HK40090342A - Detecting materials in a mixture using oligonucleotides - Google Patents

Description

Detecting materials in a mixture using oligonucleotides

Cross-references to related applications

This application claims the benefit of U.S. Provisional Patent Application No. 63/110,655, filed November 6, 2020, entitled “Detecting Materials in a Mixture Using Oligonucleotides,” the entire contents of which are incorporated herein by reference.

sequence list

This patent application includes a sequence list that has been electronically filed in ASCII format, the entire contents of which are incorporated by reference. The ASCII copy, created on October 15, 2021, is named IP-2035-PCT-SL.txt and is 1,077 bytes in size.

Background Technology

The detection of specific nucleic acid sequences present in biological samples has been used as a method for, for example, identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, studying genetic susceptibility to diseases, and measuring responses to various types of treatments. A commonly used technique for detecting specific nucleic acid sequences in biological samples is nucleic acid sequencing.

Nucleic acid sequencing methods have evolved from the chemical degradation methods used by Maxam and Gilbert and the chain elongation methods used by Sanger. Several sequencing methods are now available that allow the parallel processing of millions or even billions of nucleic acids on a single flow cell. Some platforms include bead-based and microarray formats, where silica beads are functionalized with probes, depending on the application of such formats, including sequencing, genotyping, or gene expression profiling. Some sequencing systems (whether for sequencing-by-synthesis or for genotyping) utilize substrates comprising multiple reservoirs holding different reagents for sequencing operations.

Summary of the Invention

The examples provided in this article relate to the detection of materials in a mixture using oligonucleotides. Devices using such oligonucleotides are also disclosed.

In some examples, this document provides a method for detecting materials in a mixture of materials. The method may include: providing materials; and providing oligonucleotides that have different sequences from each other. Each of these oligonucleotides may be present in and correspond to a corresponding material among these materials. The method may include obtaining a mixture of at least two of these materials. The mixture may contain oligonucleotides corresponding to those materials. The method may include sequencing these oligonucleotides in the mixture; and using the sequences of those oligonucleotides to detect materials corresponding to those oligonucleotides.

In some examples, these materials contain reagents for sequencing. In other examples, these materials are provided separately in reservoirs on the substrate.

In some examples, the sequence of each oligonucleotide in these oligonucleotides includes: (i) an index corresponding to the mixture and (ii) a barcode corresponding to the corresponding material.

In some examples, the sequence of each of these oligonucleotides includes a barcode corresponding to the respective material, and the method further includes adding an index corresponding to the mixture to each of these oligonucleotides. In some examples, the index is added after the mixture is obtained.

In some examples, the mixture is obtained using a first sequencing system. For example, the mixture may contain waste from the first sequencing system. In some examples, the oligonucleotides in the mixture are sequenced using a second sequencing system different from the first sequencing system. In some examples, these sequences of the oligonucleotides include adaptors that are compatible with the second sequencing system but incompatible with the first sequencing system.

In some examples, sequencing these oligonucleotides in the mixture involves amplifying these oligonucleotides on a surface to generate corresponding amplicon clusters on the surface. In some examples, detecting these materials involves comparing these sequences of the oligonucleotides with stored sequences. In some examples, detecting these materials involves quantifying the corresponding amounts of these oligonucleotides and correlating these amounts of the oligonucleotides with the amounts of the corresponding materials. In some examples, these oligonucleotides include sequences of single-stranded DNA, double-stranded DNA, RNA, LNA, or modified nucleotides.

In some examples, each of these materials is independently selected from the group consisting of liquids, semi-solids, and solids. In some examples, the liquid includes a solvent or reagent. In some examples, the solid includes a dry powder or powder in a liquid. In some examples, the semi-solid includes a gel.

In some examples, this document provides a method for detecting materials in a mixture. The method may include mixing at least two materials with each other to obtain a mixture. Different oligonucleotides may have different sequences and may be disposed within different materials. These sequences of the oligonucleotides can be used to detect these materials in the mixture.

In some examples, these materials contain reagents for sequencing. In some examples, these materials are provided separately in a reservoir on the substrate. In some examples, the sequence of each oligonucleotide in these oligonucleotides includes one or both of the following: (i) an index corresponding to the mixture and (ii) a barcode corresponding to the respective material.

In some examples, the mixture is obtained using a first sequencing system. For example, the mixture may contain waste from that sequencing system. In some examples, the oligonucleotides in the mixture are sequenced using a second sequencing system different from the first sequencing system. In some examples, these sequences of the oligonucleotides include adaptors that are compatible with the second sequencing system but incompatible with the first sequencing system.

In some examples, the materials are detected by comparing the sequences of these oligonucleotides with stored sequences. In some examples, the materials are detected by quantifying the corresponding amounts of these oligonucleotides and correlating these amounts with the amounts of the corresponding materials. In some examples, these oligonucleotides include sequences of single-stranded DNA, double-stranded DNA, RNA, LNA, or modified nucleotides.

In some examples, each of these materials is independently selected from the group consisting of liquids, semi-solids, and solids. In some examples, the liquid includes a solvent or reagent. In some examples, the solid includes a dry powder or powder in a liquid. In some examples, the semi-solid includes a gel.

In some examples herein, a device is provided. The device may include a substrate comprising a plurality of reservoirs. The device may include a variety of materials, each of which resides within a corresponding reservoir. The device may include a variety of oligonucleotides having different sequences from each other, each of which resides within a corresponding material.

In some examples, these materials contain reagents used for sequencing.

In some examples, the sequence of each oligonucleotide in these oligonucleotides includes one or both of the following: (i) an index corresponding to the mixture and (ii) a barcode corresponding to the corresponding material.

In some examples, the device is used with a sequencing system to obtain a mixture of two or more of these materials. For example, the mixture may contain waste from the sequencing system. In some examples, these sequences of the oligonucleotides include adaptors incompatible with the sequencing system. In some examples, these oligonucleotides include sequences of single-stranded DNA, double-stranded DNA, RNA, LNA, or modified nucleotides.

In some examples, each of these materials is independently selected from the group consisting of liquids, semi-solids, and solids. In some examples, the liquid includes a solvent or reagent. In some examples, the solid includes a dry powder or powder in a liquid. In some examples, the semi-solid includes a gel.

It should be understood that any corresponding feature/example of each aspect of this disclosure as described herein may be implemented together in any suitable combination, and any feature/example from any one or more of these aspects may be implemented together with any feature of other aspects as described herein in any suitable combination to achieve the benefits described herein.

Attached Figure Description

Figures 1A and 1B schematically illustrate exemplary equipment and operations in a processing flow for detecting materials in a mixture using oligonucleotides.

Figures 2A through 2D schematically illustrate exemplary oligonucleotides used in the processing flow described with reference to Figures 1A through 1B.

Figure 3 schematically illustrates an exemplary operation in a processing flow for detecting materials in a mixture using oligonucleotides.

Figure 4 schematically illustrates an exemplary operation in another processing flow for detecting materials in a mixture using oligonucleotides.

Figure 5 schematically illustrates another exemplary device and operation in a processing flow for detecting materials in a mixture using oligonucleotides.

Figures 6A to 6C schematically illustrate exemplary measurement results in a processing flow for detecting liquids in a mixture using oligonucleotides.

Detailed Implementation

The examples provided in this article relate to the detection of materials in a mixture using oligonucleotides. Devices using such oligonucleotides are also disclosed.

The technology of this application relates to the use of oligonucleotides mixed in small amounts with respective materials for the qualitative or quantitative determination of the presence of one or more such materials in the mixture. The oligonucleotides can be implemented in any suitable environment and in any suitable one or more materials, such as any suitable liquid, semi-solid, or solid. Exemplarily, the oligonucleotides can be used to identify possible sources of apparent contamination, for example, to detect whether any material (such as a reagent) has been intentionally or unintentionally mixed with other materials. Additionally or alternatively, the oligonucleotides can be used to quantify the effectiveness of a method for mixing two or more materials together, for example, by detecting the amount of different materials at different locations in the mixture once or multiple times. In various examples, the amount of these materials can be expressed as an absolute amount in the sample (e.g., concentration or volume), or as a relative amount in the sample (e.g., molar ratio), or qualitatively (e.g., presence or absence). In some examples, the materials can be used in oligonucleotide sequencing processes where contamination of one material by another material may adversely affect the sequencing process.

In some examples, oligonucleotides with different sequences can be mixed into different materials. Two or more of these materials can be mixed together, and therefore the mixture can also contain oligonucleotides mixed into those materials. The oligonucleotides are amplified, and then sequenced from a sample of the mixture. Using the sequences of the present oligonucleotides, it is determined which materials are mixed together, and the absolute or relative amount of such materials in the sample. For example, the sequences of these oligonucleotides can include one or more identification subsequences, which can be arbitrary but unique. For example, a first subsequence (which may be referred to herein as a “barcode”) can be unique to the particular material into which the oligonucleotides are mixed, thus allowing identification of that material when the sequence is detected. A second subsequence (which may be referred to herein as an “index”) can be unique to a particular volume of that material, thus allowing different samples (which may contain the same materials but from different volumes of those materials) to be pooled together for sequencing purposes, while allowing identification of the specific volume from which the materials in those samples originate. The second subsequence can be added after a sample of the mixture is obtained, and, for example, if the samples are not pooled together, the second subsequence can be omitted. The oligonucleotides can also include adaptors for amplification. In the example of mixing oligonucleotides into reagents used in the first sequencing process, the adapters of those oligonucleotides can be orthogonal to adapters that can be used in the first sequencing process, such that the oligonucleotides themselves are not amplified or sequenced in the first sequencing process, but are amplified and sequenced in a second sequencing process, which can be performed separately from the first sequencing process and can be performed on a different sequencing system than the first sequencing process. However, it should be understood that this technique is not limited to use in sequencing reagents and can be used to detect any quantity of material in any given mixture of materials.

First, some of the terminology used in this article will be briefly explained. Then, some exemplary methods and associated devices for detecting materials in mixtures using oligonucleotides will be described.

the term

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The use of the term “comprising” and other forms (such as “include/includes/included”) is not restrictive. The use of the term “having” and other forms (such as “have/has/had”) is not restrictive. As used herein, whether in transitional phrases or in the body of the claims, the terms “comprise(s)” and “comprising” are to be interpreted in an open-ended sense. That is, the foregoing terms should be interpreted synonymously with the phrases “at least having” or “at least including”. For example, when used in the context of a process, the term “comprising” means that the process includes at least the listed steps, but may also include additional steps. When used in the context of a compound, composition, or device, the term “comprising” means that the compound, composition, or device contains at least the listed features or components, but may also contain additional features or components.

The terms “substantially,” “approximately,” and “about” used throughout this specification are used to describe and indicate small fluctuations, such as small fluctuations caused by variations in the process. For example, they may refer to less than or equal to ±10%, less than or equal to ±5%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.2%, less than or equal to ±0.1%, or less than or equal to ±0.05%.

As used herein, “hybridization” refers to the non-covalent association of a first polynucleotide with a second polynucleotide along the length of those polymers to form a double-stranded “double helix.” For example, two DNA polynucleotide chains can associate through complementary base pairing. The strength of association between the first and second polynucleotides increases with the complementarity between the nucleotide sequences within those polynucleotides. The strength of hybridization between polynucleotides can be characterized by the melting temperature (Tm), at which 50% of the double helixes dissociate from each other.

As used herein, the term "nucleotide" is intended to refer to a molecule that contains a sugar and at least one phosphate ester group, and in some examples also contains a nucleobase. A nucleotide lacking a nucleobase can be referred to as "base-free." Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), and deoxyadenosine monophosphate (dAM). P), deoxyadenosine diphosphate (dADP), deoxynucleoside triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP).

As used herein, the term "nucleotide" is also intended to cover any nucleotide analogue that, compared to naturally occurring nucleotides, comprises modified nucleobases, sugars, and/or phosphate moieties. Exemplary modified nucleobases include inosine, xanthine, hypoxanthine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymidine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azo Cytosine, 6-azothymidine, 5-uracil, 4-thiouracil, 8-haloadenine or guanine, 8-aminoadenine or guanine, 8-thioadenine or guanine, 8-thioalkyladenine or guanine, 8-hydroxyadenine or guanine, 5-halosubstituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, etc. As is known in the art, certain nucleotide analogs cannot be incorporated into polynucleotides, such as nucleotide analogs like adenosine 5'-phosphate sulfate. Nucleotides can contain any suitable number of phosphate esters, such as three, four, five, six, or more than six phosphate esters.

As used herein, the term "polynucleotide" refers to a molecule comprising a sequence of nucleotides bonded to each other. A polynucleotide is a non-limiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and their analogues. A polynucleotide can be a single-stranded sequence of nucleotides (such as RNA or single-stranded DNA), a double-stranded sequence of nucleotides (such as double-stranded DNA), or a mixture of single-stranded and double-stranded sequences of nucleotides. Double-stranded DNA (dsDNA) includes genomic DNA as well as PCR and amplification products. Single-stranded DNA (ssDNA) can be converted to dsDNA, and vice versa. Polynucleotides can include non-naturally occurring DNA, such as enantiomeric DNA. The exact sequence of the nucleotides in a polynucleotide can be known or unknown. The following are examples of polynucleotides: genes or gene fragments (e.g., probes, primers, expression sequence tags (ESTs) or gene expression serialization analysis (SAGE) tags), genomic DNA, genomic DNA fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonuclease, cDNA, recombinant polynucleotides, synthetic polynucleotides, branched-chain polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, or amplified copies of any of the above.

As used herein, “polymerase” is intended to mean an enzyme having an active site for assembling polynucleotides by polymerizing nucleotides into polynucleotides. A polymerase can bind to an initiated single-stranded target polynucleotide and can subsequently add nucleotides to a growing primer to form a “complementary copy” polynucleotide with a sequence complementary to the target polynucleotide. Another polymerase, or the same polymerase, can then form a copy of the target nucleotide by forming a complementary copy of that complementary copy polynucleotide. Either of these copies may be referred to herein as an “amplifier.” DNA polymerases can bind to a target polynucleotide and then move down along the target polynucleotide, successively adding nucleotides to a free hydroxyl group at the 3' end of the growing polynucleotide chain (the growing amplicon). DNA polymerases can synthesize complementary DNA molecules from a DNA template, and RNA polymerases can synthesize RNA molecules from a DNA template (transcription). Polymerases can use short RNA or DNA chains (primers) to initiate chain growth. Some polymerases can displace the chain upstream of a site where they add bases to the chain. Such polymerases can be referred to as strand displacement polymerases, meaning that these polymerases have the activity of removing the complementary strand from the template strand read by the polymerase. Exemplary polymerases with strand displacement activity include, but are not limited to, large fragments of Bst (Bacillus stearothermophilus) polymerase, exo-Klenow polymerase, or sequencing T7-level exo-polymerase. Some polymerases degrade the strand preceding them, effectively replacing that strand with the following growing strand (5' exonuclease activity). Some polymerases have the activity of degrading the strand following them (3' exonuclease activity). Some useful polymerases have been modified by mutation or other means to reduce or eliminate 3' and/or 5' exonuclease activity.

As used herein, the term "primer" refers to a polynucleotide that can be added to by a free 3'OH group. Primer length can be any suitable number of bases and can include any suitable combination of natural and/or non-natural nucleotides. Target polynucleotides may include "adaptors" that hybridize with the primers (having sequences complementary to the primers) and can be amplified by adding nucleotides to the free 3'OH groups of the primers to produce complementary copy polynucleotides. Primers can be coupled to a substrate. Primers that are "complementary" to each other can hybridize to each other substantially along their entire length, while primers that are "orthogonal" to each other substantially do not hybridize to each other, and their amplicon also substantially do not hybridize to each other.

In some examples, the primers are commercially available P5 or P7 primers from Illumina, Inc. P5 and P7 primers are non-limiting examples of primers that are orthogonal to each other. In some examples, the P5 and P7 primer sequences may have the following sequences:

Paired reading segments :

P5: 5'-AATGATACGGCGACCACCGAGAUCTACAC-3' (SEQ ID NO: 1)

P7: 5'-CAAGCAGAAGACGGCATACGAG*AT-3' (SEQ ID NO: 2)

Single segment group :

P5: 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 3)

P7: 5'-CAAGCAGAAGACGGCATACGA3' (SEQ ID NO: 4)

Where G* is G or 8-oxoguanine.

In some examples, the linked oligonucleotide (such as a primer or a P5 or P7 primer) includes a linker or spacer region at its 5' end. Such linkers or spacers may be included to allow chemical or enzymatic cleavage, or to impart other desired properties, such as to enable covalent linkage with a polymer or solid support, or to act as spacers to position the cleavage site at an optimal distance from the solid support. In some cases, 10 spacer nucleotides may be positioned between the P5 or P7 primer and the polymer or solid support linker. In some examples, a polyT spacer region is used, but other nucleotides and combinations thereof may also be used. In one example, the spacer region is a 6T to 10T spacer region. In some examples, the linker includes a cleavable nucleotide containing a chemically cleavable functional group, such as vicinal diol or allyl T.

As used herein, the term "amplifier," when used to refer to a polynucleotide, is intended to mean a product that copies that polynucleotide, wherein the product has a nucleotide sequence that is substantially identical or substantially complementary to at least a portion of the nucleotide sequence of the polynucleotide. "Amplification" and "amplifying" refer to the process of preparing an amplifier of a polynucleotide. The first amplifier of the target polynucleotide can be a complementary copy. Subsequent amplifiers are copies of the target polynucleotide or formed from the first amplifier after its generation. Subsequent amplifiers can have a sequence that is substantially complementary to or substantially identical to the target polynucleotide. It should be understood that minor mutations in the polynucleotide (e.g., due to amplification artifacts) can occur during the generation of the amplifier of that polynucleotide.

As used herein, the term "substrate" refers to a material used as a carrier for the compositions described herein. Exemplary substrate materials may include glass, silica, plastic, quartz, metal, metal oxide, organosilicon (e.g., polyhedral organosquioxane (POSS)), polyacrylate, tantalum oxide, complementary metal oxide semiconductor (CMOS), or combinations thereof. An example of a POSS may be the POSS described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776–778, which is incorporated herein by reference in its entirety. In some examples, the substrate used in this application comprises a silica-based substrate, such as glass, fused silica, or other silica-containing materials. In some examples, the substrate may comprise silicon, silicon nitride, or silicone hydride. In some examples, the substrate used in this application comprises plastic materials or components such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylon, polyester, polycarbonate, and poly(methyl methacrylate). Exemplary plastic materials include poly(methyl methacrylate), polystyrene, and cyclic olefin polymer substrates. In some examples, the substrate is or comprises a silica-based material or a plastic material or a combination thereof. In certain examples, the substrate has at least one surface comprising a glass or a silicon-based polymer. In some examples, the substrate may comprise a metal. In some such examples, the metal is gold. In some examples, the substrate has at least one surface comprising a metal oxide. In one example, the surface comprises tantalum oxide or tin oxide. Acrylamide, ketones, or acrylates may also be used as substrate materials or components. Other substrate materials may include, but are not limited to, gallium arsenide, indium phosphide, aluminum, ceramics, polyimide, quartz, resins, polymers, and copolymers. In some examples, the substrate and/or substrate surface may be or comprise quartz. In some other examples, the substrate and/or substrate surface may be or comprise semiconductors such as GaAs or ITO. The foregoing list is intended to illustrate but is not limited to this application. The substrate may comprise a single material or multiple different materials. The substrate may be a composite or a laminate. In some examples, the substrate comprises an organosilicon material. The substrate may be flat, circular, spherical, rod-shaped, or any other suitable shape. The substrate may be rigid or flexible. In some examples, the substrate is a bead or a flow cell.

In some examples, the substrate includes a patterned surface. A “patterned surface” refers to an arrangement of different regions in or on an exposed layer of the substrate. For example, one or more of these regions may be features containing one or more capturing primers. These features may be separated by gap regions where no capturing primers are present. In some examples, the pattern may be an x-y format of features arranged in rows and columns. In some examples, the pattern may be a repeating arrangement of these features and/or gap regions. In some examples, the pattern may be a random arrangement of these features and/or gap regions. In some examples, the substrate includes an array of holes (recesses) in its surface. These holes may be provided by substantially vertical sidewalls. These holes can be fabricated using a variety of techniques commonly known in the art, including but not limited to photolithography, imprinting, molding, and micro-etching. Those skilled in the art will appreciate that the techniques used will depend on the composition and shape of the array substrate.

Feature portions in the patterned surface of a substrate can include pores of a patterned and covalently linked gel (such as poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide)(PAZAM)) in an array of pores (e.g., micropores or nanopores) on glass, silicon, plastic, or other suitable materials. This process produces a gel pad for sequencing that can be stable during sequencing runs with numerous cycles. The covalent linking of the polymer to the pores can help maintain the gel as a structured feature throughout various applications and the entire lifetime of the structured substrate. However, in many examples, the gel does not need to be covalently linked to the pores. For example, under certain conditions, silane-free acrylamide (SFA) not covalently linked to any part of the structured substrate can be used as the gel material.

In a specific example, the structured substrate can be fabricated by patterning a suitable material to have pores (e.g., micropores or nanopores), coating the patterned material with a gel material (e.g., PAZAM, SFA, or chemically modified variants thereof, such as the azido-SFA form), and polishing the surface of the gel-coated material (e.g., by chemical or mechanical polishing) to retain the gel within the pores, while substantially all of the gel is removed from or inactivated in the interstitial regions located between the pores on the structured substrate surface. Primers can be linked to the gel material. A solution containing multiple target polynucleotides (e.g., fragmented human genome or portions thereof) can then be contacted with the polished substrate such that individual target polynucleotides are seeded into individual pores via the interaction of primers linked to the gel material; however, due to the absence or inactivation of the gel material, the target polynucleotides will not occupy the interstitial regions. Amplification of the target polynucleotides can be confined within the pores because the absence or inactivation of the gel in the interstitial regions inhibits the outward migration of growth clusters. The process is easy to manufacture, can be scaled up, and can utilize conventional micron or nanometer manufacturing methods.

Patterned substrates can include, for example, holes etched into glass slides or chips. The etching and geometric patterning of the holes can take on a variety of different shapes and sizes, and such features can be physically or functionally separable from each other. Particularly useful substrates with such structural features include patterned substrates that allow selection of the size of solid particles (such as microspheres). An example of a patterned substrate with these characteristics is an etched substrate used in conjunction with bead array technology (Illumina, Inc., San Diego, California).

In some examples, a substrate forms at least a portion of, is located in, or is coupled to a flow cell. A flow cell may include flow chambers divided into multiple channels or sectors. Exemplary flow cells and substrates for fabricating flow cells that can be used in the methods and compositions set forth herein include, but are not limited to, flow cells and substrates commercially available from Illumina Corporation (San Diego, CA).

As used herein, the term "multiple" is intended to mean a group of two or more distinct members. Multiples can vary in size from small, medium, large to very large. Small multiples can vary in size from, for example, a few members to tens of members. Medium-sized multiples can vary, for example, from tens of members to about 100 or hundreds of members. Large multiples can vary, for example, from about hundreds of members to about 1,000 members, to thousands of members, and up to tens of thousands of members. Very large multiples can vary, for example, from tens of thousands of members to about hundreds of thousands, millions, millions, and at most or more hundreds of millions of members. Therefore, the size of a multiple can range from two to well over one hundred million members, and, as measured by the number of members, all sizes between and greater than the above example ranges. Exemplary multiples of polynucleotides include, for example, groups of about 1 × ^10⁵ or more, 5 × ^10⁵ or more, or 1 × ^10⁶ or more distinct polynucleotides. Therefore, the definition of the term is intended to include all integer values greater than two. An upper limit can be set, for example, by using the theoretical diversity of polynucleotide sequences in the sample.

As used herein, the term "target polynucleotide" is intended to mean a polynucleotide that is the target of an analysis or action. This analysis or action includes subjecting the polynucleotide to amplification, sequencing, and/or other procedures. A target polynucleotide may include a nucleotide sequence other than the target sequence to be analyzed. For example, a target polynucleotide may include one or more adaptors that side-end the target polynucleotide sequence to be analyzed, including adaptors that serve as primer binding sites.

The terms “polynucleotide” and “oligonucleotide” are used interchangeably herein. Unless otherwise specified, the different terms are not intended to indicate any particular difference in size, sequence, or other properties. For clarity of description, these terms may be used to distinguish one polynucleotide species from another when describing a particular method or composition comprising several polynucleotide species.

As used herein, the term "sequencing system" refers to a system configured to determine polynucleotide sequences. Many sequencing systems are commercially available. Exemplarily, a sequencing system may be or include the iSEQ ^™ 100 sequencing system, commercially available from Illumina, Inc. (San Diego, California). The iSEQ ^™ 100 sequencing system is a benchtop system that performs sequencing-on-synthesis using pre-filled cartridges that include reservoirs for storing different sequencing reagents. Other non-limiting examples of sequencing systems include the cBot 2, NovaSeq 6000, and MiniSeq systems, commercially available from Illumina, Inc., as well as sequencing systems from other sources.

As used herein, terms such as “mixing” and “mixed” are intended to refer to the way materials are combined with each other such that each of these materials is distributed throughout the “mixture” produced by such combination. The distribution of any suitable amount of such material throughout the mixture can be homogeneous (i.e., uniformly distributed), such that any given portion of the mixture can contain substantially the same concentration of such material as any other given portion of the mixture. Alternatively or additionally, the distribution of any suitable amount of such material throughout the mixture can be heterogeneous (i.e., non-uniformly distributed), such that any given portion of the mixture can contain a different concentration of such material than one or more other given portions of the mixture. Within a given mixture, one or more materials can be homogeneous, and one or more materials can be heterogeneous. Each material in a given mixture can maintain its own chemical identity (i.e., not chemically react with any other material in the mixture). Alternatively, two or more materials in a given mixture can chemically react with each other to form one or more new materials.

Materials of any suitable type can be mixed together to form a mixture, such as any suitable combination of liquids, solids, and/or semi-solids. The degree to which a mixture is homogeneous or heterogeneous can depend on the specific materials being mixed, for example, based on the degree to which each material is soluble in any of the other materials in the mixture (if any). For example, miscible liquids can be mixed to form a homogeneous mixture, while immiscible liquids can be mixed to form a heterogeneous mixture, such as an emulsion containing droplets of one liquid dispersed in another liquid. Additionally or alternatively, liquids can be mixed with solids, such as particulate solids (solids having particles with sizes, for example, from about 1 nm to about 1 mm, or from about 10 nm to about 100 μm, or from about 100 nm to about 10 μm). When a solid is dissolved in a liquid, the resulting mixture can be homogeneous, while when a solid is suspended in a liquid and does not dissolve in that liquid (e.g., a colloidal suspension of solid particles within that liquid), the resulting mixture can be heterogeneous. Non-limiting examples of liquids include solvents (such as water or organic solvents) and reagents (such as chemical or biological species that can react with one or more other chemical or biological species).

Alternatively or alternatively, the liquid can be mixed with a semi-solid, such as a "gel," for example, a material comprising a three-dimensional cross-linked structure (e.g., a cross-linked polymer, a cross-linked network of colloidal particles, or a cross-linked network of nanoparticles or nanostructures), wherein the liquid is disposed within the structure. When the gel dissolves in the liquid, the resulting mixture can be homogeneous, while when the gel is suspended in the liquid and does not dissolve in the liquid (e.g., a colloidal suspension in which gel particles are formed within the liquid), the resulting mixture can be heterogeneous. Alternatively or alternatively, a solid can be mixed with a semi-solid (such as a gel). When the solid dissolves in the gel, the resulting mixture can then be homogeneous, while when the solid is suspended in the gel and does not dissolve in the gel (e.g., a colloidal suspension in which solid particles are formed within the gel), the resulting mixture can then be heterogeneous. Alternatively or alternatively, a semi-solid (such as a gel) can be mixed with another semi-solid (such as a gel). When gels are miscible, for example, one gel dissolves in another gel, the resulting mixture can be homogeneous; however, when one gel is suspended in another gel and does not dissolve in the gel (e.g., forming a suspension of gel particles within the other gel), the resulting mixture can be heterogeneous. Non-limiting examples of gels include hydrogels, organic gels, and nanocomposite hydrogels.

Additionally or alternatively, the solid may be mixed with one or more other solids. Non-limiting examples of solids that can be mixed together are dry powders (e.g., agglomerated or lyophilized media, such as microspheres). Each of these solids may be flowable, i.e., capable of flowing in a manner that allows them to be miscible with each other. Depending on how and to what extent the solids are mixed with each other, the mixture of solids may be homogeneous or heterogeneous. Non-limiting examples of solids include polymers, glasses, semiconductors, metals, salts, and ceramics.

Additionally, oligonucleotides can be mixed into any suitable material, such as any suitable liquid, solid, and/or semi-solid. For example, oligonucleotides can be dissolved in a liquid, dissolved in a gel, incorporated into a solid, or coated onto the outer surface of a particulate solid. Therefore, any mixture of such materials will also contain oligonucleotides mixed into such materials. Thus, any such oligonucleotide can be amplified, sequenced, and used to detect the materials in which it is mixed, in a manner described elsewhere herein.

Any suitable method or mixer can be used, such as convection, stirring, agitation, turbulence, diffusion, or laminar mixing, to obtain a mixture of different materials. In a non-limiting example, materials can be mixed together in the waste receiver of a system (such as a sequencing system) because the system uses those materials and then disposes of them after such use.

Using oligonucleotides to detect materials in mixtures and related equipment

As described above and in more detail below, this apparatus and method can be used to detect one or more materials in a mixture of materials. Materials that can be detected using this subject matter include any suitable combination of liquids, semi-solids, and solids. For example, Figures 1A and 1B schematically illustrate exemplary apparatus and operations in a processing flow for detecting materials in a mixture using oligonucleotides. The apparatus 100 shown in Figure 1A may include a substrate 110 comprising a plurality of reservoirs, such as reservoirs 121, 122, 123, and 124. In the non-limiting example shown in Figure 1A, reservoirs 121, 122, 123, and 124 may include holes disposed within a common, integrally formed substrate 110. However, it should be understood that one or more of reservoirs 121, 122, 123, and 124, and virtually all reservoirs 121, 122, 123, and 124, may be physically separable from each other and need not be formed in a common substrate. For example, reservoirs may be physically separable from each other in a manner as described with reference to Figure 5.

In the non-limiting example shown in Figure 1A, device 100 may include a variety of materials, such as liquid 1, liquid 2, and liquid 3, but it should be understood that any other suitable type of material may be used. Each of these materials (e.g., liquids) may reside in a corresponding reservoir among these reservoirs. For example, liquid 1 may be stored in reservoir 121, liquid 2 in reservoir 122, and liquid 3 in reservoir 123. In some examples, one or more of liquids 1, 2, and 3 may contain reagents for sequencing. Illustratively, liquids 1, 2, and 3 may contain sequencing reagents selected from the group consisting of polymerases, labeled nucleotides, wash buffers, lysis reagents, and other enzymatic reagents. Illustratively, device 100 may be used in a sequencing system to obtain a mixture of two or more of these materials (e.g., liquids). Illustratively, the sequencing system may be or include the iSEQ ^™ 100 sequencing system, commercially available from Illumina Corporation (San Diego, California). The iSEQ ^™ 100 sequencing system is a benchtop system that performs sequencing-on-synthesis using pre-filled cartridges containing reservoirs that store different sequencing reagents in a manner similar to that shown in Device 100 in Figures 1A and 1B. However, it should be understood that Device 100 can be used with any other sequencing system, such as the commercially available cBot 2, NovaSeq 6000, or MiniSeq systems from Illumina, sequencing systems from other sources, and any other system in the biological, pharmaceutical, chemical, engineering, or other technical fields that actually uses one or more materials therein.

As shown in Figure 1A, device 100 may include a variety of oligonucleotides having different sequences from each other, each of which is contained in a corresponding material of these materials. For example, oligonucleotide S1 having a first sequence may be contained in liquid 1 in reservoir 121, oligonucleotide S2 having a second different sequence may be contained in liquid 2 in reservoir 122, and oligonucleotide S3 having a third different sequence may be contained in liquid 3 in reservoir 123. As described in more detail below with reference to Figures 2A to 2D, oligonucleotides S1, S2, and S3 may include adapters that can be used to amplify these oligonucleotides, and may also include unique sequences that can be used to detect liquids 1, 2, and 3, respectively, using amplified sequences of such oligonucleotides. In some examples, one or more of these oligonucleotides may be modified to be compatible with the materials provided therein. Illustratively, oligonucleotides used in liquids containing exonucleases may be modified to include a phosphate-thioester bond at the 3' end to inhibit degradation by exonucleases.

At a specific time as shown in Figure 1A, reservoir 124 may be empty. In contrast, at a specific time as shown in Figure 1B, reservoir 124 may store a mixture of two or more liquids, namely liquid 1, liquid 2, and liquid 3, as shown in Figure 1B. In some examples, reservoir 124 may be used to collect waste from processes using liquid 1, liquid 2, and/or liquid 3 (e.g., sequencing processes), and the result of such collection may include some of each of these materials. In other examples, liquid 1, liquid 2, and/or liquid 3 may be mixed together within reservoir 124, and this mixture may be used in processes (e.g., sequencing processes). The mixture within reservoir 124 may contain oligonucleotides corresponding to those materials; for example, it may contain S1, S2, and/or S3.

Samples of mixtures of at least two of these materials, such as a sample of a mixture of liquids 1, 2, and 3 as shown in Figure 1B, can be obtained, and such samples can similarly contain oligonucleotides S1, S2, and S3 as shown in the non-limiting example. The sample can have any suitable size, for example, a volume of about 1 nL to about 1 mL, or about 10 nL to about 100 μL, or about 100 nL to about 10 μL, or about 100 nL to about 100 nL, or about 1 nL to about 10 nL. The oligonucleotides in the mixture sample can be sequenced, and the sequences of those oligonucleotides can be used to detect the material corresponding to those oligonucleotides. For example, the oligonucleotides in the mixture sample can be amplified as described with reference to Figures 2A to 2D, and then sequenced. The detected sequences obtained from oligonucleotides S1, S2, and S3 can be associated, respectively, with materials 121, 122, and 123 known to contain oligonucleotides at known concentrations. For example, the detection circuitry may include a computer-readable medium and a processor configured to use information and instructions stored in the computer-readable medium. The stored information may include (i) oligonucleotide sequences, (ii) identification of materials in which the oligonucleotides having those sequences are mixed, and (iii) the concentrations of those oligonucleotides in those materials. The instructions may enable the processor to: (i) compare the detected sequences of S1, S2, and S3 with the stored oligonucleotide sequences, (ii) use such comparison to determine which oligonucleotide sequences in the stored oligonucleotide sequences correspond to the detected oligonucleotide sequences, and (iii) use the stored information to determine which materials are present in the sample for each oligonucleotide sequence in the stored oligonucleotide sequences identified as corresponding to the detected oligonucleotide sequence. Therefore, the presence of materials can be detected based on the presence of a given oligonucleotide sequence. Here, oligonucleotides S1, S2, and S3 can be used to detect the presence of corresponding materials liquid 1, liquid 2, and liquid 3 in the mixture.

Furthermore, the absolute or relative amount of material can be determined based on the quantity of a given oligonucleotide sequence. For example, a standard curve, as described below with reference to Figure 6A, can be prepared before the time required to correlate the quantity of identified reads of a specific sequence in the material with the concentration of the oligonucleotide in that material, and the slope and offset of the curve can be stored in the detection circuitry system. For example, a standard curve can be generated and stored for each of these oligonucleotides using an “internal ladder,” which may also be called a concentration ladder. More specifically, different materials can be prepared, each containing multiple oligonucleotides, each of which is present in each of these materials at different known concentrations. For example, different materials can be prepared at a specified concentration of each of these oligonucleotides, or the concentration of each of these oligonucleotides can be determined and then stored before exposure to contamination or mixing. The concentration used to generate the standard curve can be in the range of, for example, about 1 pM to about 1000 pM, or about 10 pM to about 1000 pM, or about 10 pM to about 100 pM.

A detection circuit system (e.g., a processor on a sequencing system or other type of system) can calculate the concentration of oligonucleotides in a sample of material using a standard curve (stored in a computer-readable medium) based on the amount of identified reads of the oligonucleotide sequence, for example, by comparing the amount of identified reads of the oligonucleotide sequence with the amount of identified reads in the standard curve of the oligonucleotide, and identifying the concentration of the oligonucleotide in the standard curve corresponding to that amount of identified reads in the standard curve, which corresponds to the concentration of the oligonucleotide in the sample. The concentration of the material in the sample can then be calculated using the determined concentration of the oligonucleotide in the sample and the known concentration of the oligonucleotide in the material. The detection circuit system can express the amount of material in the sample as an absolute amount (e.g., a concentration, or by multiplying the concentration by the amount of the sample being analyzed, expressed as a volume), or it can express the amount of material in the sample as a relative amount (e.g., a molar ratio, which can be calculated by dividing the concentration of one material in the sample by the concentration of one or more other materials in the sample). Materials can be detected in relatively low volumes and quantities in samples, for example, with volumes of about 0.1 nL to 500 nL, about 0.1 nL to 100 nL, about 0.1 nL to 10 nL, and about 0.1 nL to 1 nL.

Figures 2A through 2D schematically illustrate exemplary oligonucleotides for the processing flow described with reference to Figures 1A through 1B. Each oligonucleotide may include a subsequence (which may be referred to herein as a “barcode”) unique to the specific material to which the oligonucleotide is mixed, thereby allowing identification of the material upon detection of the sequence. Each oligonucleotide may also include a subsequence (which may be referred to herein as an “index”) unique to a specific volume of the material, thereby allowing the pooling of different samples (which may contain the same material but from different volumes of those materials) for sequencing purposes, while allowing identification of the specific volume from which the material in those samples originates. In the example shown, oligonucleotide S1 includes a barcode 201, a first adaptor 211, and a second adaptor 212 corresponding to the corresponding liquid 1. Similarly, oligonucleotide S2 includes a barcode 202, a first adaptor 211, and a second adaptor 212 corresponding to the corresponding liquid 2. Similarly, oligonucleotide S3 includes a barcode 203, a first adaptor 211, and a second adaptor 212 corresponding to the corresponding liquid 3. In some examples, oligonucleotides S1, S2, and S3 may comprise sequences of single-stranded DNA, double-stranded DNA, RNA, LNA, or modified nucleotides. Oligonucleotides S1, S2, and S3 may comprise nucleotides of the same type as each other (e.g., all oligonucleotides may comprise DNA, RNA, LNA, or modified nucleotides of the same type), or they may comprise nucleotides of different types as each other.

The first adaptor 211 and the second adaptor 212 may have identical sequences and can be used in the process to amplify oligonucleotides S1, S2, and S3 in the mixed sample. However, the first adaptor 211 and the second adaptor 212 may be incompatible with sequencing systems using these materials and may therefore inhibit or prevent the amplification of oligonucleotides S1, S2, and S3, but are compatible with sequencing systems that amplify and detect oligonucleotides S1, S2, and S3. That is, the first adaptor 211 and the second adaptor 212 may be orthogonal to primers used in sequencing systems using these materials and may be complementary to primers used in sequencing systems that amplify and detect oligonucleotides S1, S2, and S3. In a non-limiting example, the first adaptor 211 and the second adaptor 212 are commercially available adaptors from Illumina and can be added to barcodes 201, 202, and 203 using a tagging process, wherein transposons are used to cleave DNA into fragments and add the adaptor to both ends of the fragmented DNA. Alternatively, oligonucleotides with the desired sequence, such as those including barcodes and adaptors, can be prepared or commercially ordered.

Barcodes 201, 202, and 203 can have different sequences from each other and can therefore be used to detect materials, such as liquid 1, liquid 2, and liquid 3, and distinguish them from each other. Therefore, although some portions of the sequences of oligonucleotides S1, S2, and S3 can be identical (e.g., the first adapter 211 and the second adapter 212), the sequences of the oligonucleotides can be considered distinct from each other because barcodes 201, 202, and 203 are different from each other. The specific sequences used in barcodes 201, 202, and 203 do not need to have any specific relationship with each other other than being different, and can be virtually arbitrary. Furthermore, the lengths of barcodes 201, 202, and 203 can include any suitable number of nucleotides and do not need to have the same number of nucleotides as each other; for example, they can include about 1 to about 200 nucleotides, or about 2 to about 150 nucleotides, or about 5 to about 100 nucleotides, or about 5 to about 50 nucleotides, or about 10 to about 40 nucleotides, or about 20 to about 30 nucleotides. It should be noted that the greater the number of nucleotides in barcodes 201, 202, and 203, the more different numbers of different sequences can be prepared, and therefore the more different quantities of material can be detected using such sequences. In a non-limiting, purely illustrative example, barcodes 201, 202, and 203 are or include different fragments of a single-stranded DNA genome (icosahedral, tailless phage) of PhiX. In examples using adaptors, PhiX fragments can be generated and have adaptors 211 and 212 added thereto during the tagging process.

Additionally or alternatively, in some examples, the sequence of each oligonucleotide in oligonucleotides S1, S2, and S3 includes an index corresponding to the mixture. Such an index can be useful, for example, for pooling mixtures obtained from different samples, where the index corresponds to each such sample. In some examples, the index may be included within oligonucleotides S1, S2, and S3 before the oligonucleotides are added to their respective materials. Alternatively, the index may be added to oligonucleotides S1, S2, and S3 after samples of the mixture have been obtained. For example, Figure 2B shows amplification primers P1 and P2 that can be used to add index 220 to oligonucleotides S1, S2, and S3. Amplification primer P1 includes: a primer 231 complementary to a first surface primer in a sequencing system on which oligonucleotides S1, S2, and S3 are sequenced; an index sequence 220 corresponding to the mixture; and a primer 211' complementary to the adaptor 211 of oligonucleotides S1, S2, and S3. Amplification primer P2 comprises: primer 232 complementary to a second surface primer in a sequencing system for sequencing oligonucleotides S1, S2, and S3; an index sequence 220 corresponding to the mixture; and primer 212' complementary to an adapter 212 of oligonucleotides S1, S2, and S3. Amplification primers P1 and P2 can be mixed with a sample containing a mixture of oligonucleotides S1, S2, and S3, and both are used to amplify each of those oligonucleotides to a concentration that can be easily sequenced (e.g., using polymerase chain reaction), and provide the index sequence 220 within each of the resulting amplicons.

For example, as shown in Figure 2C, primer 211' of amplification primer P1 can hybridize with the adaptor 211 of oligonucleotides S1, S2, and S3, and primer 212' of amplification primer P2 can hybridize with the adaptor 212 of the oligonucleotide. An amplification process can then be performed that extends oligonucleotide S1 to include two examples of complement at index 220, as well as complements at primers 231 and 232; and extends amplification primers P1 and P2 so that they intersect each other and form a continuous oligonucleotide including complement at barcode 201. Each of the resulting oligonucleotides can then be further amplified (e.g., using polymerase chain reaction). Such processes can occur simultaneously for oligonucleotides S2 and S3, thereby producing an amplicon for each of these oligonucleotides in the mixture. For example, Figure 2D shows the amplicon AS1 for oligonucleotide S1, the amplicon AS2 for oligonucleotide S2, and the amplicon AS3 for oligonucleotide S3. Oligonucleotides S1, S2, and S3 can be selected to amplify at approximately the same rate as each other, and therefore the concentration of amplicon can be expected to be proportional to the concentration of oligonucleotides S1, S2, and S3. In some examples, primers 231 and 232 can hybridize with corresponding surface primers in the sequencing system and are used to further amplify amplicon at the surface to form clusters that can be sequenced appropriately, for example, using sequencing-by-synthesis.

When amplicons AS1, AS2, and AS3 are sequenced, the sequence may include: corresponding barcodes 201, 202, and 203 for oligonucleotides S1, S2, and S3, respectively, in materials (e.g., liquid 1, liquid 2, and liquid 3), in which those oligonucleotides have been provided; and an index 220 corresponding to the mixture containing those oligonucleotides. Different indices may be included in oligonucleotides obtained from other mixtures, independent of whether any oligonucleotides in those mixtures originate from the same material as each other. Therefore, when the resulting amplicons of those oligonucleotides are sequenced, the sequence may include corresponding barcodes for materials in which those oligonucleotides have been provided, and an index corresponding to the mixture containing those oligonucleotides. If desired, amplicons from different mixtures can be pooled together and sequenced together in a co-process, and the sequence of each such amplicons can be used to identify materials with the corresponding oligonucleotides added (using barcodes) and mixtures containing those oligonucleotides (using indices). This pooling avoids the need to run separate (e.g., sequential) sequencing processes and allows all materials in multiple mixtures to be detected at the same time each other.

It should be noted that the index need not be added to the oligonucleotides from the mixture, for example, in the manner described with reference to Figures 2B and 2C. Instead, the oligonucleotides can be amplified using amplification primers P1 and P2 that omit index 220 and are linked to primers 231 and 232 that can hybridize with surface primers in the sequencing system, or primers 211 and 212 for oligonucleotides S1, S2, and S3 can be directly hybridized with surface primers in the sequencing system, causing the oligonucleotides to be amplified at that surface.

It should be understood that although Figures 1A-1B and 2A-2C illustrate the use of mixtures of three materials, mixtures of any suitable quantity of materials can be similarly processed to detect materials in such mixtures using the sequence of the oligonucleotide provided within those materials. It should also be understood that not every material must contain the oligonucleotide, and not every mixture needs to contain multiple or (even one) of the materials that include the oligonucleotide. For example, one or more of these materials may contain the oligonucleotide, and a given mixture may or may not contain that material, and therefore may or may not contain the corresponding oligonucleotide. Based on the absence of the oligonucleotide in the mixture, it can be determined that the corresponding material is substantially absent from the mixture.

It should also be understood that the concentration of a given material may not necessarily be uniform throughout the entire volume of the mixture. As provided herein, the sequence of an oligonucleotide can be used to determine the amount or concentration of a given material at any given subvolume within the volume of the mixture. For example, Figure 5 schematically illustrates another exemplary apparatus and operation in a processing flow for detecting materials in a mixture using oligonucleotides. The apparatus 500 shown in Figure 5 includes a first material reservoir 521 in which material 1 having a first oligonucleotide S1 is stored, a second material reservoir 522 in which material 2 having a second oligonucleotide S2 is stored, and a third material reservoir 533 that receives material 1 from the first reservoir 521 via conduit 531 and material 2 from the second reservoir 522 via conduit 532. The third reservoir 523 can receive any suitable amount of material from any suitable number of reservoirs by any suitable structure, and any suitable amount of such material can contain the corresponding oligonucleotide. Material 1 and material 2 can be mixed with each other within the third reservoir 523 using any suitable method or mixer, such as convection, stirring, agitation, turbulence, diffusion, or laminar flow mixing. Through this mixing, materials 1 and 2 can be distributed throughout the third reservoir 523, and thus oligonucleotides S1 and S2 can be distributed throughout the third reservoir 523.

However, the mixture of materials 1 and 2 within the third reservoir 523 may not necessarily be homogeneous at all times (or virtually at all times), and the amounts of S1 and S2 at different volumes within the third reservoir 523 may vary accordingly. In fact, at any given time, the relative proportions (e.g., molar ratio) of materials 1 to 2 in two given volumes within the mixture may differ from the relative proportions at another given volume within the mixture. Similarly, at two different given times, even at the same volume within the mixture, the relative proportions (e.g., molar ratio) of materials 1 to 2 may be different at different times. To assess the extent to which materials 1 and 2 are mixed together at a specific sub-volume of the mixture at a particular time, a sample can be obtained from that sub-volume at that time. For example, samples corresponding to sub-volumes represented as A, B, C, and D in Figure 5 can be obtained. Samples can be obtained at any desired time, such as at different times from each other or at the same time from each other. Illustratively, samples may include predefined sub-volumes respectively placed at the locations where a suitable sampling device (such as a pipette (not specifically shown)) is actuated. The oligonucleotides in each of these samples can be sequenced in a manner as described with reference to Figures 1A to 1B, and the amount of those oligonucleotides is used to determine the amount of material 1 and material 2 in each of these samples.

For example, batches of materials containing known or defined amounts of oligonucleotides can be mixed using a design or method of interest. The ratio of oligonucleotides expected to be detected in a sample of the mixture is proportional to the ratio of the materials within that sample, thus providing a quantitative measurement of the partitioning or mixing event. Repeating such measurements multiple times in different samples within the same run and/or in different runs can provide a measure of variance within or between runs. Therefore, in the development of a design or method, this subject provides a sophisticated measurement tool to evaluate mixing results. For example, this measurement tool can be used to indicate which components are not present in the expected amounts in specific regions of a mixing vessel or reaction vessel, and thus identify design or method features that can be improved. After changes to the design or method, measurements can be repeated to evaluate the effects of those changes relative to previous iterations. In other examples, oligonucleotides can be used to confirm washing protocols or batch-to-batch carryover contamination, or whether equipment is used for different reagents or products.

It should also be understood that any suitable sequence of operations and order can be used to use the materials in the oligonucleotide detection mixture. For example, Figure 3 schematically illustrates exemplary operations in a processing flow for using materials in an oligonucleotide detection mixture. The processing flow 300 shown in Figure 3 may include providing materials (operation 310). For example, any suitable quantity of materials may be provided in a separate reservoir as described with reference to Figure 5, or may be provided in a reservoir disposed in a common substrate as described with reference to Figures 1A to 1B. These materials may be independently selected from the group consisting of liquids, semi-solids, and solids. In a non-limiting example, at least one of these materials contains reagents for the sequencing process.

The processing flow 300 shown in Figure 3 may further include providing oligonucleotides with different sequences from each other, each of these oligonucleotides being contained within and corresponding to a corresponding material among these materials (operation 320). For example, each of these materials may contain an oligonucleotide of known concentration, the sequence of which differs from the sequence of oligonucleotides contained in any other material among these materials. It should be noted that not every material needs to contain such oligonucleotides. In a manner described with reference to Figures 2A through 2D, the sequence of each of these oligonucleotides may include a barcode corresponding to the corresponding material. In some examples, an index corresponding to a mixture may be contained within or added to each of these oligonucleotides. In some examples, the oligonucleotides comprise single-stranded DNA; however, it should be understood that other types of oligonucleotides may be used.

The processing flow 300 shown in Figure 3 may further include obtaining a mixture of at least two of these materials, the mixture containing oligonucleotides corresponding to those materials (operation 330). In some examples, as described with reference to Figures 1A and 1B, the mixture may be obtained using a first sequencing system. The mixture may, but does not necessarily, contain waste from the first sequencing system. In other examples, as described with reference to Figure 5, the mixture may be obtained using a mixer disposed within a reservoir that receives materials from other reservoirs.

The processing flow 300 shown in Figure 3 may further include sequencing the oligonucleotides in the mixture (operation 340). Sequencing the oligonucleotides in the mixture may include amplifying the oligonucleotides on a surface to generate corresponding amplicon clusters on the surface. For example, the oligonucleotides may be amplified in a manner as described with reference to Figures 2A to 2D, for example, to add an index and/or to add primers complementary to the surface primers in the sequencing system used to sequence the oligonucleotides. Such sequencing systems may differ from sequencing systems that use fluids themselves. In some examples, the sequences of the oligonucleotides may include adaptors that are compatible with the sequencing system used to sequence the oligonucleotides but incompatible with sequencing systems that use fluids themselves.

The processing flow 300 shown in Figure 3 may also include detecting the material corresponding to those oligonucleotides using the sequences of those oligonucleotides (operation 350). For example, the sequence of the oligonucleotide can be compared with a stored sequence corresponding to the oligonucleotide provided within the respective material. In some examples, the corresponding amount of the oligonucleotide can be quantified, and such amount of oligonucleotide can be correlated with the amount of the corresponding material, for example using a standard curve as described elsewhere herein.

Figure 4 schematically illustrates an exemplary operation in another processing flow 400 for detecting materials in a mixture using oligonucleotides. The processing flow 400 shown in Figure 4 may include mixing at least two materials with each other to obtain a mixture, with different oligonucleotides having different sequences disposed within these materials (operation 410). These materials may be mixed in any suitable reservoir, such as a waste reservoir (e.g., as described with reference to Figures 1A-1B) or a mixing reservoir (e.g., as described with reference to Figure 5). In some examples, the materials may be selected from the group consisting of liquids, semi-solids, and solids. In a non-limiting example, the materials contain reagents for sequencing and/or may be provided in a substrate comprising multiple reservoirs, each material in a corresponding reservoir among these reservoirs, for example, as described with reference to Figures 1A-1B; however, it should be understood that any suitable type of material and reservoir arrangement, as well as the manner in which the materials are mixed, may be used. The oligonucleotides may include single-stranded DNA, but other suitable types of oligonucleotides may be used. In a non-limiting, purely illustrative example, the mixture is obtained using a first sequencing system.

The processing flow 400 shown in Figure 4 may include detecting materials in the mixture using the sequences of oligonucleotides (operation 420). As described with reference to Figures 2A and 2B, the sequence of each of these oligonucleotides may include an index corresponding to the mixture and/or may include a barcode corresponding to the respective material. As described, for example, with reference to Figures 1A and 1B, materials may be detected by comparing the sequences of the oligonucleotides with stored sequences. Additionally, in some examples, the materials are detected by quantifying the corresponding amounts of these oligonucleotides and correlating these amounts of the oligonucleotides with the amounts of the corresponding materials. In a non-limiting, purely illustrative example, the oligonucleotides in the mixture are sequenced using a second sequencing system different from the first sequencing system. In such examples, the sequences of the oligonucleotides may include adaptors compatible with the second sequencing system and incompatible with the first sequencing system to suppress sequencing of the oligonucleotides on the first sequencing system.

Working Example

Further examples are disclosed in more detail in the following embodiments, which are not intended to limit the scope of the claims in any way.

Figures 6A through 6C schematically illustrate exemplary measurement results in a processing flow for detecting liquids in a mixture using oligonucleotides. More specifically, a set of oligonucleotides is prepared in which 26 bases from different corresponding portions of the PhiX genome are used as barcodes, and 34 bases from a NEXTERA ^™ PCR-tagged adaptor sequence are provided as adaptors on each side, as shown in Figure 2A. A standard curve as shown in Figure 6A is prepared using the internal ladder method described above, where the percentage of identified reads is correlated with the oligonucleotide concentration.

The different oligonucleotides from these oligonucleotides were mixed into each liquid in a pre-filled cartridge of an iSEQ ^™ 100 sequencing system, commercially available from Illumina (San Diego, California). Sequencing operations using the iSEQ ^™ 100 sequencing system were performed with the different liquids in which they were used, and waste from those operations was collected, with the oligonucleotides amplified and then sequenced. The amount of each liquid in the waste sample was determined using the sequences of the oligonucleotides and the amounts of those sequences in the waste, along with a standard curve (Figure 6A) and the known concentrations of those sequences in the different liquids. More specifically, the known concentrations of the oligonucleotides in the original solutions were used to understand their corresponding dilutions in the waste pool. For example, if the corresponding concentrations of the oligonucleotides in the first and second reagents entering the waste were initially the same, and in the sample from the waste pool, the first oligonucleotide had 300 reads and the other oligonucleotide had 700 reads, then the first reagent was determined to comprise 30% of the waste solution. This concentration can be converted to volume using the total volume of the waste.

Figure 6B illustrates the volumes of different liquids in a waste sample from an exemplary process from the iSEQ ^™ 100 sequencing system, and Table 1 below lists these volumes. It should be noted that some liquids (such as HCX1, HCX2, and HCX3) each contain multiple oligonucleotides.

Table 1

As can be understood from Figure 6B and Table 1, this oligonucleotide can be used to determine the corresponding volume of each material in a mixture of several materials (e.g., a mixture of more than 10 liquids, more than 20 liquids, or more than 30 liquids) with sub-nanolithic precision (e.g., to detect volumes of about 0.1 nL to 500 nL, about 0.1 nL to 100 nL, about 0.1 nL to 10 nL, about 0.1 nL to 1 nL).

Additionally, the mixing ratios of the different liquids are determined. More specifically, Figure 6C shows the percentages of liquids HCX1, HCX2, and HCX3 obtained from a sample of the liquid mixture. As shown in Figure 6C, the “ideal” mixture of the three liquids contains 31.48% HCX1, 15.74% HCX2, and 31.48% HCX3, but the determined sample contained 29.03% HCX1, 16.49% HCX2, and 33.18% HCX3. Based on Figure 6C, it can be understood that this oligonucleotide can be used to determine the relative percentages of each material in the mixture and can be used to evaluate process improvements for bringing the actual mixture of the three materials closer to the “ideal” mixture for the desired process, for example, by increasing or decreasing the amount of one or more materials added to the process.

Additional notes

It should be understood that any corresponding feature/example of each aspect of this disclosure as described herein may be implemented together in any suitable combination, and any feature/example from any one or more of these aspects may be implemented together with any feature of other aspects as described herein in any suitable combination to achieve the benefits described herein.

While various illustrative examples have been described above, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the invention. The appended claims are intended to cover all such changes and modifications that fall within the true spirit and scope of the invention.

Claims (44)

1. A method for detecting materials in a mixture of materials, the method comprising: Provide materials; Oligonucleotides with different sequences are provided, each of the oligonucleotides being present in and corresponding to a specific material in the material; Obtain a mixture of at least two of the materials, the mixture comprising the oligonucleotides corresponding to those materials; Sequencing of the oligonucleotides in the mixture; and The material corresponding to those oligonucleotides is detected by using the sequence of those oligonucleotides. 2. The method of claim 1, wherein the material comprises reagents for sequencing. 3. The method according to claim 1 or claim 2, wherein the materials are respectively provided in a reservoir of the substrate. 4. The method according to any one of claims 1 to 3, wherein the sequence of each oligonucleotide in the oligonucleotide comprises: (i) an index corresponding to the mixture and (ii) a barcode corresponding to the respective material. 5. The method according to any one of claims 1 to 4, wherein the sequence of each oligonucleotide in the oligonucleotide includes a barcode corresponding to the respective material, and the method further comprises adding an index corresponding to the mixture to each oligonucleotide in the oligonucleotide. 6. The method of claim 5, wherein the index is added after the mixture is obtained. 7. The method according to any one of claims 1 to 6, wherein the mixture is obtained using a first sequencing system. 8. The method of claim 7, wherein the mixture comprises waste from the first sequencing system. 9. The method of claim 7 or claim 8, wherein the oligonucleotides in the mixture are sequenced using a second sequencing system different from the first sequencing system. 10. The method of claim 9, wherein the sequence of the oligonucleotide comprises an adapter compatible with the second sequencing system and incompatible with the first sequencing system. 11. The method according to any one of claims 1 to 10, wherein sequencing the oligonucleotides in the mixture comprises amplifying the oligonucleotides on a surface to generate corresponding amplicon clusters on the surface. 12. The method according to any one of claims 1 to 11, wherein detecting the material comprises comparing the sequence of the oligonucleotide with a stored sequence. 13. The method according to any one of claims 1 to 12, wherein detecting the material comprises quantifying a corresponding amount of the oligonucleotide and relating the amount of the oligonucleotide to the amount of the corresponding material. 14. The method according to any one of claims 1 to 13, wherein the oligonucleotide comprises a sequence of single-stranded DNA, double-stranded DNA, RNA, LNA, or modified nucleotides. 15. The method according to any one of claims 1 to 14, wherein each of the materials is independently selected from the group consisting of liquids, semi-solids, and solids. 16. The method of claim 15, wherein the liquid comprises a solvent or a reagent. 17. The method of claim 15 or claim 16, wherein the solid comprises dry powder or powder in a liquid. 18. The method according to any one of claims 15 to 17, wherein the semi-solid comprises a gel. 19. A method for detecting a material in a mixture of materials, the method comprising: Mix at least two materials together to obtain a mixture. Different oligonucleotides with different sequences are placed in different materials within the material; The sequence of the oligonucleotide is used to detect the material in the mixture. 20. The method of claim 19, wherein the material comprises reagents for sequencing. 21. The method according to claim 19 or claim 20, wherein the material is provided in a reservoir of the substrate. 22. The method according to any one of claims 19 to 21, wherein the sequence of each oligonucleotide in the oligonucleotide comprises one or both of the following: (i) an index corresponding to the mixture and (ii) a barcode corresponding to the respective material. 23. The method according to any one of claims 19 to 22, wherein the mixture is obtained using a first sequencing system. 24. The method of claim 23, wherein the mixture comprises waste from the sequencing system. 25. The method of claim 23 or claim 24, wherein the oligonucleotides in the mixture are sequenced using a second sequencing system different from the first sequencing system. 26. The method of claim 25, wherein the sequence of the oligonucleotide comprises an adaptor compatible with the second sequencing system and incompatible with the first sequencing system. 27. The method according to any one of claims 19 to 26, wherein the material is detected by comparing the sequence of the oligonucleotide with a stored sequence. 28. The method according to any one of claims 19 to 27, wherein the material is detected by quantifying the corresponding amount of the oligonucleotide and correlating the amount of the oligonucleotide with the amount of the corresponding material. 29. The method according to any one of claims 19 to 28, wherein the oligonucleotide comprises a sequence of single-stranded DNA, double-stranded DNA, RNA, LNA, or modified nucleotides. 30. The method according to any one of claims 19 to 29, wherein each of the materials is independently selected from the group consisting of liquids, semi-solids, and solids. 31. The method of claim 30, wherein the liquid comprises a solvent or a reagent. 32. The method of claim 30 or claim 31, wherein the solid comprises dry powder or powder in a liquid. 33. The method according to any one of claims 30 to 32, wherein the semi-solid comprises a gel. 34. An apparatus comprising: A substrate, the substrate including a plurality of storage devices; Multiple materials, each of which is contained in a corresponding reservoir in the reservoir; and Multiple oligonucleotides, wherein the multiple oligonucleotides have different sequences from each other, and each oligonucleotide is present in a corresponding material in the material. 35. The device of claim 34, wherein the material comprises reagents for sequencing. 36. The device according to claim 34 or claim 35, wherein the sequence of each oligonucleotide in the oligonucleotide comprises one or both of the following: (i) an index corresponding to the mixture and (ii) a barcode corresponding to the respective material. 37. The apparatus according to any one of claims 34 to 36, wherein the apparatus is used in a sequencing system to obtain a mixture of two or more materials of the material. 38. The device of claim 37, wherein the mixture comprises waste from the sequencing system. 39. The device of claim 37 or claim 38, wherein the sequence of the oligonucleotide includes an adaptor incompatible with the sequencing system. 40. The device according to any one of claims 34 to 39, wherein the oligonucleotide comprises a sequence of single-stranded DNA, double-stranded DNA, RNA, LNA, or modified nucleotides. 41. The apparatus according to any one of claims 34 to 40, wherein each of the materials is independently selected from the group consisting of liquids, semi-solids, and solids. 42. The apparatus of claim 41, wherein the liquid comprises a solvent or a reagent. 43. The device according to claim 41 or claim 42, wherein the solid comprises dry powder or powder in a liquid. 44. The device according to any one of claims 41 to 43, wherein the semi-solid comprises a gel.