Patent Description:
In general, genetic sequencing involves determining the order of nucleotides or nucleic acids in a length of genetic material, such as a fragment of DNA or RNA. Increasingly longer sequences of base pairs are being analyzed, and the resulting sequence information may be used in various bioinformatics methods to logically fit fragments together so as to reliably determine the sequence of extensive lengths of genetic material from which the fragments were derived. Automated, computer-based examination of characteristic fragments have been developed, and have been used in genome mapping, identification of genes and their function, evaluation of risks of certain conditions and disease states, and so forth. In certain applications, for example, modified target nucleic acids are hybridized to sequencing or amplification primers on the surface of an array, amplified, and their genetic sequences determined.

In other examples, individual DNA and RNA probes may be attached at small locations in a geometric grid (or randomly) on an array support. A test sample, e.g., from a known person or organism, may be exposed to the grid, such that complementary fragments hybridize to the probes at the individual sites in the array. The array can then be examined by scanning specific frequencies of light over the sites to identify which fragments are present in the sample, by fluorescence of the sites at which the fragments hybridized.

Proper functionality and reproducibility of the nucleic acid-functionalized array depends on consistent display of the nucleic acid sequences bound thereto. The present application is directed to quality control compositions, arrays, and methods for ensuring consistent deposition of nucleic acid sequences on the array surface and retention of the sequences following any subsequent manufacturing and storage of the arrays.

The following prior art may be useful in understanding the invention. <CIT> describes an array including a solid support having a surface, the surface having a plurality of wells, the wells containing a gel material, the wells being separated from each other by interstitial regions on the surface, the interstitial regions segregating the gel material in each of the wells from the gel material in other wells of the plurality; and a library of target nucleic acids in the gel material, wherein the gel material in each of the wells comprises a single species of the target nucleic acids of the library. Methods for making and using the array are also provided. <CIT> describes methods for capturing and amplifying target polynucleotides on a solid surface, in particular in a well in a microarray, wherein the microarray may comprise a) a substrate comprising at least one well, a surface surrounding the well and an inner well surface; b) a first layer covering the inner well surface and comprising at least one first capture primer pair; and c) a second layer covering the first layer and the surface surrounding the well. Alternatively, the microarray may comprise a) a substrate comprising at least one well, a surface surrounding the well and an inner well surface; and b) a layer covering the inner well surface and comprising at least one first capture primer pair and at least one second capture primer pair. In particular kinetic exclusion amplification is used in creating monoclonal populations of the nucleic acids in the wells. The application also discloses a method for modifying an immobilized capture primer comprising: a) contacting a substrate comprising a plurality of immobilized capture primers with a plurality of template nucleic acids to produce one or more immobilized template nucleic acids, wherein the plurality of immobilized capture primers comprises a first plurality of primers comprising a <NUM>'-terminal universal capture region Y, e.g. primer P5, and a second plurality of primers comprising a <NUM>'-terminal universal capture region Z, e.g. primer P7; and wherein each template nucleic acid is flanked by a <NUM>'-terminal and a <NUM>'-terminal universal capture region Y or Z and comprises one or more, e.g. SapI, restriction sites and a target-specific capture region between the one or more restriction sites and the <NUM>'-terminal universal capture region; and b) extending one or more immobilized capture primer. Finally, the application discloses a method for modifying an immobilized capture primer comprising: a) contacting a substrate comprising a plurality of immobilized capture primers with a plurality of different seed nucleic acids to produce a plurality of different immobilized seed nucleic acids; b) extending two or more of the immobilized capture primers to produce a plurality of different immobilized extension products complementary to two or more of the plurality of different immobilized seed nucleic acids; and c) activating one immobilized extension product of the plurality of different immobilized extension products, to form an activated capture primer. <CIT> relates to a substrate with a surface comprising a silane or a silane derivative covalently attached to optionally substituted cycloalkene or optionally substituted heterocycloalkene for direct conjugation with a functionalized molecule of interest, such as a polymer, a hydrogel, an amino acid, a nucleoside, a nucleotide, a peptide, a polynucleotide, or a protein. The silane or silane derivative may contain optionally substituted norbornene or norbornene derivatives. Also described is a method for preparing a functionalized surface and the use in DNA sequencing and other diagnostic applications are also disclosed. <CIT> describes methods for targeted sequencing of polynucleotide such as a method of sequencing a target polynucleotide with fewer probes; and a method of sequencing a target polynucleotide with longer reads. Locus-specific, ligation-assisted sequencing/genotyping method and ligation-captured sequencing methods are also provided.

The present invention provides an array comprising a support comprising a plurality of discrete wells, a gel material positioned in each of the plurality of discrete wells, and a quality control tracer comprising a cleavable nucleotide sequence comprising a cleavage site and a detectable fluorescent label; wherein the cleavable nucleotide sequence comprises a grafted region with a first end and a second end, where the first end is grafted to the gel material and the second end is linked to a cleavable region that is linked to the detectable fluorescent label and comprises the cleavage site; wherein the cleavable nucleotide sequence is grafted to the gel material at its <NUM>' end and the detectable fluorescent label is attached at or near the <NUM>' end of the cleavable nucleotide sequence; wherein said quality control tracer is grafted to the gel material in each of the plurality of discrete wells; and wherein either (i) the portion of the cleavable nucleotide sequence which remains attached to the gel material after cleavage is a non-reactive nucleotide sequence, and thus will not actively participate in a particular DNA or RNA synthesis that is being performed or interfere with sequencing, and the quality control tracer is present in a predetermined ratio with an unlabeled primer nucleotide sequence grafted to the gel material in each of the plurality of discrete wells; or (ii) the portion of the cleavable nucleotide sequence which remains attached to the gel material after cleavage comprises a primer nucleic acid sequence.

The present invention also provides a method of determining the density and/or distribution of a primer nucleotide sequence grafted to a support comprising: providing an array of the invention as above; detecting a signal from the detectable fluorescent label; and wherein: (i) the portion of the cleavable nucleotide sequence which remains attached to the gel material after cleavage is a non-reactive nucleotide sequence, and thus will not actively participate in a particular DNA or RNA synthesis that is being performed or interfere with sequencing, and the quality control tracer is present in a predetermined ratio with the unlabeled primer nucleotide sequence grafted to the gel material in each of the plurality of discrete wells, determining the density and/or distribution of the quality control tracer based at least in part on the signal from the detectable label and determining the density and/or distribution of the grafted primer nucleotide sequence based at least in part on the determined density and/or distribution of the quality control tracer and the predetermined ratio with the unlabeled primer nucleotide sequence; or (ii) the portion of the cleavable nucleotide sequence which remains attached to the gel material after cleavage comprises the primer nucleic acid sequence, determining the density and/or distribution of the grafted primer nucleotide sequence based at least in part on the signal from the detectable fluorescent label.

In another aspect of the invention there is provided a method of grafting a quality control tracer to a gel material in a plurality of discrete wells on a support wherein the quality control tracer comprises a cleavable nucleotide sequence comprising a cleavage site and a detectable fluorescent label, wherein the cleavable nucleotide sequence comprises a grafted region with a first end and a second end, where the first end is grafted to the gel material and the second end is linked to a cleavable region that is linked to the detectable fluorescent label and comprises the cleavage site; wherein the cleavable nucleotide sequence is grafted to the gel material at its <NUM>' end and the detectable fluorescent label is attached at or near the <NUM>' end of the cleavable nucleotide sequence; wherein said quality control tracer is grafted to the gel material in each of the plurality of discrete wells; and wherein either: (i) the portion of the cleavable nucleotide sequence which remains attached to the gel material after cleavage is a non-reactive nucleotide sequence, and thus will not actively participate in a particular DNA or RNA synthesis that is being performed or interfere with sequencing, and the quality control tracer is present in a predetermined ratio with an unlabeled primer nucleotide sequence grafted to the gel material in each of the plurality of discrete wells; or (ii) the portion of the cleavable nucleotide sequence which remains attached to the gel material after cleavage comprises the primer nucleic acid sequence; detecting the quality control tracer using fluorescence, and based at least in part on the fluorescence, determining a density, or a distribution, or the density and the distribution, of the primer nucleotide sequence grafted to the gel material wherein either: (i) the portion of the cleavable nucleotide sequence which remains attached to the gel material after cleavage is a non-reactive nucleotide sequence, and thus will not actively participate in a particular DNA or RNA synthesis that is being performed or interfere with sequencing, and the quality control tracer is present in a predetermined ratio with the unlabeled primer nucleotide sequence grafted to the gel material in each of the plurality of discrete wells, determining the density and/or distribution of the quality control tracer based at least in part on the signal from the detectable label and determining the density and/or distribution of the grafted primer nucleotide sequence based at least in part on the determined density and/or distribution of the quality control tracer and the predetermined ratio with the unlabeled primer nucleotide sequence; or (ii) the portion of the cleavable nucleotide sequence which remains attached to the gel material after cleavage comprises the primer nucleic acid sequence, determining the density and/or distribution of the grafted primer nucleotide sequence based at least in part on the signal from the detectable fluorescent label; and cleaving the fluorescent label from the primer nucleotide sequence after the density, or the distribution, or the density and the distribution, of the primer nucleotide sequence is determined.

The present invention further provides a method of co-grafting a quality control tracer and a primer nucleotide sequence to the array of the present invention wherein the portion of the cleavable nucleotide sequence which remains attached to the gel material after cleavage is a non-reactive nucleotide sequence, and thus will not actively participate in a particular DNA or RNA synthesis that is being performed or interfere with sequencing, and the quality control tracer is present in a predetermined ratio with the unlabeled primer nucleotide sequence grafted to the gel material in each of the plurality of discrete wells, the method comprising: combining the quality control tracer and the primer nucleotide sequence at a predetermined ratio to form a grafting mix; exposing the grafting mix to a gel material in a well on a support; and incubating the grafting mix and the gel material, thereby co-grafting the quality control tracer and the primer nucleotide sequence to the gel material, detecting the grafted quality control tracer by detecting a signal from the fluorescent detectable label; and based at least in part on the detected fluorescent signal and the predetermined ratio, determining a density, or a distribution, or the density and the distribution, of the primer nucleotide sequence grafted to the gel material (<NUM>); and removing the detectable fluorescent label from the cleavable nucleotide sequence via enzymatic cleavage or chemical cleavage at the cleavage site.

Specific embodiments of the invention are set forth in the dependent claims. Associated methods are also described herein to aid in the understanding of the invention, but these do not form part of the claimed invention. Examples described herein which do not fall under the definition of the claims do not form part of the present invention.

The present invention is described further below by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, components.

It is to be understood that terms used herein will take on their ordinary meaning in the relevant art unless specified otherwise. Several terms used herein and their meanings are set forth below.

The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). The alkyl group may have <NUM> to <NUM> carbon atoms. Example alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like. As an example, the designation "C1-<NUM> alkyl" indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, isobutyl, sec-butyl, and t-butyl.

The alkyl may be substituted with a halide or halogen, which means any one of the radio-stable atoms of column <NUM> of the Periodic Table of the Elements, e.g., fluorine, chlorine, bromine, or iodine. This group is referred to as an "alkyl halide".

As used herein, "alkenyl" or "alkene" refers to a straight or branched hydrocarbon chain containing one or more double bonds. The alkenyl group may have <NUM> to <NUM> carbon atoms. Example alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like. The alkenyl group may be designated as, for example, "C2-<NUM> alkenyl," which indicates that there are two to four carbon atoms in the alkenyl chain.

As used herein, "alkynyl" or "alkyne" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. The alkyne group may have <NUM> to <NUM> carbon atoms. The alkyne group may be designated, for example, as "C2-<NUM> alkynyl," which indicates that there are two to four carbon atoms in the alkyne chain.

An "amido" functional group refers to
<CHM>
where R is any group that can attach to a fluorescent lable, R' is H, and R" is any group that can attached to a nucleotide sequence.

An "amino" functional group refers to an -NRaRb group, where Ra and Rb are each independently selected from hydrogen, C1-<NUM> alkyl, C2-<NUM> alkenyl, C2-<NUM> alkynyl, C3-<NUM> carbocyclyl, C6-<NUM> aryl, <NUM>-<NUM> membered heteroaryl, and <NUM>-<NUM> membered heterocyclyl, as defined herein).

As used herein, "aryl" refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent carbon atoms) containing only carbon in the ring backbone. When the aryl is a ring system, every ring in the system is aromatic. The aryl group may have <NUM> to <NUM> carbon atoms, which may be designated as C6-<NUM>. Examples of aryl groups include phenyl, naphthyl, azulenyl, and anthracenyl.

An "azide" or "azido" functional group refers to -N<NUM>.

As used herein, the term "attached" refers to the state of two things being joined, fastened, adhered, connected or bound to each other. For example, a nucleic acid can be attached to a material, such as the gel material, 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.

As used herein, "carbocyclyl" means. When the carbocyclyl is a ring system, two or more rings may be joined together in a fused, bridged or spiro-connected fashion. Carbocyclyls may have any degree of saturation, provided that at least one ring in a ring system is not aromatic. Thus, carbocyclyls include cycloalkyls, cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have <NUM> to <NUM> carbon atoms (i.e., C3-<NUM>).

As used herein, "cycloalkenyl" or "cycloalkane" means a carbocyclyl ring or ring system having at least one double bond, wherein no ring in the ring system is aromatic. Examples include cyclohexenyl or cyclohexene and norbornene or norbornyl
<CHM>
Also as used herein, "heterocycloalkenyl" or "heterocycloalkene" means a carbocyclyl ring or ring system with at least one heteroatom in ring backbone, having at least one double bond, wherein no ring in the ring system is aromatic.

As used herein, "cycloalkynyl" or "cycloalkyne" means a carbocyclyl ring or ring system having at least one triple bond, wherein no ring in the ring system is aromatic. An example is cyclooctyne
<CHM>
Another example is bicyclononyne (i.e., a bicyclic ring system, such as
<CHM>
Also as used herein, "heterocycloalkynyl" or "heterocycloalkyne" means a carbocyclyl ring or ring system with at least one heteroatom in ring backbone, having at least one triple bond, wherein no ring in the ring system is aromatic.

The term "chemical cleavage," as used herein, refers to a chemical reaction that removes the quality control tracer or a portion thereof from a support.

As used herein, the term "cleavable nucleotide sequence" refers to a single stranded nucleic acid sequence that can be broken at an excision site or at a linker molecule.

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.

As used herein, the term "enzymatic cleavage" refers to a process that utilizes an endonuclease or an exonuclease to remove the quality control tracer or a portion thereof from the support.

The term "excision site," as used herein, refers to a nucleotide or a base of a nucleotide (i.e., nucleobase) that is targeted by an enzyme. A quality control tracer or a portion thereof can be cleaved at the excision site. Such cleavage may involve a single enzymatic step or multiple enzymatic steps (e.g., base modification or excision followed by cleavage).

The term "fluorescent label," as used herein, refers to a fluorophore that is chemically attached to a nucleotide sequence. The fluorescent label may be attached to the <NUM>' terminus of the nucleotide sequence, to a cleavable nucleobase of a non-reactive nucleotide sequence, or to a linker molecule that is attached to the nucleotide sequence.

As used herein, the term "gel material" is intended to mean a semi-rigid material that is permeable to liquids and gases. Typically, the gel material is a hydrogel that can swell when liquid is taken up and can contract when liquid is removed by drying.

As used herein, the term "grafted" is intended to mean covalently bound and not attached solely via non-covalent interactions, such as hybridization. In some instances, the quality control tracer, cleavable nucleotide sequence, non-reactive nucleotide sequence, and/or primer or primer nucleotide sequence are grafted to the gel material by formative of covalent bonds between functional groups on the tracer or sequence with functional groups in the gel material. As used herein, the term "co-graft" refers to grafting of more than one entity.

As used herein, "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent atoms) that contain(s) one or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur, in the ring backbone. When the heteroaryl is a ring system, every ring in the system is aromatic. The heteroaryl group may have <NUM>-<NUM> ring members.

As used herein, "heterocyclyl" means a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring backbone. Heterocyclyls may be joined together in a fused, bridged or spiro-connected fashion. Heterocyclyls may have any degree of saturation provided that at least one ring in the ring system is not aromatic. In the ring system, the heteroatom(s) may be present in either a non-aromatic or aromatic ring. The heterocyclyl group may have <NUM> to <NUM> ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms). The heterocyclyl group may be designated as "<NUM>-<NUM> membered heterocyclyl" or similar designations. In some examples, the heteroatom(s) are O, N, or S.

The term "hydrazine" or "hydrazinyl" as used herein refers to a -NHNH<NUM> group.

As used herein, the term "hydrazone" or "hydrazonyl" as used herein refers to a group Ra(Rb)C=N-NH<NUM>, in which Ra and Rb are previously defined herein.

As used herein, "hydroxyl" is an -OH group.

As used herein, the term "interstitial region" refers to an area in a substrate/support or on a surface that separates other areas of the substrate or surface. For example, an interstitial region can separate one feature of an array from another feature of the array. The two features that are separated from each other can be discrete, i.e., lacking contact with each other. In another example, an interstitial region can separate a first portion of a feature from a second portion of a feature. In many examples, the interstitial region is continuous whereas the features are discrete, for example, as is the case for a plurality of wells defined in an otherwise continuous surface. The separation provided by an interstitial region can be partial or full separation. Interstitial regions may have a surface material that differs from the surface material of the features defined in the surface. For example, features of an array can have an amount or concentration of gel material and a quality control tracer that exceeds the amount or concentration present at the interstitial regions. In some examples, gel material and quality control tracer(s) may not be present at the interstitial regions.

The term "linker molecule," as used herein, refers to a molecule that includes, at one end, a functional group that can attach to the detectable or fluorescent label and, at the other end, a functional group that can attach to a nucleotide sequence. The attachment points are covalent bonds. Similarly, the term "linked," as used herein, refers to two entities that are connected via one or more covalent bonds, either directly or via a linker molecule.

"Nitrile oxide," as used herein, means a "RaC≡N+O-" group in which Ra is selected from hydrogen, C1-<NUM> alkyl, C2-<NUM> alkenyl, C2-<NUM> alkynyl, C3-<NUM> carbocyclyl, C6-<NUM> aryl, <NUM>-<NUM> membered heteroaryl, and <NUM>-<NUM> membered heterocyclyl. Examples of preparing nitrile oxide include in situ generation from aldoximes by treatment with chloramide-T or through action of base on imidoyl chlorides [RC(Cl)=NOH].

"Nitrone," as used herein, means a "RaRbC=NRc+O-" group in which Ra and Rb are previously defined herein and Rc is selected from C1-<NUM> alkyl, C2-<NUM> alkenyl, C2-<NUM> alkynyl, C3-<NUM> carbocyclyl, C6-<NUM> aryl, <NUM>-<NUM> membered heteroaryl, and <NUM>-<NUM> membered heterocyclyl, as defined herein.

As used herein, a "nucleotide" includes a nitrogen containing heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are monomeric units of a nucleic acid sequence. In RNA, the sugar is a ribose, and in DNA a deoxyribose, i.e. a sugar lacking a hydroxyl group that is present at the <NUM>' position in ribose. The nitrogen containing heterocyclic base (i.e., nucleobase) can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof. The C-<NUM> atom of deoxyribose is bonded to N-<NUM> of a pyrimidine or N-<NUM> of a purine.

The "non-reactive nucleotide sequence" referred to herein, when in accordance with the invention, is any nucleic acid sequence that does not actively participate in a particular DNA or RNA synthesis that is being performed. In some examples, the non-reactive nucleotide sequence may make up a portion of a quality control tracer. For example, the non-reactive nucleotide sequence may be a poly T sequence or a poly A sequence that is part of a cleavable nucleotide sequence that also includes an excision site. For another example, the non-reactive nucleotide sequence may be orthogonal to the primer nucleotide sequence(s) that is/are being used, and thus the non-reactive nucleotide sequence will not participate in the DNA or RNA synthesis which utilizes the primer nucleotide sequence(s).

As used herein, "predetermined ratio" refers to a ratio between two compounds in a mixture. The ratio is determined before the mixture is prepared. The ratio is the ratio of concentrations or molarities of the components in the mixture, or is the ratio of volumes of solutions of the two components that are blended to make the mixture. In some aspects, the "predetermined ratio" refers to the ratio of tracer and primer on the gel material, and in such cases, the ratio is based on the ratio of components reacted with the gel material and optionally takes into account any differential reactivity of the two components. In some aspects, the predetermined ratio for a mixture of the tracer and the primer is set at a percent volume of the primer (or primer mixture, where more than one primer sequence is used).

As used herein, the "primer nucleotide sequence" is defined as a single stranded nucleic acid sequence (e.g., single strand DNA or single strand RNA) that serves as a starting point for DNA or RNA synthesis. The <NUM>' terminus of the sequencing primer may be modified to allow a coupling reaction with a gel material. The sequencing primer length can be any number of bases long and can include a variety of non-natural nucleotides. In an example, the sequencing primer is a short strand, including from <NUM> bases to <NUM> bases.

The term "untagged," as used herein, means that a nucleotide sequence does not have a fluorescent label attached thereto.

A "quality control tracer" includes a nucleotide sequence and a fluorescent label attached to the nucleotide sequence. The fluorescent label of the quality control tracer is capable of being detected in a quality control method. The fluorescent label of the quality control tracer is also capable of being removed, and the remaining portion of the tracer is either capable of participating in a sequencing method or is non-reactive during a sequencing method.

As used herein, a "site" refers to a location defined on or in a support where the gel material and a quality control tracer may be attached.

The terms "substrate" and "support" are used interchangeably herein, and refer to a surface in which or on which the site is located. The support is generally rigid and is insoluble in aqueous liquid. The support may be inert to a chemistry that is used to modify the gel material. For example, a support can be inert to chemistry used to attach the quality control tracer, to the gel material in a method set forth herein. Examples of suitable supports include glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene (such as TEFLON® from Chemours), cyclic olefins/cyclo-olefin polymers (COP) (such as ZEONOR® from Zeon), polyimides, etc.), nylon, ceramics, silica or silica-based materials, siloxanes, silicon and modified silicon, carbon, metals, inorganic glasses, and optical fiber bundles.

A "thiol" functional group refers to -SH (e.g.,
<CHM>.

As used herein, the terms "tetrazine" and "tetrazinyl" refer to six-membered heteroaryl group comprising four nitrogen atoms. Tetrazine can be optionally substituted.

"Tetrazole," as used herein, refer to five-membered heterocyclic group including four nitrogen atoms. Tetrazole can be optionally substituted.

As used herein, the term "well" refers to a discrete concave feature in a support having a surface opening that is completely surrounded by interstitial region(s) of the support surface. Wells can have any of a variety of shapes at their opening in a surface including, as examples, round, elliptical, square, polygonal, star shaped (with any number of vertices), etc. The cross-section of a well taken orthogonally with the surface can be curved, square, polygonal, hyperbolic, conical, angular, etc..

Examples of the arrays disclosed herein include several sites, each of which has an example of a quality control tracer attached to a gel material. The quality control tracer includes either a primer nucleotide sequence or is present in a predetermined ratio with the primer nucleotide sequence, and thus may be used in a quality control method to determine the density and/or distribution of the primer nucleotide sequence. The quality control tracer includes a fluorescent label that can be used in the quality control method and that can be cleaved from the tracer so that it does not interfere with sequencing. The inclusion of the quality control tracer in some instances alleviates the need to perform hybridization and dehybridization for quality control purposes. The examples disclosed herein enable the density and/or distribution of primer nucleotide sequences on a support to be assessed without having to load sequencing reagents and samples and without having to perform initial steps in a sequencing workflow.

The aspects of the invention and examples set forth herein and recited in the claims can be understood in view of the above definitions.

Referring now to <FIG>, an example of the array <NUM> is depicted. In general, the array <NUM> includes a substrate or support <NUM> and lines or flow channels <NUM> across the support <NUM>. Each of the flow channels <NUM> includes multiple sites <NUM>, which are separated from one another by interstitial regions <NUM>. At each site <NUM>, at least quality control tracers <NUM> are attached to the gel material (<NUM>, <NUM>', for example, in <FIG>). In some instances, in addition to the quality control tracers <NUM>, separate primer nucleotide sequence(s) <NUM>, <NUM>' are also attached to the gel material <NUM>.

The array <NUM> illustrated in <FIG> and discussed herein may be disposed in or formed as a part of a flow cell, which is a chamber including a solid surface across which various carrier fluids, reagents, and so forth may be flowed. In an example, the flow cell may include the array <NUM> bonded to a top substrate through a sealing material (e.g., black polyimide or another suitable bonding material). The bonding takes place in bonding regions of the support <NUM>, the sealing material, and the top substrate. The bonding regions may be located between the flow channels so that the sealing material physically separates one flow channel <NUM> from an adjacent flow channel <NUM> (to prevent cross-contamination) and may be located at the periphery of the flow cell (to seal the flow cell from external contamination). It is to be understood, however, that the bonding regions and the sealing material may be located in any desired region depending on the implementation. Bonding may be accomplished via laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma activation bonding, glass frit bonding, or others methods known in the art.

Other examples of flow cells and related fluidic systems and detection platforms that can be integrated with the array <NUM> and/or readily used in the methods of the present disclosure are described, for example, in <NPL>), <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; and <CIT>, and <CIT>.

In some applications, the flow cell is used to perform controlled chemical or biochemical reactions in a reaction automation device, such as in a nucleotide sequencer. Ports <NUM> may be drilled through the support <NUM>. By connecting to ports <NUM>, the reaction automation device may control the flow of reagent(s) and product(s) in the sealed flow channels <NUM>. The reaction automation device may, in some applications, adjust the pressure, temperature, gas composition and other environmental conditions of the flow cell. Further, in some applications, ports <NUM> may be drilled in the top substrate or in both the support <NUM> and the top substrate. The reactions taking place in sealed flow channels <NUM> may be monitored through the top substrate and/or the support <NUM> by imaging or measurements of heat, light emission and/or fluorescence.

It is to be understood that the particular orientation of the flow channels <NUM>, the sites <NUM>, etc. may differ from those illustrated in <FIG>. In some examples, the sites <NUM> are contiguous and thus need not be separated by interstitial regions <NUM>.

The array <NUM> of <FIG>, and examples of how the array <NUM> can be made, will now be described in more detail in reference to <FIG>.

<FIG> depicts the support <NUM> having sites <NUM> defined therein and separated by interstitial regions <NUM>. This support <NUM> has a patterned surface. A "patterned surface" refers to an arrangement of different regions (i.e., sites <NUM>) in or on an exposed layer of the solid support <NUM>. For example, one or more of the sites <NUM> can be features where one or more quality control tracers <NUM>, and, in some instances, separate primer nucleotide sequence(s) <NUM> are present. The features can be separated by the interstitial regions <NUM>, where quality control tracers <NUM> and separate primer nucleotide sequence(s) <NUM> are not present. Many different layouts of the sites <NUM> may be envisaged, including regular, repeating, and non-regular patterns. In an example, the sites <NUM> are disposed in a hexagonal grid for close packing and improved density. Other layouts may include, for example, rectilinear (i.e., rectangular) layouts, triangular layouts, and so forth. As examples, the layout or pattern can be an x-y format of sites <NUM> that are in rows and columns. In some other examples, the layout or pattern can be a repeating arrangement of sites <NUM> and/or interstitial regions <NUM>. In still other examples, the layout or pattern can be a random arrangement of sites <NUM> and/or interstitial regions <NUM>. The pattern may include spots, pads, wells, posts, stripes, swirls, lines, triangles, rectangles, circles, arcs, checks, plaids, diagonals, arrows, squares, and/or cross-hatches. Still other examples of patterned surfaces that can be used in the examples set forth herein are described in <CIT>; <CIT>; and <CIT>, and <CIT>,.

The layout or pattern may be characterized with respect to the density of the sites <NUM> (i.e., number of sites <NUM>) in a defined area. For example, the sites <NUM> may be present at a density of approximately <NUM> million per mm<NUM>. The density may be tuned to different densities including, for example, a density of at least about <NUM> per mm<NUM>, about <NUM>,<NUM> per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, or more. Alternatively or additionally, the density may be tuned to be no more than about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM>,<NUM> per mm<NUM>, about <NUM> per mm<NUM>, or less. It is to be further understood that the density of sites <NUM> on the support <NUM> can be between one of the lower values and one of the upper values selected from the ranges above. As examples, a high density array may be characterized as having sites <NUM> separated by less than about <NUM>, a medium density array may be characterized as having sites <NUM> separated by about <NUM> to about <NUM>, and a low density array may be characterized as having sites <NUM> separated by greater than about <NUM>.

The layout or pattern may also or alternatively be characterized in terms of the average pitch, i.e., the spacing from the center of the site <NUM> to the center of an adjacent interstitial region <NUM> (center-to-center spacing). The pattern can be regular such that the coefficient of variation around the average pitch is small, or the pattern can be non-regular in which case the coefficient of variation can be relatively large. In either case, the average pitch can be, for example, at least about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or more. Alternatively or additionally, the average pitch can be, for example, at most about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or less. The average pitch for a particular pattern of sites <NUM> can be between one of the lower values and one of the upper values selected from the ranges above. In an example, the sites <NUM> have a pitch (center-to-center spacing) of about <NUM>.

In some examples, the sites <NUM> are wells <NUM>', and thus the support <NUM> includes an array of wells <NUM>' in a surface thereof. The wells <NUM>' (or other sites <NUM> with different configurations, such as shape, cross-section, etc.) may be fabricated using a variety of techniques, including, for example, photolithography, nanoimprint lithography, stamping techniques, embossing techniques, molding techniques, microetching techniques, printing techniques, etc. As will be appreciated by those in the art, the technique used will depend on the composition and shape of the support <NUM>.

The wells <NUM>' may be micro wells (having at least one dimension on the micron scale, e.g., about <NUM> to about <NUM>) or nanowells (having at least one dimension on the nanoscale, e.g., about <NUM> to about <NUM>). Each well <NUM>' may be characterized by its volume, well opening area, depth, and/or diameter.

Each well <NUM>' can have any volume that is capable of confining a liquid. The minimum or maximum volume can be selected, for example, to accommodate the throughput (e.g. multiplexity), resolution, analyte composition, or analyte reactivity expected for downstream uses of the array <NUM>. For example, the volume can be at least about <NUM>×<NUM>-<NUM> µm<NUM>, about <NUM>×<NUM>-<NUM> µm<NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, or more. Alternatively or additionally, the volume can be at most about <NUM>×<NUM><NUM> µm<NUM>, about <NUM>×<NUM><NUM> µm<NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, or less. It is to be understood that the gel material <NUM> can fill all or part of the volume of a well <NUM>'. The volume of the gel material <NUM> in an individual well <NUM>' can be greater than, less than or between the values specified above.

The area occupied by each well opening on a surface can be selected based upon similar criteria as those set forth above for well volume. For example, the area for each well opening on a surface can be at least about <NUM>×<NUM>-<NUM> µm<NUM>, about <NUM>×<NUM>-<NUM> µm<NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, or more. Alternatively or additionally, the area can be at most about <NUM>×<NUM><NUM> µm<NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM>×<NUM>-<NUM> µm<NUM>, or less.

The depth of each well <NUM>' can be at least about <NUM>, about <NUM>, about <NUM>, about <NUM>, or more. Alternatively or additionally, the depth can be at most about <NUM>×<NUM><NUM> µm, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or less.

In some instances, the diameter of each well <NUM>' can be at least about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or more. Alternatively or additionally, the diameter can be at most about <NUM>×<NUM><NUM> µm, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or less.

In the array <NUM> that is formed, the gel material <NUM> is positioned in each of the discrete wells <NUM>'. Positioning the gel material <NUM> in each well <NUM>' may be accomplished by first coating the patterned surface of the support <NUM> with the gel material <NUM>, as shown in <FIG>, and then removing the gel material <NUM>, for example via chemical or mechanical polishing, from at least the interstitial regions <NUM> on the surface of the structured support <NUM> between the wells <NUM>'. These processes retain at least some of the gel material <NUM> in the wells <NUM>' but remove or inactivate at least substantially all of the gel material <NUM> from the interstitial regions <NUM> on the surface of the structured support <NUM> between the wells <NUM>'. As such, these processes create gel pads <NUM>' (<FIG>) used for sequencing that can be stable over sequencing runs with a large number of cycles.

Particularly useful gel materials <NUM> will conform to the shape of the site <NUM> where it resides. Some useful gel materials <NUM> can both (a) conform to the shape of the site <NUM> (e.g., well <NUM>' or other concave feature) where it resides and (b) have a volume that does not at least substantially exceed the volume of the site <NUM> where it resides.

One example of a suitable gel material <NUM> includes a polymer represented by Formula (I):
<CHM>
wherein:.

In the structure of Formula (I), one of ordinary skill in the art will understand that the "n" and "m" subunits are recurring subunits that are present in a random order throughout the polymer. One of ordinary skill will also recognize that other monomeric components may be present in the polymer.

A particular example of a gel material <NUM> is poly(N-(<NUM>-azidoacetamidylpentyl)acrylamide-co-acrylamide ("PAZAM") (described, for example, <CIT> and <CIT>), which comprises the structure shown below:
<CHM>
wherein n is an integer in the range of <NUM>-<NUM>,<NUM>, and m is an integer in the range of <NUM>-<NUM>,<NUM>. As with Formula (I), one of ordinary skill in the art will recognize that the "n" and "m" subunits are recurring units that are present in random order throughout the polymer structure.

The molecular weight of the PAZAM may range from about <NUM> kDa to about <NUM> kDa, or may be, in a specific example, about <NUM> kDa.

In some examples, PAZAM is a linear polymer. In some other embodiments, PAZAM is a lightly cross-linked polymer. In other examples, PAZAM comprises branching.

Other examples of suitable gel materials <NUM> include those having a colloidal structure, such as agarose; or a polymer mesh structure, such as gelatin; or a cross-linked polymer structure, such as polyacrylamide polymers and copolymers, silane free acrylamide (SFA, see, for example, <CIT>, or an azidolyzed version of SFA. Examples of suitable polyacrylamide polymers may be formed from acrylamide and an acrylic acid or an acrylic acid containing a vinyl group as described, for example, in <CIT> or from monomers that form [<NUM>+<NUM>] photo-cycloaddition reactions, for example, as described in <CIT> or <CIT>.

The gel material <NUM> may be a preformed gel material. Preformed gel materials may be coated using spin coating, or dipping, or flow of the gel under positive or negative pressure, or techniques set forth in <CIT>. Dipping or dip coating may be a selective deposition technique, depending upon the support <NUM> and the gel material <NUM> that are used. As an example, the patterned support <NUM> is dipped into a preformed gel material <NUM>, and the gel material <NUM> may fill the sites <NUM> selectively (i.e., the gel material <NUM> does not deposit on the interstitial regions <NUM>), and polishing (or another removal process) may not be necessary.

Preformed PAZAM may be coated on the patterned support <NUM> using spin coating, or dipping, or flow of the gel under positive or negative pressure, or techniques set forth in <CIT>. The attachment of PAZAM may also take place by chemical reaction to form a covalent bond, or via a surface initiated atom transfer radical polymerization (SI-ATRP) to a silanized surface.

In some examples, the support surface is treated with an alkene-derivatized silane, wherein the alkene portion may be linear, branched, or cyclic. In some examples, the silane reagent is (RO)<NUM>Si-Linker-Alkene, and in other examples, the silane reagent is (RO)<NUM>Si-C<NUM>-<NUM>alkylene-cycloalkene, and in other examples, the silane reagent is (RO)<NUM>Si-CH<NUM>CH<NUM>-norbornene, where each R is a C<NUM>-<NUM>alkyl or is methyl or ethyl. The gel material <NUM>, such as PAZAM, is covalently bound to the alkene-derivatized silanes under thermal or uv conditions.

In other examples, the support <NUM> surface may be pre-treated with an amino-derivatized silane, such as an aminopropyl-trialkoxysilane (APTS), for example <NUM>-aminopropyltrimethoxysilane (APTMS) or <NUM>-aminopropyl-triethoxysilane (APTES) to covalently link silicon to one or more oxygen atoms on the surface (without intending to be held by mechanism, each silicon may bond to one, two or three oxygen atoms). This chemically treated surface is baked to form an amine group monolayer. The amine groups are then reacted with Sulfo-HSAB to form an azido derivative. UV activation at <NUM> with <NUM> J/cm<NUM> to <NUM> J/cm<NUM> of energy generates an active nitrene species, which can readily undergo a variety of insertion reactions with the PAZAM.

Other examples for coating PAZAM on the support <NUM> are described in <CIT>, and include ultraviolet (UV) mediated linking of PAZAM monomers to an amine-functionalized surface, or a thermal linkage reaction involving an active group (acryloyl chloride or other alkene or alkyne-containing molecule) with subsequent deposition of PAZAM and application of heat.

The gel material <NUM> may be formed by applying a liquid that subsequently forms the gel material <NUM>. An example of applying liquid that subsequently forms the gel material <NUM> is the coating of an array of sites <NUM> with silane-free acrylamide and N-[<NUM>-(<NUM>-bromoacetyl) aminopentyl]acrylamide (BRAPA) in liquid form and allowing the reagents to form a gel by polymerization on the surface. Coating of an array in this way can use chemical reagents and procedures as set forth in <CIT>.

The gel material <NUM> may be covalently linked to the support <NUM> (at the sites <NUM>) or may not be covalently linked to the support <NUM>. The covalent linking of the polymer to the sites <NUM> is helpful for maintaining the gel in the structured sites <NUM> throughout the lifetime of the array <NUM> during a variety of uses. However, as noted above and in many examples, the gel material <NUM> need not be covalently linked to the sites <NUM>. For example, silane free acrylamide, SFA, is not covalently attached to any part of the support <NUM>.

As mentioned above, <FIG> illustrates the removal of the gel material <NUM> from the interstitial regions <NUM>. Removal may be accomplished via polishing. Polishing may be a mechanical and/or chemical treatment process.

Mechanical polishing can be carried out by applying abrasive forces to the surface of the solid support <NUM> (having the gel material <NUM> thereon). Example methods include abrasion with a slurry of beads, wiping with a sheet or cloth, scraping, or the like. It will be understood that beads used for polishing may or may not be spherical, and can have irregular shapes, polygonal shapes, ovoid shapes, elongated shapes, cylindrical shapes, etc. The surface of the beads can be smooth or rough. Any of a variety of particles can be useful as beads for polishing. One example of polishing includes using a lintless (cleanroom grade) wipe coated with a <NUM> silica bead slurry (<NUM>% w/v in water) to remove the gel material <NUM> from the interstitial regions <NUM>. A polishing wheel/grinder can also be used with this slurry. Mechanical polishing can also be achieved using a fluid jet or gas (e.g., air or inert gas such as Argon or Nitrogen) jet to remove gel from interstitial regions <NUM>.

Chemical polishing techniques, such as hydrolysis or radical-based degradation of acrylamide (e.g. via exposure to benzoyl peroxide or dilute hydrogen peroxide) may be used. During this form of polishing, the chemicals can be provided in a solid, liquid, gas or plasma state. Accordingly, plasma polishing can be useful in some examples.

Polishing can also involve a combination of chemical and mechanical polishing methods, where a chemical slurry containing a colloidal suspension of particles is used to mechanically exfoliate and then chemically dissolve displaced portions of gel material <NUM> from interstitial regions <NUM>.

Other methods to polish or clean the interstitial regions <NUM> include adhesive based techniques, for example, techniques wherein a rigid, planar adhesive film with affinity to the gel material <NUM> is applied, thereby making intimate contact (e.g., via chemical linkage) with the gel material <NUM> in interstitial regions <NUM>. The mechanical removal/peeling of this adhesive film will result in the mechanical removal of the gel material <NUM> from interstitial regions <NUM>, while leaving gel material <NUM> in the sites <NUM>.

In one example, thiophosphate-grafted SFA can be removed from interstitial regions <NUM> on a support <NUM> surface as follows: a water-dampened Whatman wipe can be dabbed into aluminum oxide (~<NUM>, <NUM>) or steel beads, and then the formed slurry can be rubbed on the surface of the support (having the thiophosphate-grafted SFA thereon), in small concentric circles, using even pressure, and then a clean water-wet Whatman wipe can be used to remove the slurry and the thiophosphate-grafted SFA from the surface.

The mechanical and chemical polishing methods exemplified herein for removing gel material <NUM> from interstitial regions <NUM> can also be used to inactivate gel material at interstitial regions <NUM>, whether or not the gel material <NUM> is removed. For example, the gel material <NUM> can be inactivated with respect to the ability to attach the quality control tracers <NUM> and separate primer nucleotide sequence(s) <NUM>, <NUM>'.

After the gel material <NUM> is positioned in each well <NUM>', the quality control tracers <NUM>, and, in some instances separate primer nucleotide sequence(s) <NUM>, <NUM>' are grafted to the gel material <NUM>. The attachment technique that is used, and whether separate, primer nucleotide sequences <NUM>, <NUM>' are included will depend, in part, upon the quality control tracer <NUM> that is utilized. Various examples of the quality control tracer <NUM> are shown in <FIG> and in <FIG>. Each example will now be described.

<FIG> depicts an example of the quality control tracer 22A that is included on the gel material <NUM>/gel pad <NUM>' with separate primer nucleotide sequence(s) <NUM>, <NUM>'. In this example, the quality control tracer 22A is a cleavable nucleotide sequence <NUM> tagged, at its <NUM>' end, with a fluorescent label <NUM>, and the primer nucleotide sequence(s) <NUM>, <NUM> are untagged primer nucleotide sequence(s).

The cleavable nucleotide sequence <NUM> of <FIG> may include a functional group at its <NUM>' end that is capable of attaching to the gel material <NUM>. Examples of this functional group include an alkyne, a norbornyl (or other cycloalkenyls), a copper free click moiety (e.g., dibenzocyclooctyne (DIBO), other cycloalkynes, or others), and a thiol. This functional group may be selected based upon the gel material <NUM> that is used. For example, alkynes, norbornyls, and copper free click moieties may react with azides of PAZAM via click reactions, and thiols may react with SFA. In some embodiments, the functional group is an alkyne.

The cleavable nucleotide sequence <NUM> of <FIG> may also include a functional group at its <NUM>' end that is capable of attaching to the fluorescent label <NUM>. An example of this functional group, which can be used during oligonucleotide synthesis, is <NUM>'-Dimethoxytrityl-<NUM>-[N-(trifluoroacetylaminohexyl)-<NUM>-acrylimido]-<NUM>'-deoxyUridine,<NUM>'-[(<NUM>-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (i.e., amino modifier C6 dT). Other examples include <NUM>'-Dimethoxytrityl-N-dimethylformamidine-<NUM>-[N-(trifluoroacetylaminohexyl)-<NUM>-acrylimido]-<NUM>'-deoxyCytidine,<NUM>'-[(<NUM>-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (amino modifier C6 dC) and the use of solid supports such as (<NUM>-Dimethoxytrityloxymethyl-<NUM>-fluorenylmethoxycarbonylamino-hexane-<NUM>-succinoyl)-long chain alkylamino-CPG. The functional group at the <NUM>' end may be selected based upon the fluorescent label <NUM> that is used.

The detectable or fluorescent label <NUM> may be any suitable fluorophore that can attach to the cleavable nucleotide sequence <NUM>, e.g., at the <NUM>' end (or at or near the <NUM>' end). Examples of suitable fluorescent labels <NUM> include Texas Red® (a sulfonyl chloride dye, ThermoFisher Scientific), Cy7®, Cy7. <NUM>® or sulfo-Cyanine7 NHS ester (cyanine dyes from Lumiprobe), a red wavelength dye, such as TEX™ <NUM> (Exiqon), fluorescent dyes , such as those in the Alexa Fluor® series (ThermoFisher Scientific), Atto dyes (e.g., Atto <NUM>, the structure of which is:
<CHM>
the Atto-Tec series, from Atto-Tec), FAM™ dyes (derivatives of fluorescein, Integrated DNA Technologies), xanthene fluorophores, such as CAL Fluor® dyes (e.g., CAL Fluor® Gold <NUM>, CAL Fluor® Orange <NUM>, CAL Fluor® Red <NUM>, CAL Fluor® Red <NUM>, and CAL Fluor® Red <NUM>, from LGC Biosearch Technologies), indocarbocyanine dyes, such as Quasar® dyes (e.g., Quasar® <NUM>, Quasar® <NUM>, and Quasar® <NUM>, from LGC Biosearch Technologies), or any other suitable fluorophore known to those of ordinary skill in the art. Other examples include Dylight™<NUM> (an amine reactive dye), or a fluorophore with an emission maximum of approximately <NUM>. In some examples, the fluorescent label is a xanthene fluorophore. In some examples, the xanthene fluorophore has an emission maximum in the range of <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>. In other examples, the xanthene fluorophore has an emission maximum in the range of <NUM> to <NUM>. In others , the xanthene fluorophore emits in the red region of the spectrum. In others , the xanthene fluorophore has an emission maximum of approximately <NUM>, or <NUM>, or <NUM>. Other suitable detectable labels include non-fluorescent labels, such as plasmonic nanoparticles (detected by, e.g., SPR sensing) or quantum dots. In some examples, the detectable label is derivatized with an amino-reactive group such as an NHS to allow for coupling to an oligonucleotide sequence.

The fluorescent label <NUM> may be attached to the cleavable nucleotide sequence <NUM> using any suitable method, such as template directed ligation, polymerase-mediated oligonucleotide elongation, chemical synthesis, etc. The attachment of the fluorescent label <NUM> may take place during nucleotide synthesis (e.g., using mutant DNA polymerases which allow for synthesis of a complementary, fluorophore-labeled DNA or using fluorescent label-modified monomers during solid phase oligonucleotide synthesis) or after nucleotide synthesis (e.g., via coupling chemistry to conjugate the label <NUM> onto a previously installed functional group located at the <NUM>' position).

In this example of the invention the cleavable nucleotide sequence <NUM> includes a cleavable portion <NUM> and a remaining portion <NUM>. The cleavable portion <NUM> includes the fluorescent label <NUM> (and any functional group used to attached the fluorescent label <NUM>), any sequence of nucleotides, and an excision site <NUM>. As such, the cleavable portion <NUM> of the cleavable nucleotide sequence <NUM> can be removed when the sequence <NUM> is exposed to an enzyme that targets the nucleotide located at the excision site <NUM>. The portion <NUM> of the cleavable nucleotide sequence <NUM> that remains attached to the gel material <NUM> after enzymatic cleavage is a non-reactive nucleotide sequence, and thus will not participate in or interfere with a sequencing operation that is to be performed or is being performed. The remaining portion may be a short poly T or poly A sequence, or may be a sequence that is orthogonal to the primer nucleotide sequence(s) <NUM>, <NUM>' also attached to the gel material <NUM>.

The quality control tracer 22A in <FIG> is used (e.g., in examples of the quality control method disclosed herein) in combination with separate primer nucleotide sequence(s) <NUM>, <NUM>'. Examples of suitable primer nucleotide sequence(s) <NUM>, <NUM>' include forward amplification primers or reverse amplification primers for hybridization to a complementary sequence and amplification of a sequence. Some specific examples of suitable primer nucleotide sequence(s) <NUM>, <NUM>' include P5 and/or P7 primers. The P5 and P7 primers are used on the surface of commercial flow cells sold by Illumina, Inc. , for sequencing on HiSeq®, HiSeqX®, MiSeq®, NextSeq®, Genome Analyzer®, and other instrument platforms. The P5 and P7 primers, as well as other sequencing primers <NUM>, <NUM>', may be modified at the <NUM>' end with a group that is capable of reacting with a functional group of the gel material <NUM>. One example of a suitable functional group is bicyclo[<NUM>. <NUM>] non-<NUM>-yne (BCN), which can react with an azide of the gel material <NUM>. Other examples of terminated primers include a tetrazine terminated primer, a norbornene terminated primer, an alkyne terminated primer, an amino terminated primer, an epoxy or glycidyl terminated primer, a thiophosphate terminated primer, and a triazolinedione terminated primer. Examples of the P5 and P7 primers, which may be alkyne terminated, include the following:.

and derivatives thereof. In some examples, the P7 sequence includes a modified guanine at the G* position, e.g., an <NUM>-oxo-guanine. In other examples, the * indicates that the bond between the G* and the adjacent <NUM>' A is a phosphorothioate bond. In some examples, the P5 and/or P7 primers include unnatural linkers.

Optionally, one or both of the P5 and P7 primers can include a poly T tail. The poly T tail is generally located at the <NUM>' end of the sequence (e.g., between the <NUM>' terminal base and the alkyne unit), but in some cases can be located at the <NUM>' end. The poly T sequence can include any number of T nucleotides, for example, from <NUM> to <NUM>.

While the P5 and P7 primers are given as examples, it is to be understood that any suitable amplification primers can be used in the examples presented herein. One of skill in the art will understand how to design and use primer nucleotide sequence(s) <NUM>, <NUM>' that are suitable for capture and amplification of nucleic acids as presented herein.

An example of a quality control tracer 22A is orthogonal to the P5 and P7 primer nucleotide sequence(s) <NUM>, <NUM>'. In some examples, the quality control tracer includes a uracil excision site.

In some examples, the quality control tracer includes one of the following sequences:.

where U is cleavage site. In some cases, U is a uracil excision site.

In some examples, the quality control tracer comprises a polyT sequence at the <NUM>' end of the sequence. In some examples, the polyT region comprises <NUM> to <NUM>, or <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> T nucleotides. In some examples, the polyT region comprises <NUM>, <NUM>, or <NUM> T bases. In other examples, the polyT region comprises <NUM> T nucleotides. In some examples, the sequence is:.

In some examples, the quality control tracer includes Texas Red® as the fluorescent label. In some examples, the quality control tracer is:.

In some examples, these tracers also include a polyT region between the alkyne and the rest of the sequence, as described herein.

In some examples, the quality control tracer is:.

In some examples, these tracers also include a polyT region at the <NUM>' end between the alkyne and the rest of the sequence, as described herein.

In some examples, the quality control tracer comprises an alkyne terminus, for example, a <NUM>'-hexyne terminus. Suitable termini are used to attach the tracer to the gel material.

In some examples, the quality control tracer is:
<NUM>'-alkyne-TTTTTTACATACATACATACATACAUACATACA[Amino C6 dT-xanthene fluorophore]-<NUM>' (SEQ. In some aexamples, the alkyne is a hexyne. In some examples, the U cleavage site is a uracil excision site. In some examples, the xanthene fluorophore has an emission maximum in the range of <NUM> to <NUM>. In other examples, the xanthene fluorophore emits in the red region of the spectrum. In other examples, the xanthene fluorophore has an emission maximum of approximately <NUM>, or <NUM>, or <NUM>. In some examples, the fluorophore is Texas Red®.

In some examples, where the quality control tracer comprises both a primer sequence and the detectable label, the quality control tracer includes one of the following sequences:.

In some examples, the quality control tracer includes a polyT region as described above. In some examples, the quality control tracer comprises one of the following sequences:.

In some examples, the quality control tracer includes a terminus that allows for grafting to the gel material. In some examples, the terminus is an alkyne or a hexyne moiety. Thus, the quality control tracer comprises one of the following sequences:.

In some examples, the quality control tracer comprises a xanthene fluorophore as described herein, or Texas Red. In some examples, the quality control tracer comprises one of the following sequences:.

When the quality control tracer 22A in <FIG> is used, it is to be understood that it is present in a predetermined ratio with respect to the separate primer nucleotide sequence(s) <NUM>, <NUM>'. The predetermined ratio can be used in the quality control method disclosed herein to indirectly determine the density and/or distribution of the separate primer nucleotide sequence(s) <NUM>, <NUM>'.

To form the example shown in <FIG>, sequential grafting or co-grafting may be used.

Sequential grafting may be accomplished by exposing the support <NUM> (having the gel material <NUM> in the sites <NUM>) to a solution or mixture containing the quality control tracer 22A and incubating, and then to a solution or mixture containing the primer nucleotide sequence(s) <NUM>, <NUM>' and incubating. Alternatively, sequential grafting may be accomplished by exposing the support <NUM> (having the gel material <NUM> in the sites <NUM>) to a solution or mixture containing the primer nucleotide sequence(s) <NUM>, <NUM>' and incubating, and then to a solution or mixture containing the quality control tracer 22A and incubating.

Co-grafting may be accomplished by exposing the support <NUM> (having the gel material <NUM> in the sites <NUM>) to a solution or mixture containing the quality control tracer 22A and the primer nucleotide sequence(s) <NUM>, <NUM>', and then incubating. Exposure of the support <NUM> to this solution or mixture may be accomplished by depositing a mixture of the quality control tracer 22A and the primer nucleotide sequence(s) <NUM>, <NUM>' onto the support <NUM>. In an example, the solution or mixture may be drawn across the gel material <NUM> coated support <NUM> (shown in <FIG>).

In any of the grafting examples used to form the example shown in <FIG>, incubation takes place at a predetermined temperature which depends, in part, upon the quality control tracer 22A and the primer nucleotide sequence(s) <NUM>, <NUM>' used. As examples, incubation may be accomplished at a temperature ranging from about <NUM>° to about <NUM>.

Also in any of the grafting examples used to form the example shown in <FIG>, the solution may include the quality control tracer 22A and/or the primer nucleotide sequence(s) <NUM>, <NUM>', water, a buffer, and a catalyst. The quality control tracer 22A and/or the primer nucleotide sequence(s) <NUM>, <NUM>', whether present in the same solution/mixture or separate solutions/mixtures, may be present at any desired predetermined ratio.

<FIG> depicts another example of the quality control tracer 22B that is included on the gel material <NUM>/gel pad <NUM>' with separate primer nucleotide sequence(s) <NUM>, <NUM>'. In this example, the quality control tracer 22B is a non-reactive nucleotide sequence <NUM> with the fluorescent label <NUM> attached to a cleavable nucleobase, and the primer nucleotide sequence(s) <NUM>, <NUM> are untagged primer nucleotide sequence(s).

The non-reactive nucleotide sequence <NUM> of this example of the quality control tracer 22B remains at least substantially intact after the quality control method is performed and while sequencing is performed, and thus the sequence <NUM> is orthogonal to the primer nucleotide sequence(s) <NUM>, <NUM>' also attached to the gel material <NUM>.

The non-reactive nucleotide sequence <NUM> of <FIG> may include a functional group at its <NUM>' end that is capable of attaching to the gel material <NUM>. Examples of this functional group include an alkyne, a norbornyl (or other cycloalkenyls), a copper free click moiety (e.g., dibenzocyclooctyne (DIBO), other cycloalkynes, or others), and a thiol. This functional group may be selected based upon the gel material <NUM> that is used. For example, alkynes, norbornyls, and copper free click moieties may react with azides of PAZAM via click reactions, and thiols may react with SFA.

In this example of the quality control tracer 22B, the fluorescent label <NUM> is attached to a cleavable nucleobase of the sequence <NUM>. The nucleobase to which the fluorescent label <NUM> is attached is one that can be cleaved by an exonuclease, which catalyzes the excision of the particular base, while leaving the phosphodiester backbone intact.

In this example, the fluorescent label <NUM> may be attached near (e.g., within <NUM> bases of) but not directly at, the <NUM>' end of the sequence <NUM>. Any of the previously described fluorescent labels <NUM> may be used, as long as the selected label can covalently attach to a desirable nucleobase of the non-reactive nucleotide sequence <NUM>.

The quality control tracer 22B in <FIG> is used in combination with separate primer nucleotide sequence(s) <NUM>, <NUM>'. Examples of suitable primer nucleotide sequence(s) <NUM>, <NUM>' include forward amplification primers or reverse amplification primers for hybridization to a complementary sequence and amplification of a sequence. Some specific examples of suitable primer nucleotide sequence(s) <NUM>, <NUM>' include the previously described P5 or P7 primers.

When the quality control tracer 22B in <FIG> is used, it is to be understood that it is present in a predetermined ratio with respect to the primer nucleotide sequence(s) <NUM>, <NUM>'. The predetermined ratio can be used in an example of the quality control method disclosed herein to indirectly determine the density and/or distribution of the primer nucleotide sequence(s) <NUM>, <NUM>'.

To form the example shown in <FIG>, sequential grafting or co-grafting may be used as previously described, except that the quality control tracer 22B is used instead of the quality control tracer 22A. In any of the grafting examples used to form the example shown in <FIG>, incubation takes place at a predetermined temperature which depends, in part, upon the quality control tracer 22B and the primer nucleotide sequence(s) <NUM>, <NUM>' used. Also in any of the grafting examples used to form the example shown in <FIG>, the solution may include the quality control tracer 22B and/or the primer nucleotide sequence(s) <NUM>, <NUM>', water, a buffer, and a catalyst. The quality control tracer 22B and/or the primer nucleotide sequence(s) <NUM>, <NUM>', whether present in the same solution/mixture or separate solutions/mixtures, may be present at any desired predetermined ratio.

<FIG> depicts still another example of the quality control tracer 22C that is included on the gel material <NUM>/gel pad <NUM>' with separate primer nucleotide sequence(s) <NUM>, <NUM>'. In this example, the quality control tracer 22C is a non-reactive nucleotide sequence <NUM>' with the fluorescent label <NUM> attached to a cleavable nucleobase (near the <NUM>' end) and with an excision site <NUM>, and the primer nucleotide sequence(s) <NUM>, <NUM> are untagged primer nucleotide sequence(s).

Since this example of the quality control tracer 22C includes the fluorescent label <NUM> attached to a cleavable nucleobase, an exonuclease may be used to remove the fluorescent label <NUM> after the quality control method has been performed. In this example, the non-reactive nucleotide sequence <NUM>' remains at least substantially intact after the quality control method has been performed, and thus the non-reactive nucleotide sequence <NUM>' may be orthogonal to the primer nucleotide sequence(s) <NUM>, <NUM>' also attached to the gel material <NUM>.

Moreover, since this example of the quality control tracer 22C also includes the excision site <NUM>, enzymatic cleavage may be used to remove a cleavable portion <NUM>' of the non-reactive sequence <NUM>' after the quality control method has been performed. In this example, the cleavable portion <NUM>' includes any sequence of oligonucleotides, the fluorescent label <NUM> attached to a nucleobase along the sequence, and the excision site <NUM>. The portion <NUM>' of the non-reactive nucleotide sequence <NUM>' that remains attached to the gel material <NUM> after enzymatic cleavage is also a non-reactive nucleotide sequence, and thus will not participate in or interfere with a sequencing operation that is to be performed or is being performed. The remaining portion <NUM>' may be a short poly T or poly A sequence, or may be a sequence that is orthogonal to the primer nucleotide sequence(s) <NUM>, <NUM>' also attached to the gel material <NUM>.

The non-reactive nucleotide sequence <NUM>' of <FIG> may include a functional group at its <NUM>' end that is capable of attaching to the gel material <NUM>. Examples of this functional group include an alkyne, a norbornyl (or other cycloalkenyls), a copper free click moiety (e.g., dibenzocyclooctyne (DIBO), other cycloalkynes, or others), and a thiol. This functional group may be selected based upon the gel material <NUM> that is used. For example, alkynes, norbornyls, and copper free click moieties may react with azides of PAZAM via click reactions, and thiols may react with SFA.

In this example, the fluorescent label <NUM> may be attached near (e.g., within <NUM> bases of) but not directly at, the <NUM>' end of the sequence <NUM>'. Any of the previously described fluorescent labels <NUM> may be used, as long as the selected label can covalently attach to a desirable nucleobase of the non-reactive nucleotide sequence <NUM>'.

The quality control tracer 22C in <FIG> is used in combination with separate primer nucleotide sequence(s) <NUM>, <NUM>'. Examples of suitable primer nucleotide sequence(s) <NUM>, <NUM>' include forward amplification primers or reverse amplification primers for hybridization to a complementary sequence and amplification of a sequence. Some specific examples of suitable primer nucleotide sequence(s) <NUM>, <NUM>' include the previously described P5 or P7 primers.

When the quality control tracer 22C in <FIG> is used, it is to be understood that it is present in a predetermined ratio with respect to the primer nucleotide sequence(s) <NUM>, <NUM>'. The predetermined ratio can be used in an example of the quality control method disclosed herein to indirectly determine the density and/or distribution of the primer nucleotide sequence(s) <NUM>, <NUM>'.

To form the example shown in <FIG>, sequential grafting or co-grafting may be used as previously described, except that the quality control tracer 22C is used instead of the quality control tracer 22A. In any of the grafting examples used to form the example shown in <FIG>, incubation takes place at a predetermined temperature which depends, in part, upon the quality control tracer 22C and the primer nucleotide sequence(s) <NUM>, <NUM>' used. Also in any of the grafting examples used to form the example shown in <FIG>, the solution may include the quality control tracer 22C and/or the primer nucleotide sequence(s) <NUM>, <NUM>', water, a buffer, and a catalyst. The quality control tracer 22C and/or the primer nucleotide sequence(s) <NUM>, <NUM>', whether present in the same solution/mixture or separate solutions/mixtures, may be present at any desired predetermined ratio.

<FIG> depicts an example of the quality control tracer 22D that is included on the gel material <NUM>/gel pad <NUM>' without separate primer nucleotide sequence(s) <NUM>, <NUM>'. In this example, the quality control tracer 22D is a cleavable nucleotide sequence <NUM>' tagged, at its <NUM>' end, with the fluorescent label <NUM>. The fluorescent label <NUM> may be any of the previously described fluorophores.

This example of the cleavable nucleotide sequence <NUM>' includes a cleavable portion <NUM>" and a remaining portion <NUM>". The cleavable portion <NUM>" includes the fluorescent label <NUM>, any sequence of nucleotides, and an excision site <NUM> near the <NUM>' end. The cleavable portion <NUM>" may also include a functional group that attaches the fluorescent label <NUM> to the sequence of nucleotides. An example of this functional group is <NUM>'-Dimethoxytrityl-<NUM>-[N-(trifluoroacetylaminohexyl)-<NUM>-acrylimido]-<NUM>'-deoxyUridine,<NUM>'-[(<NUM>-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (i.e., amino modifier C6 dT). Other examples include <NUM>'-Dimethoxytrityl-N-dimethylformamidine-<NUM>-[N-(trifluoroacetylaminohexyl)-<NUM>-acrylimido]-<NUM>'-deoxyCytidine,<NUM>'-[(<NUM>-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (amino modifier C6 dC) and the use of solid supports such as (<NUM>-Dimethoxytrityloxymethyl-<NUM>-fluorenylmethoxycarbonylamino-hexane-<NUM>-succinoyl)-long chain alkylamino-CPG. This functional group may be selected based upon the fluorescent label <NUM> that is used. The cleavable portion <NUM>" of the cleavable nucleotide sequence <NUM>' can be removed when the sequence <NUM>' is exposed to an enzyme that targets the nucleotide located at the excision site <NUM>.

The portion <NUM>" of the cleavable nucleotide sequence <NUM>' that remains attached to the gel material <NUM> after enzymatic cleavage is a primer nucleotide sequence <NUM>, <NUM>'. The primer nucleotide sequence <NUM>, <NUM>' that remains after the cleavable portion <NUM>" is removed will participate in a sequencing operation that is to be performed or is being performed. Any example of the primer nucleotide sequence(s) <NUM>, <NUM>' disclosed herein may be used in the quality control tracer 22D.

The cleavable nucleotide sequence <NUM>' of <FIG> may include a functional group at its <NUM>' end that is capable of attaching to the gel material <NUM>. Examples of this functional group include an alkyne, a norbornyl (or other cycloalkenyls), a copper free click moiety (e.g., dibenzocyclooctyne (DIBO), other cycloalkynes, or others), and a thiol. This functional group may be selected based upon the gel material <NUM> that is used. For example, alkynes, norbornyls, and copper free click moieties may react with azides of PAZAM via click reactions, and thiols may react with SFA.

An example of the P5 primer tagged, at its <NUM>' end, with the fluorescent label, Texas Red®, is:
<NUM>'-alkyne-AATGATACGGCGACCACCGAGAUCTACA[Amino C6 dT-Texas Red]-<NUM>' (SEQ.

The quality control tracer 22D in <FIG> can be used in an example of the quality control method disclosed herein to directly determine the density and/or distribution of the primer nucleotide sequence(s) <NUM>, <NUM>' that is part of the tracer 22D.

To form the example shown in <FIG>, grafting of one type of quality control tracer 22D (each of which includes the same primer nucleotide sequence(s) <NUM> or <NUM>') may be used, or co-grafting of different types of quality control tracers 22D (e.g., some of which include the primer nucleotide sequence <NUM> and others of which include a different primer nucleotide sequence <NUM>') may be used. Grafting or co-grafting may be performed as previously described, except that the quality control tracer 22D is used instead of the quality control tracer 22A.

Still other examples of the quality control tracer 22E are shown in <FIG>. In these examples, the quality control tracer 22E is a cleavable nucleotide sequence tagged, at its <NUM>' end, with the fluorescent label <NUM>. Each of these examples of the cleavable nucleotide sequence includes a nucleotide sequence X, a linker molecule <NUM>, and the fluorescent label <NUM>.

In accordance with the invention, the nucleotide sequence X is a non-reactive nucleotide sequence (such as sequence <NUM>) or a primer nucleotide sequence (such as sequence <NUM> or <NUM>'). When the nucleotide sequence X is the non-reactive nucleotide sequence, the quality control tracer 22E may be used in combination with separate primer nucleotide sequence(s) <NUM>, <NUM>' (similar to the examples shown in <FIG>. When the nucleotide sequence X is the primer nucleotide sequence, the quality control tracer 22E may be used alone (i.e., without separate primer nucleotide sequence(s) <NUM>, <NUM>') (similar to the example shown in <FIG>).

The nucleotide sequence X of <FIG> may include a functional group at its <NUM>' end that is capable of attaching to the gel material <NUM>. Examples of this functional group include an alkyne, a norbornyl (or other cycloalkenyls), a copper free click moiety (e.g., dibenzocyclooctyne (DIBO), other cycloalkynes, or others), and a thiol. This functional group may be selected based upon the gel material <NUM> that is used. For example, alkynes, norbornyls, and copper free click moieties may react with azides of PAZAM via click reactions, and thiols may react with SFA.

The linker molecule <NUM> includes, at one end, a functional group that can attach (directly or indirectly) to the fluorescent label <NUM> and, at the other end, a functional group that can attach (directly or indirectly) to the nucleotide sequence X (e.g., at the <NUM>' end or at a particular nucleobase in the sequence X). The linker molecule <NUM> of the quality control tracer 22E provides a chemical linkage that can undergo a cleavage reaction that removes at least the fluorescent label <NUM> from the quality control tracer 22E after an example of the quality control method has been performed. The cleavage reaction that is performed depends upon the linker molecule <NUM> that is used. Example linker molecules <NUM> include a diol, a disulfide, a silane, an azobenzene, a photocleavable group, and an azido.

A diol is any chemical compound containing two hydroxyl groups. Linear or cyclic diols may be used. Examples of suitable diols are vicinal diols, where the hydroxyl groups are attached to adjacent carbon atoms. Vicinal diols are cleavable under oxidative conditions, such as exposure to periodate (such as sodium periodate) or under enzymatic conditions. An example of a suitable diol is the tartaric acid-derived diol shown as 34A in <FIG>. In this example, the amine groups at each end of the diol attach to respective moieties, one of which is attached to the <NUM>' end of nucleotide sequence X and the other of which is attached to the fluorescent label <NUM> via any suitable attachment site (e.g., alcohol, amine, carboxylic acid). The diol may be cleaved by exposing the quality control tracer 22E to sodium periodate (NaIO<NUM>) which can oxidatively cleave the diol into two aldehydes.

A disulfide has the general structure R<NUM>-S-S-R<NUM>, where R<NUM> and R<NUM> may be any of alkyl or aryl groups. Disulfide bonds are cleaved under reducing conditions. An example of a suitable disulfide is shown as 34B in <FIG>. In this example, the amine group at one end of the disulfide is attached via a linker such as an alkyl chain to the nucleotide sequence X at the <NUM>' end and the amine group at the other end of the disulfide forms an amido linkage (
<CHM>
) where R is -(CH<NUM>)<NUM>-O-fluorescent label <NUM>, R' is H, and R" is -(CH<NUM>)<NUM>-S-S-(CH<NUM>)<NUM>-(C=O)-NH-(CH<NUM>)<NUM>-X. The fluorescent label <NUM> is attached via any suitable attachment site (e.g., alcohol, amine, carboxylic acid). In an example, the disulfide may be cleaved by exposing the quality control tracer 22E to reducing conditions, such as a thiol (R<NUM>SH, where R<NUM> is may be any suitable alkyl group) or a tertiary phosphine (R<NUM><NUM>P, where R<NUM> is any suitable alkyl group). The reaction of the thiol with the disulfide will break the disulfide bond and create a new disulfide and a thiol derived from the original disulfide. The reaction of the tertiary phosphine with the disulfide is a bimolecular nucleophilic substitution (SN<NUM>) reaction that will break the disulfide bond and create two sulfur-containing products.

A silane, as used herein and as shown at 34C in <FIG>, includes a silicon atom bonded to two oxygen atoms and two R groups (e.g., each of which may be alkyl or aryl groups). In this example, the oxygen atoms of the silane attach, respectively, to a linker such as an alkyl chain which is attached to the nucleotide sequence X at the <NUM>' end and to the fluorescent label <NUM> via any suitable attachment site (e.g., alcohol, amine, carboxylic acid). The silane may be cleaved at one of the Si-O bonds by exposing the quality control tracer 22E to an acid (shown as H+ in <FIG>) or by treatment with fluoride ion (not shown in <FIG>).

An azobenzene is a chemical compound composed of two phenyl rings linked by a N=N double bond. Different functional groups may extend from the phenyl rings at the para positions relative to the N=N double bond. These functional groups may be the same or different, and examples include an amido group, an alkyl amine, or hydroxyl groups. An example of a suitable azobenzene is shown as 34D in <FIG>. In this example, the functional groups include an amido group and ethylamine. The amido group is attached to a linker such as an alkyl chain which is attached to the nucleotide sequence X at the <NUM>' end, and the amine group of the ethylamine forms an amido linkage (
<CHM>
) where R is -(CH<NUM>)<NUM>-O-fluorescent label <NUM>, R' is H, and R" is -(CH<NUM>)<NUM>-azobenzene-(C=O)-NH-(CH<NUM>)<NUM>-X. The fluorescent label <NUM> is attached via any suitable attachment site (e.g. alcohol, amine, carboxylic acid). In an example, the azobenzene may be cleaved by exposing the quality control tracer 22E to sodium dithionate (Na<NUM>S<NUM>O<NUM>). The reaction of the sodium dithionate with the azobenzene reduces the azobenzene to two aniline groups.

A photocleavable group is a non-nucleotide moiety that includes a photo cleavage site, where cleavage occurs by irradiation with a predetermined wavelength of light for a predetermined time. The photocleavable group can be used as an intermediary to attach any available phosphoramidite modification at the <NUM>' end of the nucleotide sequence X to a functional group attached to the fluorescent label <NUM>. An example of a suitable photocleavable group, an ortho-nitrobenzyl group, is shown as 34E in <FIG>. In this example, photocleavable group includes amine groups at either end. In this example, the amine group at one end of the photocleavable group is attached to a linker such as an alkyl chain which is attached to the nucleotide sequence X, and the amine group at the other end of the disulfide forms an amido linkage attached to the fluorescent label <NUM> through -(CH<NUM>)<NUM>-O-. The fluorescent label <NUM> is attached via any suitable attachment site (e.g. alcohol, amine, carboxylic acid). In an example, at least the fluorescent label <NUM> is cleaved from the quality control tracer 22E at the photocleavable site by exposing the quality control tracer 22E to light of a predetermined wavelength for a predetermined time.

As mentioned herein, an azido is any molecule including the group N<NUM>. An example of a suitable azido is shown as the O-azidoalkyl group 34F in <FIG>. In this example, the amine group at one end of the azido is attached to a linker such as an alkyl chain which is attached to the nucleotide sequence X at the <NUM>' end, and the amine group at the other end of the azido forms an amido linkage (
<CHM>
) where (in this example) R is -(CH<NUM>)<NUM>-O-fluorescent label <NUM>, R' is H, and R" is -(CH<NUM>)<NUM>-O-(CN<NUM>)-(CH<NUM>)-(O(CH<NUM>)<NUM>)<NUM>-(CH<NUM>)-(C=O)-NH-(CH<NUM>)<NUM>-X. The fluorescent label <NUM> is attached via any suitable attachment site (e.g. alcohol, amine, carboxylic acid). In an example, the azido may be cleaved by exposing the quality control tracer 22E to a tertiary phosphine (R<NUM><NUM>P, where R<NUM> is an appropriately substituted alkyl or aryl group). The tertiary phosphine and the azido undergo the Staudinger reduction reaction.

Each of the linker molecules <NUM> disclosed herein can undergo a chemical cleavage reaction that removes at least the fluorescent label <NUM> from the quality control tracer 22E after an example of the quality control method has been performed. The remaining portion may be a non-reactive nucleotide sequence (that will not participate in sequencing) or a primer nucleotide sequence (that will participate in sequencing).

When the nucleotide sequence X of the quality control tracer 22E is non-reactive, the quality control tracer 22E may be sequentially grafted or co-grafted with separate primer nucleotide sequence(s) <NUM>, <NUM>' as previously described herein. When the nucleotide sequence X of the quality control tracer 22E is a primer nucleotide sequence(s) <NUM>, <NUM>', one type of quality control tracer 22E (each of which includes the same primer nucleotide sequence(s) <NUM> or <NUM>') may be grafted, or different types of quality control tracers 22E (e.g., some of which include the primer nucleotide sequence <NUM> and others of which include a different primer nucleotide sequence <NUM>') may be co-grafted. Grafting or co-grafting may be performed as previously described, except that an example of the quality control tracer 22E is used instead of the quality control tracer 22A.

Referring back to <FIG>, an example of the as-grafted quality control tracer(s) <NUM> with separate primer nucleotide sequence(s) <NUM>, <NUM>' is depicted. In examples of the tracer <NUM> that include the primer nucleotide sequence(s) <NUM>, <NUM>' (e.g., tracer 22D and some examples of tracer 22E), separate primer nucleotide sequence(s) <NUM>, <NUM>' will not be present with the tracer(s) <NUM>.

The array <NUM> disclosed herein may be used in a quality control method. An example of the quality control method is depicted in <FIG>. The method <NUM> generally includes grafting a quality control tracer <NUM> to a gel material <NUM> in a well <NUM>' on a support <NUM>, the quality control tracer being selected from the group consisting of a cleavable nucleotide sequence <NUM>, <NUM>' tagged, at its <NUM>' end, with a fluorescent label <NUM> and a non-reactive nucleotide sequence <NUM>, <NUM>' with the fluorescent label <NUM> attached to a cleavable nucleobase (as shown at reference numeral <NUM>); detecting the quality control tracer <NUM> using fluorescence (as shown at reference numeral <NUM>); and based at least in part on the fluorescence, determining a density, or a distribution, or the density and the distribution, of a primer nucleotide sequence <NUM>, <NUM>' grafted to the gel material <NUM> (as shown at reference numeral <NUM>).

The quality control method may be performed after the quality control tracer <NUM>, alone or in combination with separate primer nucleotide sequence(s) <NUM>, <NUM>', is/are grafted to the gel material <NUM> and prior to completion of flow cell manufacturing (e.g., prior to the bonding of the array <NUM> to a top substrate flow cell). The quality control method may also or alternatively be performed by an end-user of the fully manufactured flow cell. When the quality control method is performed by the end-user of the flow cell, it is to be understood that the method may be performed prior to loading any sequencing workflow reagents and DNA sample onto the flow cell. Because each quality control tracer 22A, 22B, 22C, 22D, 22E includes the detectable fluorescent label <NUM>, hybridization of fluorescently-labeled complementary nucleotides does not need to be performed as part of the quality control method disclosed herein.

In the method <NUM>, the grafting of the various examples of the quality control tracer 22A, 22B, 22C, 22D, 22E may be accomplished as previously described.

Each of the example quality control tracers 22A, 22B, 22C, 22D, 22E includes the fluorescent label <NUM>, which will emit light of longer wavelength(s) when exposed to incident radiation of shorter wavelength(s). As such, detecting the quality control tracer <NUM> may be accomplished by exposing the array <NUM> (or flow cell including the array <NUM>) to radiation emitted by a laser. After laser excitation, the emitted fluorescence (in terms of intensity) from the fluorescent label <NUM> of each quality control tracer 22A, 22B, 22C, 22D, 22E is captured via a suitable fluorescence detector.

In accordance with the invention, when the quality control tracer 22A, 22B, 22C, or some examples of tracer 22E are utilized in a predetermined ratio with separate primer nucleotide sequence(s) <NUM>, <NUM>', the fluorescence intensity from these quality control tracers 22A, 22B, 22C, 22E and the predetermined ratio may be used to assess or indirectly determine the density and/or distribution of the primer nucleotide sequence(s) <NUM>, <NUM>'. The fluorescence intensity indicates the density and/or distribution of the quality control tracers 22A, 22B, 22C, 22E, and the predetermined ratio enables the data for the quality control tracers 22A, 22B, 22C, 22E to be correlated to the primer nucleotide sequence(s) <NUM>, <NUM>'.

When the quality control tracer 22D or other examples of tracer 22E (which include primer nucleotide sequence(s) <NUM>, <NUM>') are utilized, the fluorescence results alone may be used to assess or directly determine the density and/or distribution of the primer nucleotide sequence(s) <NUM>, <NUM>'. These examples of the quality control tracer 22D, 22E include the sequences <NUM>, <NUM>' as part of the tracer 22D, 22E, and thus the fluorescence intensity alone indicates the density and/or distribution of the primer nucleotide sequence(s) <NUM>, <NUM>'.

After completion of the quality control method (e.g., at the end-user portion of the workflow), the fluorescent label <NUM> may be cleaved from quality control tracers 22A, 22B, 22C, 22D, 22E using the methods described herein.

As mentioned above in reference to <FIG>, quality control tracer 22A includes the excision site <NUM>. As such, cleavage of the fluorescent label <NUM> in this example tracer 22A may be accomplished via enzymatic cleavage. The quality control tracer 22A is exposed to an enzyme that targets the nucleotide located at the excision site <NUM>. The enzyme may be introduced prior to initiating sequencing, or may be introduced as part of a cluster generation process of a sequencing workflow. Examples of suitable enzymes include exonucleases (which remove successive nucleotides from an end of the sequence), endonucleases (which cleave phophodiester bonds within a sequence), base excision repair enzymes (which remove specific bases to form an apurinic/apyrimidinic (AP) site, which can then be cleaved by an AP endonuclease), restriction enzymes (which scan the quality control tracer 22A for a particular sequence of <NUM> to <NUM> nucleotides at which to cut the single-stranded sequence), etc. Some specific examples are shown in Table <NUM> below.

In Table <NUM>, * represents the fluorescent label <NUM>, N represents nucleotides, and the excision site is in bold.

In one example in Table <NUM>, the excision site <NUM> is a uracil (dU) that is targeted by the USER enzyme. The USER enzyme is a mixture of uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase endonuclease VIII. UDG catalyzes the excision of the uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact. The lyase activity of endonuclease VIII breaks the phosphodiester backbone at the <NUM>' and <NUM>' sides of the abasic site so that base-free deoxyribose is released. As such, cleavage of tracer 22A at excision site <NUM> removes a portion <NUM> of the sequence <NUM> and the fluorescent label <NUM> from the support <NUM>. In the other example in Table <NUM>, the excision site <NUM> is an oxidized purine (e.g., oxoG) that is targeted by FPG. FPG recognizes and removes the oxidized guanine. The FPG acts as both an N-glycosylase and an AP-lyase.

The portion <NUM> of the quality control tracer 22A that remains attached to the gel material <NUM> after enzymatic cleavage is a non-reactive nucleotide sequence, and thus will not participate in or interfere with a sequencing operation that is to be performed or is being performed.

As mentioned above in reference to <FIG>, quality control tracer 22B includes fluorescent label <NUM> attached to a cleavable nucleobase of the sequence <NUM>. The quality control tracer 22B is exposed to an exonuclease that catalyzes the excision of the particular base by phosphodiester cleavage. The exonuclease may be introduced prior to initiating sequencing, or may be introduced as part of a cluster generation process of a sequencing workflow. As an example, Exonuclease I catalyzes the excision of a uracil base having the fluorescent label <NUM> attached thereto.

The sequence <NUM> of the quality control tracer 22B that remains attached to the gel material <NUM> after nucleobase cleavage is non-reactive, and thus will not participate in or interfere with a sequencing operation that is to be performed or is being performed.

As mentioned above in reference to <FIG>, quality control tracer 22C includes the fluorescent label <NUM> attached to a cleavable nucleobase of the sequence <NUM>' as well as the excision site <NUM>. As such, cleavage of the fluorescent label <NUM> in this example tracer 22C may be accomplished via any of the previously described examples of enzymatic cleavage. In one example, an exonuclease may be used to catalyze the excision of the base having the fluorescent label <NUM> attached thereto. In another example, an enzyme that targets the nucleotide located at the excision site <NUM> may be used to catalyze the excision of the portion <NUM>' of the sequence <NUM>'. In these examples, the enzyme may be introduced prior to initiating sequencing, or may be introduced as part of a cluster generation process of a sequencing workflow.

The portion <NUM>' of the sequence <NUM>' of the quality control tracer 22C that remains attached to the gel material <NUM> after nucleobase or portion <NUM>' cleavage is non-reactive, and thus will not participate in or interfere with a sequencing operation that is to be performed or is being performed.

As mentioned above in reference to <FIG>, quality control tracer 22D includes the excision site <NUM>. As such, cleavage of the fluorescent label <NUM> in this example tracer 22D may be accomplished via any of the previously described examples of enzymatic cleavage. In this example, the portion <NUM>" of the sequence <NUM>' that remains attached to the gel material <NUM> after portion <NUM>" cleavage is a primer nucleotide sequence <NUM>, <NUM>', and thus will participate in a sequencing operation that is to be performed or is being performed.

As mentioned above in reference to <FIG>, the various examples of the quality control tracer 22E include the linker molecule <NUM>. Cleavage of the fluorescent label <NUM> in these example tracers 22E may be accomplished via chemical cleavage by exposing the tracer 22E to a chemical that is suitable for cleaving the linker molecule <NUM> present in the tracer 22E. Various examples of the linker molecules 34A, 34B, 34C, 34D, 34E, 34F and associated cleavage chemicals are described in reference to <FIG>. In these examples, the cleavage chemical may be introduced prior to initiating sequencing.

The portion of the quality control tracer 22E that remains attached to the gel material <NUM> after chemical cleavage will depend upon the nucleotide sequence X and where the chemical cleavage takes place. The remaining portion may include a non-reactive nucleotide sequence (which will not participate or interfere with sequencing) or a primer nucleotide sequence (which will participate in sequencing).

While not shown in the figures, another example of the method includes incorporating an example of the quality control tracer (e.g., 22A, 22B, 22C, and some examples of 22E) into a grafting mix with a primer nucleotide sequence <NUM>, <NUM>' at a predetermined ratio; exposing the grafting mix to a gel material <NUM> in a well <NUM>' on a support <NUM>; incubating the grafting mix, thereby co-grafting the quality control tracer 22A, 22B, 22C, and some examples of 22E and the primer nucleotide sequence <NUM>, <NUM>' to the gel material <NUM>; detecting the quality control tracer using fluorescence; and based at least in part on the fluorescence and the predetermined ratio, determining a density, or a distribution, or the density and the distribution, of the primer nucleotide sequence <NUM>, <NUM>' grafted to the gel material <NUM>.

A variety of sequencing approaches or technologies, including techniques often referred to as sequencing-by-synthesis (SBS), sequencing-by-ligation, pyrosequencing, and so forth may be performed after the fluorescent label <NUM> is cleaved. With any of these techniques, since the gel material <NUM> and attached primer nucleotide sequences <NUM>, <NUM>' are present in the sites <NUM> and not on the interstitial regions <NUM>, amplification will be confined to the various sites <NUM>.

Briefly, a sequencing by synthesis (SBS) reaction may be run on a system such as the HiSeq®, HiSeqX®, MiSeq® or NextSeq® sequencer systems from Illumina (San Diego, CA). A set of target DNA molecules to be sequenced is hybridized to the bound primer nucleotide sequences <NUM>, <NUM>' (and not to any non-reactive nucleotide sequences) and then amplified by bridge amplification or by kinetic exclusion amplification. Denaturation leaves single-stranded templates anchored to the gel material <NUM>, and several million dense clusters of double-stranded DNA are generated (i.e., cluster generation). The sequencing reactions are then carried out. The data area aligned and compared to a reference, and sequencing differences are identified.

To further illustrate the present invention, an example is given herein.

The following quality control tracer was used:
<NUM>'-Hexyne-poly T tail-CATCTAGGCATCTAAGCATCAAUCTTACA[Amino C6 dT-Texas Red]-<NUM>' (SEQ.

To evaluate an example of the QC tracer for quality control of P5/P7 primer grafting onto a substrate surface, P5/P7 primer grafting mixes were prepared with a <NUM>% spike of a QC tracer and without the QC tracer spike (control). Substrate surfaces grafted with the P5/P7 primer mix that included the <NUM>% spike of QC tracer were evaluated by measuring tracer fluorescence. The control surfaces (without QC tracer spike) were evaluated by hybridization-based TET QC. TET is a dye labeled oligonucleotide having complementary sequence to the P5/P7 primers. TET is hybridized to the P5/P7 primers on a surface, the excess TET is washed away, and the attached dye concentration is measured by fluorescence detection.

<FIG> respectively show a fluorescence image of grafted control substrate surfaces hybridized with complementary dye-containing oligonucleotides (TET QC) and a fluorescence image of grafted substrate surfaces that include QC tracers. <FIG> shows a plot of QC tracer fluorescence versus TET fluorescence for the grafted surfaces of <FIG>. The data in <FIG> show that fluorescence intensity from the QC tracer correlates with P5/P7 density on the substrate surface.

To evaluate cleavage of grafted QC tracers from the surface of a flow cell during a standard cluster generation process, flow cells grafted with different QC tracers were used. <FIG> shows an initial fluorescence image, which shows flow cells grafted with QC tracers before cluster generation. <FIG> shows a post-clustering fluorescence image, which shows flow cells grafted with QC tracers after cluster generation. The QC tracers (at <NUM>) were:.

Referring to the initial fluorescence image of <FIG>, the dark areas in the flow cells are the signal from the QC tracers; lighter areas are control lanes (i.e., no QC tracers were present). Referring to post-clustering fluorescence image of <FIG>, the signal from the QC tracers is substantially reduced indicating cleavage of the QC tracers during cluster formation.

<FIG> show a plot of QC tracer fluorescence post-grafting and a plot of QC tracer fluorescence post-clustering, respectively, for the grafted flow cells of <FIG>. The data show that residual fluorescence after cluster formation is minimal. The cleavage efficiency ranges from about <NUM>% to about <NUM>%.

To evaluate the effect of QC tracer grafting and subsequent cleavage on downstream sequencing metrics, the flow cells of <FIG> were used for sequencing.

<FIG> show a plot of the read <NUM> (R1) fluorescence intensity and a plot of the read <NUM> (R2) fluorescence intensities, respectively, for the red channel after one sequencing cycle (C1). The data show that the C1 intensities in the red channel are minimally impacted by the use of QC tracers.

<FIG> show a plot of the read <NUM> (R1) fluorescence intensity and a plot of the read <NUM> (R2) fluorescence intensities, respectively, for the green channel after one sequencing cycle (C1). The data show that the C1 intensities in the green channel are minimally impacted by the use of QC tracers.

<FIG> show a plot of read <NUM> (R1) sequence alignment and plot of read <NUM> (R2) sequence alignment, respectively, to a reference genome. The data show that there is little or no impact by the use of QC tracers on sequence alignment metrics.

<FIG> show a plot of read <NUM> (R1) sequencing error rates and plot of read <NUM> (R2) sequencing error rates, respectively. The data show that there is little or no impact by the use of QC tracers on sequencing error rate metrics.

Reference throughout the specification to "one example", "another example", "an example", and so forth, means that a particular element (e.g., feature, structure, and/or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any example may be combined in any suitable manner in the various examples unless the context clearly dictates otherwise.

Claim 1:
An array (<NUM>), comprising:
a support (<NUM>) comprising a plurality of discrete wells (<NUM>');
a gel material (<NUM>, <NUM>') positioned in each of the plurality of discrete wells (<NUM>'); and
a quality control tracer (<NUM>) comprising a cleavable nucleotide sequence (<NUM>) comprising a cleavage site and a detectable fluorescent label (<NUM>);
wherein the cleavable nucleotide sequence (<NUM>) comprises a grafted region with a first end and a second end, where the first end is grafted to the gel material (<NUM>) and the second end is linked to a cleavable region that is linked to the detectable fluorescent label and comprises the cleavage site (<NUM>);
wherein the cleavable nucleotide sequence (<NUM>) is grafted to the gel material (<NUM>) at its <NUM>' end and the detectable fluorescent label (<NUM>) is attached at or near the <NUM>' end of the cleavable nucleotide sequence (<NUM>);
wherein said quality control tracer is grafted to the gel material in each of the plurality of discrete wells (<NUM>'); and
wherein either:
(i) the portion (<NUM>) of the cleavable nucleotide sequence (<NUM>) which remains attached to the gel material (<NUM>) after cleavage is a non-reactive nucleotide sequence, and thus will not actively participate in a particular DNA or RNA synthesis that is being performed or interfere with sequencing, and the quality control tracer (<NUM>) is present in a predetermined ratio with an unlabeled primer nucleotide sequence (<NUM>, <NUM>') grafted to the gel material (<NUM>) in each of the plurality of discrete wells (<NUM>); or
(ii) the portion (<NUM>) of the cleavable nucleotide sequence (<NUM>) which remains attached to the gel material (<NUM>) after cleavage comprises a primer nucleic acid sequence.