NTP; and detecting the label of labeled NTP hybridized during extension to detect the presence of the target nucleic acid.

17. A method of detecting nucleic acids in a sample, the method comprising: providing a nucleic acid microarray formed by the methods of claims 1 to 10; adding target nucleic acids to the microarray surface such that the target nucleic acids are captured by a respective capture probe having a complementary sequence; hybridizing fluorescent barcodes to the captured target nucleic acids, the fluorescent barcodes including fluorescent labeled nucleic strands, which are complementary to the captured target nucleic acids; and detecting the fluorescent properties of each probe cluster to determine the identity of captured target nucleic acids.

18. A method of forming a next generation sequencing (NGS) library using a nucleic acid detecting microarray, such as a nucleic acid detecting microarray formed by the methods of claims 1 to 10, the method comprising: providing on a surface of a microarray nucleic strands containing surface primer domain (Surface primer 1), sample barcode (or index) domain, and a probe domain complementary to NGS targets (Probe 1); amplified the nucleic acid strands to increase the binding affinity of NGS targets to the probe; capturing NGS target strands in a sample with the probes; extending probes with the captured NGS target; removing captured NGS strands from the microarray;hybridizing second surface primer (Surface primer 2) strands to the extended end of the nucleic strands provided on the surface, wherein the Surface primer 2 strands have a probe domain on the 3 ’ end that hybridizes to the end of the targeted region on the strands on the surface (Probe 2) and an optional index domain; extending the Surface primer 2 strands to form strands containing domains of, from 5’ end to 3’ end, Surface primer2, Index 2, Probe 2, target, Index 1, Surface primer 1; and denaturing and collecting the formed strands form a targeted library for NGS.

Images