Next-generation sequencing for protein measurement

Abstract

A method for detecting and quantifying a target molecule, such as a protein, in a biological sample is provided. The disclosed method includes capturing the target molecule with an aptamer, replacing the aptamer with an aptamer-identifying sequence, and sequencing the aptamer-identifying sequence using next-generation sequencing technology.

Description

[Technical field]

[0001] Related Applications

[0002] The following specifications and materials are incorporated herein in their entirety for all purposes: U.S. Provisional Patent Application No. 63 / 294,964, filed December 30, 2021.

[0003] Technical Field The present disclosure relates to systems and methods for quantitatively measuring proteins in biological samples. More specifically, the disclosed embodiments relate to capturing target proteins with specially designed aptamers, creating an elution of the aptamers that have captured the target proteins, and then replacing the aptamers in the elution with "reporter" DNA molecules that can be sequenced more easily than the aptamers themselves.

[0004] Overview Traditionally, various attempts to evaluate gene activity and / or decipher biological processes, such as disease processes or pharmacological biological processes, have focused on genomics. However, proteomics can provide additional information about the biological functions of cells and organisms. Proteomics involves the qualitative and quantitative measurement of gene activity by detecting and quantifying expression at the protein level, rather than at the gene level. Proteomics also includes the study of events that are not genetically encoded, such as post-translational modifications of proteins and interactions between proteins.

[0005] It is now possible to obtain vast amounts of genomic information. DNA chips have become practical as molecular arrays for this purpose, and the price of direct DNA sequencing continues to fall significantly. Similarly, the demand for high-throughput proteomics is also increasing. For health monitoring, proteomics is far superior to genomics because the genome is static and only represents medical potential, whereas the proteome changes dynamically in response to and may even define the medical condition of the patient. However, whereas proteins are difficult to detect and quantify, nucleic acids are relatively easy to detect and quantify, at least in part because proteins have more complex and more diverse biological functions than DNA. For this reason, many efforts are underway to measure mRNA (messenger RNA) concentrations as a proxy for protein concentrations. However, mRNA concentrations have been shown to not correlate well with protein concentrations. Proteomics appears to depend on the ability to detect proteins directly.

[0006] One method of detecting and quantifying the presence of specific proteins in biological samples is through the use of protein capture SOMAmer® (Slow Off-rate Modified Aptamer) reagents. SOMAmer reagents are composed of chemically modified nucleotides that greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the SOMAmer reagents are selected. Assays using SOMAmer reagents measure native proteins in complex matrices by converting each individual protein concentration to a corresponding SOMAmer reagent concentration, which is then quantified by standard DNA techniques such as microarrays or qPCR.

[0007] SOMAmer reagents are single-stranded DNA-based protein affinity reagents that contain chemically modified nucleotides that mimic amino acid side chains, expanding the chemical diversity of standard aptamers and enhancing the specificity and affinity of protein-nucleic acid interactions. These modified nucleotides are incorporated into a nucleic acid library that is used in an iterative selection and amplification process called SELEX (Systematic Evolution of Ligands by EXponential enhancement), from which SOMAmer reagents are selected. By using a SELEX-type process, SOMAmer reagents can be generated to capture proteins that were resistant to selection by unmodified nucleic acids (ACTG traditional aptamers). SOMAmer reagents can also be adapted to select for the desired properties of specificity and slow off-rates, as well as to mimic the assay conditions in which the reagents will be used.

[0008] SOMAmer-based assays convert the presence of protein in a sample into a specific SOMAmer-based DNA signal. A SOMAmer-protein binding step is followed by a series of partitioning and washing steps in which the relative protein concentration is converted into a measurable nucleic acid signal that is quantified using DNA detection techniques such as hybridization of fluorescently labeled SOMAmers to custom DNA microarrays. Upon laser scanning of the microarray, the readout in relative fluorescence units (RFU) is directly proportional to the amount of target protein in the initial sample.

[0009] Quantification of protein detection using microarray hybridization has various drawbacks. These drawbacks include limited scalability, constant assay costs, and limited commercial sources of microarrays. Therefore, it is desirable to develop an alternative method to quantify SOMAmer molecules in the eluate after capture. Summary of the Invention

[0010] The present disclosure provides systems, devices, and methods for protein detection and quantification using a parallel sequencing technology known as next generation sequencing or NGS. More specifically, the present disclosure relates to a hybridization capture (HC) technology in which SOMAmer elution molecules that signal protein capture are replaced with "reporter" DNA molecules that contain SOMAmer-specific identification tags or "SOMA IDs," which can then be sequenced using NGS technology.

[0011] In some embodiments, the present disclosure provides systems and methods for quantifying the abundance of a target protein in a biological sample, comprising: capturing the target protein by exposing the biological sample to a plurality of aptamers, each configured to bind to a specific protein; isolating an aptamer that captures one of the target proteins in an aptamer-containing eluate; and forming a plurality of trimolecular complexes by exposing the aptamers in the eluate to a plurality of capture probes, each configured to hybridize to a specific aptamer, wherein each trimolecular complex comprises one of the aptamers from the eluate and a first portion of the aptamer hybridized to the first portion of the aptamer. forming a first capture probe comprising a portion hybridized to a second portion of the aptamer, a DNA primer region, and a second capture probe comprising an aptamer ID sequence corresponding to the aptamer; separating the trimolecular complex from capture probes that are not bound to the aptamer; dissociating the capture probes in the trimolecular complex from their corresponding aptamers; amplifying the aptamer ID sequence in the dissociated capture probe; sequencing the aptamer ID sequence by next generation sequencing; and determining the abundance of the target protein in the biological sample based on data obtained from sequencing the aptamer ID sequence.

[0012] In some embodiments, the present disclosure relates to systems and methods for quantifying the abundance of two or more target proteins in a biological sample, comprising: capturing the target proteins by exposing the biological sample to a plurality of aptamers, each configured to capture a particular protein; forming an aptamer-containing eluate by isolating an aptamer that captured one of the target proteins in the biological sample; forming a plurality of trimolecular complexes, each comprising a particular aptamer present in the aptamer-containing eluate, a first probe hybridized to a corresponding first portion of the particular aptamer, and a portion hybridized to a corresponding second portion of the particular aptamer, at least one DNA primer region, and a second probe comprising an aptamer ID sequence corresponding to the particular aptamer; amplifying the aptamer ID sequence; sequencing the aptamer ID sequence; and quantifying the abundance of the target proteins based on the sequenced aptamer ID sequence.

[0013] In some embodiments, the present disclosure relates to systems and methods for detecting a target protein in a biological sample, comprising: combining the biological sample with a plurality of aptamers, each configured to capture a specific protein, thereby capturing the target protein with an aptamer; forming a trimolecular complex comprising the aptamer that captured the target protein, a first probe comprising a portion hybridized to a corresponding first portion of the aptamer that captured the target protein, and a second probe comprising a portion hybridized to a corresponding second portion of the aptamer that captured the target protein, at least one DNA primer region, and an aptamer ID sequence corresponding to the aptamer that captured the target protein; amplifying the aptamer ID sequence; and sequencing the aptamer ID sequence to identify the aptamer ID sequence, thereby identifying the aptamer that captured the target protein and the target protein.

[0014] In some embodiments according to aspects of the present disclosure, the aptamers used to capture the target proteins can be directly sequenced, for example using next generation sequencing techniques, without the use of trimolecular complexes to convert the aptamers into simpler sequences.

[0015] In some embodiments according to aspects of the present disclosure, the aptamer used to capture the target protein may be a SOMAmer.

[0016] In some embodiments according to aspects of the present disclosure, the aptamer-containing eluate may be divided into groups before and / or after exposure to the hybridized probe regions. In some instances, some or all of the eluate groups may be diluted to a desired extent.

[0017] In some embodiments according to aspects of the present disclosure, a quantitative spike reporter may be added at any desired assay stage to correct for complementary changes in analyte count proportionality.

[0018] The features, functions, and advantages may be achieved independently in various embodiments of the present disclosure or may be combined in yet other embodiments, further details of which can be seen by reference to the following description and drawings. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is a schematic diagram of a trimolecular complex comprising an aptamer and two probes hybridized to the aptamer, according to an embodiment of the present disclosure. [Figure 2A] 2 is a flow chart showing steps of an exemplary method for making the hybridization probes necessary to make the trimolecular complex of FIG. 1 in accordance with an embodiment of the present teachings. [Figure 2B] 2 is a flow chart illustrating steps of an exemplary method for making the trimolecular complex of FIG. 1 in accordance with an embodiment of the present teachings. [Figure 3A]2 is a flow chart showing steps of an exemplary method for performing a next generation sequencing assay using a trimolecular complex, such as the complex shown in FIG. 1, in accordance with an embodiment of the present teachings. [Figure 3B] 2 is a flow chart showing steps of an exemplary method for performing a next-generation sequencing assay including capturing a target protein and then forming a trimolecular complex, such as the complex shown in FIG. 1, in accordance with an embodiment of the present teachings. [Figure 4] FIG. 1 is a flow diagram showing the steps and by-products of an exemplary next-generation sequencing assay including four hybridization groups, in accordance with an embodiment of the present teachings. [Diagram 5] FIG. 1 is a flow diagram showing the steps and by-products of an exemplary next-generation sequencing assay including a single hybridization group and four PCR groups, in accordance with an embodiment of the present teachings. [Figure 6] FIG. 1 is a flow diagram showing the steps and by-products of an exemplary next-generation sequencing assay including four hybridization groups and four PCR groups, in accordance with an embodiment of the present teachings. [Figure 7] 1 is a graph showing the results of a hypothetical simplified two-analyte assay of two separate samples in histogram format. [Figure 8] 8 is a graph showing the results of the assay of FIG. 7 where both samples were spiked with a quantitative spike (qSpike) control reporter added at the same concentration, in accordance with an embodiment of the present teachings. [Figure 9] 1 is a graph showing, in histogram format, the raw results of a three analyte SOMAmer assay of eight separate samples at different analyte concentrations, with four different qSpike reporters added to each sample, in accordance with an embodiment of the present teachings. [Figure 10] 10 is a graph showing normalized results of the assay of FIG. 9 in histogram form in accordance with an embodiment of the present teachings. [Figure 11]FIG. 1 is a flow diagram showing several steps and by-products of an exemplary next generation sequencing assay including four PCR groups and a qSpike control reporter added to each group, in accordance with an embodiment of the present teachings. [Figure 12] 1 is a graph of relative fluorescence units (RFU) versus temperature overlaid with experimental single-phase thermal melting data and a two-state theoretical model curve fit for a SOMAmer probe duplex in accordance with an embodiment of the present teachings. [Figure 13] 1 is a graph of relative fluorescence units (RFU) versus temperature overlaid with experimentally obtained biphasic thermal melting data and a biphasic theoretical model curve fit for a SOMAmer probe duplex in accordance with an embodiment of the present teachings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Various aspects and examples of hybridization-capture, next-generation sequencing assay systems for protein detection and quantification, and related methods are described below and illustrated in the associated figures. Unless otherwise specified, a protein assay according to the teachings of the present invention, and / or various components thereof, may contain at least one of the structures, components, functions, and / or variations described, illustrated, and / or incorporated herein. Furthermore, unless specifically excluded, the process steps, structures, components, functions, and / or variations described, illustrated, and / or incorporated in connection with the teachings of the present invention may be included in other similar devices and methods, including interchangeable between the disclosed embodiments. The following description of various examples is merely illustrative in nature and is not intended to limit the disclosure, its application, or use. Furthermore, the advantages provided by the examples and embodiments described below are exemplary in nature, and not all examples and embodiments provide the same advantages or the same degree of advantages.

[0021] The Detailed Description includes the following sections immediately following; (1) Definitions; (2) Overview; (3) Examples, Components, and Alternatives; (4) Advantages, Features, and Benefits; and (5) Conclusion. The Examples, Components, and Alternatives section is further divided into subsections, each of which is appropriately labeled.

[0022] definition Unless otherwise stated, the following definitions apply in this document:

[0023] "Comprising," "including," and "having" (and their conjugations) are used interchangeably to mean including, but not necessarily limited to, and are open-ended terms that are not intended to exclude additional, unrecited elements or method steps.

[0024] Terms such as "first," "second," and "third" are used to distinguish or identify various members of a group, etc., and are not intended to denote a sequential or numerical limitation.

[0025] "AKA" means "also known as" and may be used to indicate an alternative or corresponding term for a given element or elements.

[0026] Orientational terms such as "up," "down," "vertical," "horizontal," and the like, are to be understood in the context of the particular object of interest. For example, the object may be oriented about defined X, Y, and Z axes. In these examples, the XY plane defines the horizontal, with up being defined as the positive Z direction and down being defined as the negative Z direction.

[0027] "Providing" in the context of a method may include receiving, obtaining, purchasing, manufacturing, producing, processing, pre-processing, and / or the like, such that the object or material provided is in a condition and configuration for performing other steps.

[0028] "NGS" stands for "next generation sequencing."

[0029] "HC" stands for "hybridization-capture."

[0030] "SOMAmer" refers to the "Slow Off-rate Modified Aptamer" reagent developed and manufactured by SomaLogic Operating Co., Inc. ("SomaLogic"), Boulder, Colorado.

[0031] "SOMAmer ID sequence" or "SOMA ID" or "reporter" refers to a portion of a trimolecular complex that contains a SOMAmer-specific DNA strand that can be sequenced using NGS technology.

[0032] "Quantitative Spike" or "Reporter Spike" or "qSpike" refers to an amplifiable reporter that is used to normalize complementary read counts between samples, allowing true signal changes to be recognized.

[0033] In this disclosure, one or more publications, patents, and / or patent applications may be incorporated by reference. However, such materials are incorporated only to the extent that there is no inconsistency between the incorporated materials and the descriptions and drawings set forth herein. In the event of any such inconsistency, including any inconsistency in terminology, the present disclosure will control.

[0034] overview In general, the present disclosure relates to a method for detecting and quantifying target molecules, such as proteins, in biological samples. The disclosed method may include capturing the target molecule with an aptamer, replacing the aptamer with an aptamer-identifying sequence, and then sequencing the aptamer-identifying sequence using next-generation sequencing technology. Alternatively, the disclosed method may include directly sequencing the aptamer after capturing the target molecule with the aptamer.

[0035] Examples, Components, and Alternatives The following sections describe selected aspects of protein detection and quantification using aptamers such as SOMAmer reagents, and related systems and / or methods, in which the SOMAmer is displaced via hybridization capture with a reporter DNA molecule containing a SOMAmer-specific segment that can be sequenced using next-generation sequencing technology. The examples in these sections are for illustrative purposes and should not be construed as limiting the scope of the disclosure. Each section may include one or more separate embodiments or examples, and / or contextual or related information, functions, and / or structures.

[0036] A. Exemplary Aptamers This section describes SOMAmers (Slow Off-rate Modified Aptamers), which are exemplary examples of aptamers suitable for use in conjunction with embodiments of the systems and methods described herein.

[0037] Through a method known as "Systematic Evolution of Ligands by EXponential enrichment," sometimes referred to as the SELEX process, it has been discovered that nucleic acids have three-dimensional structural diversity, much like proteins. The SELEX process is a method for in vitro evolution of nucleic acid molecules for a particular desired activity. Here, we describe SELEX for generating nucleic acid molecules that bind highly specifically to a target molecule. The SELEX process provides any class of products, called nucleic acid ligands or aptamers, each with a unique sequence and the property of specifically binding to a desired target compound or molecule. Each nucleic acid capture agent identified in SELEX is a specific ligand for a given target compound or molecule. The SELEX process is based on the unique insight that nucleic acids have sufficient capacity to form a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can act as targets.

[0038] The SELEX method applied to high affinity binding applications involves selecting from a mixture of candidate oligonucleotides, and using the same general selection scheme, stepwise repeating binding, partitioning, and amplification to achieve virtually any desired criteria for binding affinity and selectivity.The SELEX method preferably includes starting with a mixture of nucleic acids that contain any segment of randomized sequence, contacting the mixture with a target under conditions that favor binding, partitioning unbound nucleic acids from nucleic acids that specifically bind to the target molecule, dissociating the nucleic acid-target complex, amplifying the nucleic acids that have been dissociated from the nucleic acid-target complex to generate a mixture enriched in nucleic acid ligands, and then repeating the binding, partitioning, dissociating, and amplifying steps for a desired number of cycles to generate highly specific high affinity nucleic acid ligands for the target molecule.In this way, aptamers that are suitable for binding to virtually any target protein can be discovered.

[0039] More specifically, SOMAmers are protein-binding aptamers discovered through a refinement of the SELEX process, and generally have dissociation rates (t 1 / 2 ), which is the average time it takes for half of the protein-aptamer complex to dissociate. In addition, SOMAmers contain modified nucleosides that provide different built-in functionalities. These functionalities may include tags for immobilization, labels for detection, means to facilitate or control separation, amino acid-like side chains to provide better affinity with proteins, and / or the like. Modifications to improve affinity with proteins are usually chemical groups attached to the 5-position of the pyrimidine base. Functionalizing the 5-position with a protein-like moiety (e.g., benzyl, 2-naphthyl) expands the chemical diversity of the SOMAmer, which allows for high affinity binding to an even wider range of target molecules. Furthermore, some polymerases can still transcribe DNA with modifications at these positions, thus allowing the amplification required for the SELEX process.

[0040] It should be noted that although binding aptamers, including SOMAmers, are generally discovered by the SELEX process, there may be other means for selecting them.For example, as computer modeling of molecular interactions improves, it may be possible to directly calculate the ideal nucleic acid sequence for aptamers and the relevant chemical modifications for SOMAmers to generate capture reagents that are specific for a given target molecule.Other chemical techniques for screening aptamers and SOMAmers are also possible, other than SELEX.

[0041] Assays aimed at the detection and quantification of physiologically important molecules in biological and other samples are important tools in scientific research and the health care field. SOMAmers can each bind to a target molecule in a sample very specifically and with very high affinity. After appropriate washing and partitioning steps, first to remove unbound proteins and then to remove unbound SOMAmers, the SOMAmers are eluted from the resulting SOMAmer-protein complex. The SOMAmer eluate is then contacted with a microarray containing the complement of the SOMAmer, allowing the determination of the presence, absence, amount, and / or concentration of the target molecule in the sample.

[0042] B. Exemplary Hybridization Capture Assay Approaches This section describes a target hybridization-capture (HC) assay in which the SOMAmer signal from the assay eluate is displaced by a "reporter" DNA molecule that contains a SOMAmer-specific identification sequence or "SOMA ID" for sequencing.

[0043] Prior to the HC assay described in this section, a SOMAmer binding step has already been performed to obtain an eluate containing a SOMAmer reagent, which is indicative of the presence of the corresponding target protein in the sample. For example, but not limited to, the following steps may be performed to obtain a SOMAmer-containing eluate: (1) Protein-specific SOMAmer reagents labeled with a 5' fluorescent dye, a photocleavable linker, and biotin are immobilized on streptavidin (SA)-coated beads and incubated with one or more samples containing complex mixtures of proteins; (2) SOMAmer-target protein complexes are formed on the beads; (3) The beads are washed to remove unbound proteins and bound proteins are tagged with biotin. (4) SOMAmer-protein complexes are released from the beads by photolysis of the linker with UV light; (5) Incubation in a buffer containing a polyanionic competitor prevents rebinding of dissociated proteins, thereby kinetically enriching complexes specific for target proteins that bind with a slow off-rate compared to fast off-rate interactions representing binding of the target protein to its corresponding SOMAmer; (6) the SOMAmer-protein complexes are recaptured, via the biotin-tagged proteins, onto a second set of streptavidin-coated beads, followed by an additional wash step that facilitates further removal of non-specifically bound SOMAmer reagents; and (7) The SOMAmer reagents are released from the beads in a denaturing buffer to form a SOMAmer-containing eluate that is suitable for quantitative analysis.

[0044] Turning now to the hybridization-capture approach, which is the primary focus of this section, Figure 1 illustrates generally a trimolecular complex, generally designated 100, that can be used in a next generation sequencing assay to identify a target protein. Complex 100 includes a post-assay SOMAmer, SOMAmer 102, which is one of the SOMAmers remaining in the SOMAmer-containing eluate after exposure to the biological sample and (for example) the other steps described above. In other words, the presence of SOMAmer 102 in the post-assay eluate indicates the presence of the corresponding target protein (or other target molecule) in the sample.

[0045] Complex 100 further comprises a first probe 104 and a second probe 106. First probe 104 comprises a hybridization region H1 that is complementary to the left portion of SOMAmer 102 in FIG. 1. Second probe 106 comprises a hybridization region H2 that is complementary to the right portion of SOMAmer 102 in FIG. 1, common primer regions P1 and P2, and a unique SOMAmer identifying sequence I that corresponds to SOMAmer 102 as described in more detail below. s or "SOMA ID". The positions of H1 and H2 may be reversed, and the common primer regions P1 and P2, and the unique SOMAmer identification sequence I in H2 are included. S With appropriate inversion, H1 hybridizes to the right portion of SOMAmer102 and H2 hybridizes to the left portion of SOMAmer102.

[0046] Hybridization regions H1 and H2 are configured to specifically bind to their respective complementary portions of the corresponding SOMAmers and can be designed to have similar melting temperatures (Tm) to achieve uniform hybridization under a given set of assay conditions. The hybridization regions of complex 100 can be designed and produced, for example, according to the following steps.

[0047] First, the target region of the probe is determined on the SOMAmer. In the case of truncated SOMAmers, the complete SOMAmer sequence including 5 bases of the fixed region used for amplification in SELEX from each end of the random region may be used as the target region. In the case of full-length SOMAmers, the SOMAmer may be truncated in silico (i.e., computationally) to any desired length, such as a 50-mer, and then the hybridization complement is determined.

[0048] Next, a boundary is determined to divide the target region into two parts. In some embodiments, in order to achieve similar melting temperatures in the two hybridization regions, the melting temperature of a 25mer (for example) duplex between SOMAmer and both H1 and H2 is determined computationally, and then the boundary modification between the two regions is carried out stepwise until the melting temperature is maximally balanced between the H1-SOMAmer duplex and the H2-SOMAmer duplex. In other examples, different melting temperatures may be purposefully selected, for example, a first melting temperature (such as 45°C) for the H1 probe and a second melting temperature (such as 35°C) for the H2 probe.

[0049] Hybridization region can also be length-constrained.For example, both H1 and H2 can be set to a minimum length of 18mer.Similarly, for example, H2 can be set to a maximum length of 30mer, and ensure that the combined length of H2 and the remaining reporter portion of the second probe for subsequent synthesis remains less than, for example, 100 bases.Under these constraints, hybridization region H1 and H2 can be generated by computer.

[0050] Common primer regions P1 and P2, and SOMAmerID sequence I of the second probe 106 S Various factors may be considered when generating the amplicons. For example, sequencing amplicon design for counting applications must find the right balance between the desire for short, inexpensive reads and the need for sequences with sufficient length and information content to serve as identifier sequences, such as SOMAmer IDs used for counting, and barcode sequences used for multiplexing. For these reasons, when scaling content, the real estate length of the reporter region, which ultimately becomes the largest part of the sequencing template, may be limited. For example, the length of the primer regions P1 and P2 may be limited to 24 mers, and the SOMAmer ID sequence I Smay be limited in length to 15 mers, with an edit distance of at least 5, and no homopolymer longer than 2 mers in length. Other length constraints and choices are possible for both the primer region and the SOMAmer ID sequence.

[0051] Figure 2A is a flow chart illustrating steps of an exemplary method 200 for making hybridization probes H1 (104) and H2 (106) used to form the trimolecular complex of Figure 1. In step 202, a set of SOMAmer sequences to be used in the assay is provided.

[0052] In step 204, hybridization probe regions H1 and H2 are generated. These hybridization probe regions may be computationally determined under various length and / or other constraints, for example, as described above. Also, as previously described, in some instances, the hybridization regions may be distributed from a single SOMAmer complementary structure based on factors such as the balance of the melting temperatures of each region.

[0053] In step 206, each SOMAmer is assigned a unique SOMAmer ID ( S ) region is created. The SOMAmer ID region can be designed in a variety of ways. For example, S The regions can be designed "by eye" (e.g., to have maximum edit distance) or can be computer generated in combination with computer generation of hybridization regions H1 and H2. After the library of SOMAmer ID regions is generated, SOMAmerIDs are assigned randomly or in any other suitable manner to SOMAmers to create a unique reporter corresponding to each SOMAmer.

[0054] In step 208, universal primer regions P1 and P2 are created. The universal primers may be designed for stability and to reduce the risk of downstream bias. For example, in some examples, the primers may be 24-mer or 25-mer in length with an estimated melting temperature of about 70° C. In some examples, the primers may terminate with a guanine (G) at the 3′ end for stability. In some examples, the primers may be evaluated with an oligonucleotide (oligo) analyzer to reduce the potential risk of dimer formation. In some examples, the primers may be further adjusted to avoid non-specific interactions with functional oligos used in known sequencing technologies.

[0055] In step 210, first and second SOMAmer-specific probes (sometimes referred to as "capture probes") are generated. The first probes each contain a hybridization region H1 with one or more elements suitable for binding to assay beads, e.g., biotin for binding to streptavidin-coated beads. The first probes may also contain additional elements, such as a photocleavable linker. The second probes each contain a hybridization region H2, universal primer regions P1 and P2, and a SOMAmer ID sequence I. S As part of creating the second probe, a SOMAmer ID region may be added to the universal primer to create an amplifiable reporter.

[0056] Figure 2B is a flow chart illustrating steps of an exemplary method 250 for generating a trimolecular complex, such as the trimolecular complex of Figure 1. In step 252, a SOMAmer-containing eluate is provided, where the SOMAmers in the eluate indicate the presence of one or more target proteins or other target molecules in one or more biological samples that have been exposed to the SOMAmer library, as described above.

[0057] In step 254, the set or library of SOMAmer-specific capture probes produced by method 200 is combined with the post-assay SOMAmer-containing eluate. In one example, 25 μl of SOMAmer-containing eluate is combined with 25 μl of probe-containing solution to result in a hybridization volume of 50 μl. To facilitate hybridization, the capture probes may have a concentration equal to or higher than the concentration of the SOMAmers in the eluate. For example, a suitable concentration of probes may fall in the range of 0.05 nM to 5.0 nM, such as 0.5 nM (nM = nanomoles per liter).

[0058] In some examples, in optional step 253 of method 250, the SOMAmer-containing eluate may be divided and selectively diluted prior to hybridization, and then recombined prior to sequencing. More specifically, the post-assay eluate may be divided into two or more dilution groups (e.g., four dilution groups) based on the expected relative abundance of the SOMAmers within each group. The least concentrated (most diluted) sample may contain the most abundant SOMAmers in the eluate. Conversely, the most concentrated (least diluted) sample may contain the least abundant SOMAmers in the eluate. In this manner, the SOMAmer count may be "smoothed out" to improve accuracy and precision in detecting less abundant SOMAmers. Each dilution group may be hybridized separately by exposure to a corresponding subset of probes. Further details regarding the use of dilution groups are provided in subsequent sections of this disclosure.

[0059] Furthermore, equalization may be achieved by introducing a fixed ratio of capture tagged and capture untagged H1 probes for a particular high abundance SOMAmer in the eluate, and SOMAmers that form trimolecular complexes with the capture untagged H1 probes are removed during a wash step, as detailed below in method 300.

[0060] In step 256, the first and second probes hybridize to the SOMAmer, forming a trimolecular complex, each of which includes (i) the SOMAmer, (ii) the first probe bound to the SOMAmer via hybridization region H1, and (iii) the second probe bound to the SOMAmer via hybridization region H2. The second probes each include a SOMAmer ID sequence I S which can be sequenced to indicate the presence of the corresponding SOMAmer in the eluate, and thus the presence of the corresponding protein captured by that SOMAmer in the original biological sample. Hybridization of the capture probe with the SOMAmer can be carried out using any suitable technique, such as suitable thermal cycling, and may include additives to enhance the rate of hybridization.

[0061] Figure 3A is a flow chart illustrating steps of an exemplary method 300 for performing a next generation sequencing assay using trimolecular complexes, such as those depicted in Figure 1 and produced by methods such as that of Figure 2B. In step 302, hybridized trimolecular complexes (e.g., each having a structure such as that of complex 100 and produced according to a method such as method 250) corresponding to a desired library of post-assay SOMAmers are provided for a sequencing assay.

[0062] In step 304, the trimolecular complex is captured on the magnetic beads. For example, the complex may be captured via binding of biotin bound to the hybridization region H1 of the first probe with streptavidin on the beads. Capture may be achieved using any suitable technique. For example, in one example, the hybridization volume may be combined with 30 μl of a solution containing beads at a concentration of 20 mg / ml, and then mixed in a thermomixer at 1200 rpm at a temperature of 45° C. for 30 minutes.

[0063] In step 306, the solution containing the bead capture probes is washed one or more times to remove unbound H2 probe reporters, i.e. probes that are not hybridized to the corresponding SOMAmers. For example, washing may be performed with a suitable buffer, such as 20 mM phosphate buffer containing 1 mM EDTA and 0.05% SDS (sodium dodecyl sulfate). The washing steps may be performed statically, dynamically using a thermomixer, or a combination of both types in sequence. In one example, there may be two static 5 minute wash phases and two 10 minute dynamic wash phases at 1200 rpm. In either case, after washing, the resulting solution should contain trimolecular complexes bound to the beads, with at most a small amount of unbound H2 probe / reporter remaining.

[0064] In certain embodiments of SOMAmer elution leveling, or dynamic range compression, trimolecular complexes that do not have a bead capture tag on H1 are removed as well as unbound H2 probe reporters in step 306. These complexes result in fewer copies of these SOMAmers in the final NGS sequencing, thereby lowering the counts on these high abundance SOMAmers.

[0065] In step 308, the bound trimolecular complex is eluted from the bound beads, for example by exposure to solvent, heat, or any other suitable elution method. As part of this step, the components of the complex may dissociate, resulting in separated SOMAmers and probes. In one example, the complex is eluted by adding 85 μl of 20 mM NaOH to the eluate containing the bound complex, followed by mixing in a thermomixer at 1200 rpm for 3 minutes and mixing for 5 minutes to distribute the complex. The solution containing the eluted complex may then be combined with 20 μl of HCL.

[0066] In some examples, the elution solution (i.e., eluate) resulting from step 308 may be split and / or diluted into two or more groups, such as four dilution or primer amplification groups, in optional step 309 of method 300. As previously described in the context of method 250, the use of multiple dilution or primer amplification groups (which may not be diluted in some examples) corresponding to subsets of SOMAmers with different expected abundances helps to smooth out the relative abundance of the overall set of SOMAmers or compress a broad distribution to increase precision and accuracy in detecting relatively rare target molecules. Such splitting into groups can be done before hybridization (as in step 253 of method 20), after hybridization (as in step 309 described herein), or both. Further details of possible dilution and recombination techniques are provided in later sections of this disclosure.

[0067] In step 310, the solution(s) resulting from step 308 and optional step 309 are prepared for next generation sequencing (NGS), which involves sequencing the associated SOMAmer ID sequence I S The preparation for NGS may include PCR amplification of a reporter region containing a common primer (P1 and P2 in FIG. 1) with the NGS adapter. Preparation for NGS may also include attaching adapter sequences and / or barcode sequences for demultiplexing when eluates from multiple samples are combined prior to sequencing. The creation of adapter and barcode sequences and attachment to the reporter region may be accomplished by any suitable method known in the art, as is common in preparing samples for next generation sequencing. In some examples, the barcode sequence may be added as part of the first preparation step, and the NGS adapter may be added as part of the second preparation step.

[0068] In optional step 311, any groups that remain separated after step 310 may be recombined in preparation for sequencing.

[0069] In step 312, the prepared sample(s) are sequenced using next-generation sequencing technology. In some examples, the prepared sample may be sequenced using a next-generation sequencing platform developed by Illumina, Inc. (San Diego, California). However, the method disclosed in the present invention is also suitable for use with other NGS sequencing platforms.

[0070] After NGS, the data obtained by sequencing may be analyzed or otherwise processed in an optional step 314 to determine the concentration of an analyte, such as a target protein, in the original biological sample. Generally, this analysis involves demultiplexing the sequencing data using barcodes corresponding to each original sample (if multiple samples are multiplexed), counting reporter sequences, and scaling and / or normalizing the data to extract accurate results. For analytical purposes, the sequencing data may be written to a data file in a standard format, such as the ADAT format developed by SomaLogic. Possible quantitative analysis methods are discussed in more detail below.

[0071] 3B is a flow chart illustrating the steps of an exemplary method 350 for performing a next-generation sequencing assay, including capturing a target protein with an aptamer, forming a trimolecular complex from the aptamer, and using the trimolecular complex as a basis for identifying the captured target protein. It should be noted that any of the steps of method 350 may be similar to the corresponding steps of the above-mentioned methods (i.e., methods 200 and 300), and therefore the same details may not be described again.

[0072] In step 352 of method 350, a target protein is captured by exposing the biological sample to a plurality of aptamers, e.g., SOMAmers, each configured to bind to a specific protein. By exposing the sample to a library of many such SOMAmers, multiple target protein species can be detected in a single assay.

[0073] In step 354, the aptamers that have captured one of the target proteins are isolated in an aptamer-containing eluate. Forming an aptamer-containing eluate may be accomplished, for example, in the so-called SomaScan assay process implemented by SomaLogic, which includes binding aptamers to assay beads, capturing proteins with aptamers, washing away unbound proteins, tagging the bound proteins with biotin, releasing the aptamers from the beads, capturing the tagged proteins on new beads, removing unbound aptamers, denaturing the aptamers from the captured proteins, and then separating the aptamers into an eluate.

[0074] In optional step 356, the aptamer-containing eluate may be divided into multiple groups, which may be dilution groups, as described in more detail below with respect to FIGS.

[0075] In step 358, multiple trimolecular complexes are formed by exposing the aptamers in the eluate (or each eluate dilution group) to multiple capture probes, each configured to hybridize to a specific aptamer. For example, each complex may have a structure similar to that of complex 100 shown in FIG. 1. Thus, each trimolecular complex includes (i) a specific one of the aptamers from the eluate, (ii) a first capture probe that includes a portion hybridized to a first portion of the aptamer, and (iii) a second capture probe that includes a portion hybridized to a second portion of the aptamer and further includes one or more DNA primer regions and an aptamer ID sequence that corresponds to the specific aptamer. When separate dilution groups are formed, each dilution group is exposed to a different set of capture probes that correspond to a different subset of aptamers. Optionally, some of the H1 probes may lack a bead capture tag to further equalize the SOMAmer count.

[0076] In step 360, the groups formed in step 356 (if any) may be recombined.

[0077] In step 362, the trimolecular complex is separated from the capture probe that is not bound to the aptamer. For example, the hybridized complex may be captured on magnetic beads and then washed to remove unbound probe.

[0078] In step 364, the capture probes in the trimolecular complexes are dissociated from their corresponding aptamers. This may involve eluting the complexes from the beads, but in any case, the result of step 362 is that the capture probes are no longer bound to the aptamers.

[0079] In step 366, the eluate containing unbound capture probes may optionally be divided and / or diluted (perhaps a second time, as discussed below with respect to FIG. 6) to generate multiple PCR groups.

[0080] In step 368, the aptamer ID sequences in the dissociated capture probes of the eluate are amplified, for example, via PCR amplification of the DNA primer region(s) and associated ID sequences. At this stage, further preparation for NGS, for example, attachment of adapter sequences and / or demultiplexing of barcode sequences, may be performed.

[0081] In step 370, the separated PCR groups (if any) may be recombined.

[0082] In step 372, next-generation sequencing technology is used to sequence the aptamer ID sequence. In some examples, this can be accomplished using a next-generation sequencing platform developed by Illumina, Inc. (San Diego, California).

[0083] In step 374, the data obtained by sequencing the aptamer ID sequence may be used to determine the abundance of the target protein in the original biological sample.

[0084] C. Exemplary dilution groups or dynamic range compression for next generation sequencing This section describes possible methods of diluting assay eluates to obtain higher assay efficiency, reproducibility, performance, and / or production feasibility in next-generation sequencing systems in accordance with embodiments of the present teachings; see Figures 4-6.

[0085] First, it should be appreciated that it is possible to perform NGS on SOMAmer-containing eluates in a single elution solution without splitting the eluate into dilution groups, i.e., never split, diluted, or recombined. Such an assay is within the scope of the teachings of the present invention and has the advantage that less eluate is required, requiring only one hybridization plate for every 96 samples. However, such an assay has challenges with sensitivity and accuracy, for example, because the abundance of SOMAmers in the original eluate may range in extremes, perhaps spanning several orders of magnitude. For example, the target protein in the sample may have a concentration in the fM-μM range (i.e., a range of about 9 orders of magnitude), resulting in a concentration of SOMAmers in the eluate spanning 5 or more orders of magnitude. Performing an assay on such an eluate may result in overcounting of more abundant SOMAmers, and a corresponding undercounting of less abundant SOMAmers. It may therefore be desirable to smooth or compress the range of abundance of SOMAmers before sequencing and counting.

[0086] The disclosed systems and methods address this issue by splitting SOMAmers into subpopulations prior to counting, which, in combination with dilution, results in count smoothing or dynamic range compression in the subpopulations when the subpopulations are recombined for sequencing and counting. In some examples, the set of SOMAmer probes is split into subpopulations based on abundance in the SOMAmer elution (a first group with rare SOMAmers, a second group with less rare SOMAmers, a third group with more abundant SOMAmers, etc.). The dynamic range of each subpopulation is smaller, and in some examples much smaller, than the dynamic range of the original (unsplit) elution. As described below, the dilution groups can be formed before and / or after hybridization of the SOMAmers with the probes, i.e., before and / or after the formation of trimolecular complexes suitable for NGS.

[0087] In addition to leveling by dilution, high abundance SOMAmers may be "leveled" by introducing H1 probes that do not contain a bead capture tag along with the tag-containing probes. The ratio of bead capture tagged H1 probes to untagged H1 probes reduces the trimolecular complexes captured in step 304 of method 300 and step 358 of method 350 by an amount corresponding to the ratio. For example, a 10:1 ratio of untagged to tagged probes will capture only 10 percent of the trimolecular complexes, reducing the counts in the NGS output by an order of magnitude compared to an assay without the untagged probe. The ratio of untagged to tagged probes can vary from SOMAmer to SOMAmer depending on the expected counts of each SOMAmer.

[0088] 1. Four hybridization groups 4 illustrates the steps and by-products of an exemplary NGS assay, generally designated 400, involving four hybridization groups. In step 402, a SOMAmer-containing eluate 404 is provided. Eluate 404 contains SOMAmers resulting from prior exposure to a biological sample and separation from target molecules, as previously described.

[0089] In step 406, the eluate 404 is divided into four equal parts or aliquots 408, 410, 412, and 414. In the assay of Figure 4, aliquot 408 is diluted in a ratio of 1:16, i.e., 1 part eluate to 16 parts buffer; aliquot 410 is diluted in a ratio of 1:4; and aliquots 412 and 414 are each diluted in a ratio of 1:2. In some examples, the aliquots may not be diluted.

[0090] In step 416, the four aliquots are combined with groups of hybridization capture probes 418, 420, 422, and 424, labeled "Group 1," "Group 2," "Group 3," and "Group 4," respectively. In this example, capture probe group 418 is combined with the most dilute eluate and therefore contains capture probes configured to bind to the most prevalent SOMAmers in the eluate. Similarly, capture probe group 420 contains probes configured to bind to the next most prevalent SOMAmers, and capture probe groups 422 and 424 each contain probes configured to combine with a different subset of relatively low abundance SOMAmers. Thus, the result of step 416 are four separate solutions, each configured to, upon hybridization, result in a set of trimolecular compounds comprising a SOMAmer and a corresponding probe hybridized to the SOMAmer. Each compound may be generally similar to compound 100 of FIG. 1.

[0091] Any of the four groups of hybridization capture probes may contain a fixed ratio of H1 probes with bead capture tags and H1 probes without tags for some subset of SOMAmers in each group to further smooth out the counts.

[0092] In step 426, the four solutions produced in step 416 are hybridized, captured onto beads, washed, and eluted, respectively, which may be accomplished, for example, as previously described with respect to steps 304, 306, and 308 of the method 300 shown in FIG.

[0093] In step 428, the separate elution solutions resulting from step 426 are recombined into a single elution solution 430. In some examples, the separate solutions may be recombined in different volumes, resulting in further dilution of the relatively more abundant capture groups (i.e., corresponding to the more abundant SOMAmers and the more abundant target species in the original biological sample). This results in a normalized combined solution with less overall variation in the concentrations of the different trimolecular compounds, which may be analyzed with fewer sequencing "reads." For example, the combination of dilution and normalization reduces the number of required reads per sample from approximately 200 million to less than 5 million, thereby enabling multiplexing of a large number of samples per sequencing run, reducing the cost per sample while achieving acceptable precision as measured by the coefficient of variation (CV) of the results.

[0094] In step 432, which may be considered as a combination of the above steps 310, 312, and 314 of method 300, the solution 430 from step 428 is prepared for next generation sequencing (NGS), sequenced, and the results written to a data file and optionally analyzed. SThe preparation may include PCR amplification of a reporter region containing a common primer (P1 and P2 in FIG. 1) along with a common primer. As mentioned above, the preparation may also include attaching an adapter sequence and / or a barcode sequence for demultiplexing into a trimolecular compound. The prepared solution is then sequenced using an NGS technology, such as the next generation sequencing platform developed by Illumina, Inc. (San Diego, California) or other NGS sequencing platforms. After NGS, the data obtained by sequencing may be analyzed or otherwise processed to determine the concentration of an analyte, such as a target protein, in the original biological sample. This may include demultiplexing the sequencing data using the barcodes corresponding to each original sample (if multiple samples are combined), counting the reporter sequences, and scaling and / or normalizing the data to extract accurate results. The sequencing data may be written to a data file in a standard format, such as the ADAT format developed by SomaLogic.

[0095] 2. Single hybridization group with four PCR groups 5 illustrates the steps and by-products of an exemplary NGS assay, generally designated 500, with one hybridization group and four PCR groups. In step 502, a SOMAmer-containing eluate 504 is provided. Eluate 504 contains SOMAmers resulting from prior exposure to a biological sample and separation from target molecules, as described above.

[0096] In step 506, the eluate 504 is combined with a complete set of hybridization capture probes 508, i.e., a set of probes configured to combine with all SOMAmers in the eluate. The set of probes may also contain a fixed ratio of tagged and untagged H1 probes, as described above.

[0097] In step 510, the solution obtained in step 506 is hybridized, captured on beads, washed, and eluted. This may be accomplished as described with respect to steps 304, 306, and 308 of method 300 shown in FIG. 3A. However, in this example, instead of only one set, four sets of universal primers may be used, with each set of primers associating with a different set of SOMAmer IDs corresponding to a set of SOMAmers that fall into a particular expected concentration range. In other words, step 510 results in four separate groups of tri-molecular compounds corresponding to different abundance groups of SOMAmers, and therefore different abundance groups of target molecules in the SOMAmer eluate from the original biological sample, each of which includes different PCR primers and can therefore be amplified separately.

[0098] In step 512, the eluate resulting from step 510 is divided into four equal portions or aliquots 514, 516, 518, and 520. Each aliquot may be optionally diluted to any desired extent at this stage in order to normalize the expected concentration of SOMAmer ID sequences amplified in the next step. However, FIG. 5 does not show any dilution in step 512.

[0099] In step 522, the individual eluates from step 512 are prepared for next generation sequencing (NGS), which involves PCR amplification of the reporter regions. However, in this case, a different set of primers and associated reporter regions is amplified in each individual eluate, resulting in amplification of just a known subset of the SOMAmer ID sequences in each eluate.

[0100] In step 524, the separate solutions containing the amplified SOMAmer ID sequences in each aliquot are combined into a single elution solution 526. In some examples, the separate solutions can be recombined in different volumes to obtain a desired degree of dilution of the relatively abundant SOMAmer ID sequences and a normalized combined solution with less overall variation in SOMAmer ID concentrations, which can be analyzed with relatively fewer reads.

[0101] In step 528, the solution 526 is further prepared for next generation sequencing (NGS), sequenced, and the results written to a data file and analyzed as desired. Preparation of the amplified eluate may include attaching adapter sequences and / or barcode sequences for demultiplexing to the reporter regions of the trimolecular compounds. The prepared solution may then be sequenced using NGS technology, and the data obtained from the sequencing may then be analyzed or otherwise processed to determine the concentration of the target analyte in the original biological sample, as described above.

[0102] 3. Four hybridization groups and four PCR groups Figure 6 shows the steps and by-products of an exemplary NGS assay, generally designated 600, with four hybridization groups and four PCR groups, thus combining aspects of assays 400 and 500 of Figures 4-5. Step 602 provides a SOMAmer-containing eluate 604 that contains SOMAmers resulting from prior exposure to a biological sample and separation from target molecules.

[0103] In step 606, the eluate 604 is divided into four equal portions or aliquots 608, 610, 612, and 614. These aliquots may optionally be diluted to various degrees, and in some instances, the aliquots may not be diluted.

[0104] In step 616, the four aliquots are each combined with a group of hybridization capture probes 618, 620, 622, and 624, labeled "Group 1," "Group 2," "Group 3," and "Group 4," respectively, each of which is configured to bind to a different subset of SOMAmers in the eluate, and then the aliquots are individually hybridized. Thus, the result of step 616 is four separate solutions, each solution containing a set of trimolecular compounds comprising a SOMAmer and a corresponding probe hybridized to the SOMAmer. Each compound may be generally similar to compound 100 of FIG. 1.

[0105] Each of the four hybridization capture probe groups may optionally contain a fixed ratio of untagged and tagged H1 probes to further smooth out the counts.

[0106] In step 626, the four hybridized solutions produced by step 616 are combined into one elution solution 628, which is then sequentially captured by beads, washed, and eluted. This may be accomplished, for example, as described above with respect to steps 304, 306, and 308 of method 300 shown in Figure 3A. As described above, each hybridized solution may or may not be diluted before recombining, and the differential volumes of each group may be used to compress analyte variation prior to bead capture and washing.

[0107] In step 630, the eluate resulting from step 626 is divided into four equal portions or aliquots 632, 634, 636, and 638. Each aliquot may be optionally diluted to any desired extent at this stage to normalize the expected concentration of SOMAmer ID sequences amplified in the next step. However, FIG. 6 does not show any dilution in step 630.

[0108] In step 640, the individual eluates from step 630 are prepared for next generation sequencing (NGS), which involves PCR amplification of the reporter regions. Similar to assay 500 of Figure 5, a different set of primers and associated reporter regions are amplified in each individual eluate, resulting in amplification of a subset of the SOMAmer ID sequences in each eluate.

[0109] In step 642, the separate solutions containing the amplified SOMAmer ID sequences in each aliquot are recombined into a single elution solution 644. In some examples, the separate solutions can be recombined in different volumes to obtain a normalized combined solution with a desired degree of dilution of the relatively abundant SOMAmer ID sequences and less overall variation in SOMAmer ID concentrations, which can be analyzed with relatively fewer reads.

[0110] In step 646, the solution 644 is further prepared for NGS, sequenced, and the results written to a data file and analyzed as desired. Preparation of the amplified eluate may include attaching adapter sequences and / or barcode sequences for demultiplexing to the reporter regions of the trimolecular compounds. The prepared solution may then be sequenced using NGS technology, and the data obtained from the sequencing may then be analyzed or otherwise processed to determine the concentration of the target analyte in the original biological sample.

[0111] D. Quantitative spike normalization for PCR groups In NGS-based systems, signal is measured as sequence read counts, where the read counts of all analytes in a given sample are measured in the same sequencing mix with a fixed or finite set of total reads. As a proportion of the same mixture, the NGS read counts of all analytes measured in a given sample influence each other, such that the signal count observed per analyte is the "net result" of the increases and decreases of all analytes measured in a "zero-sum game" per sample with a fixed total reads. More specifically, in an NGS system with a fixed total number of reads, any increase in the count of one analyte results in a corresponding decrease in the counts of the other analytes, with the decrease being distributed according to the fractional total reads of each analyte.

[0112] FIG. 7 graphically represents this "zero-sum game" by showing a simplified two-analyte assay of two separate samples in histogram format, where the vertical axis represents the total number of read counts for each analyte, which is fixed at 2 million. In sample 1, analyte A and analyte B have equal counts. In sample 2, analyte A appears to have increased by 500,000 counts and analyte B appears to have decreased proportionately by 500,000 counts. However, because the total number of reads is finite, it is not possible to know from FIG. 7 whether the difference in counts between analyte A and analyte B in sample 2 is due to an increase in analyte A, a decrease in analyte B, or a combination of both.

[0113] FIG. 8 illustrates how the introduction of a reference reporter or quantitative spike control reporter ("qSpike") can be used to normalize complementary read counts across samples, thereby allowing for the identification of actual signal changes. In the NGS assay depicted in FIG. 8, a qSpike reporter is physically added (spiked) into a two analyte system containing analytes A and B at the same known concentration in all samples. In this example, the qSpike reporter happens to be at the same concentration as analytes A and B in sample 1. In sample 2, as before, an increase in analyte A and a decrease in analyte B are observed. However, a decrease in qSpike can be observed in sample 2 compared to sample 1, which is known to have had the same concentration of qSpike added. By scaling / adjusting all of the analytes in sample 2 to bring the spikes back to the expected concentrations, it becomes possible to clearly identify an increase in analyte A compared to analyte B, as shown by the "Sample 2 qSpike Adjusted" histogram in FIG. 8.

[0114] In a more practical NGS assay, a mixture of multiple qSpike reference reporters may be used to correct for complementary changes in analyte count proportionality. For example, an assay according to the teachings of the present invention may use a mixture of four unique H2 reporters, i.e., four unique amplifiable reporters that form part of the second probe introduced after elution of the trimolecular complex, for example, in step 308 of assay 300 shown in FIG. 3A. Alternatively, a specific qSpike SOMAmer may be introduced in the eluate, and the appropriate qSpike reporter is contained in a library of SOMAmer-specific probes. The qSpike reporters or SOMAmers may be provided at different relative concentrations.

[0115] 9-10 are histograms showing the raw and qSpike adjusted results of these assays, respectively, with legend references having the following meanings: QSpike-H = High-concentration qSpike reporter QSpike-MH = Medium-high concentration qSpike reporter QSpike-ML = Medium-low concentration qSpike reporter QSpike-L = low concentration qSpike reporter Apo E2, Transferrin, Kininogen HMW=SOMAmer Analytes

[0116] In the assay depicted in Figures 9-10, the three SOMAmers ApoE2, transferrin, and kininogen HMW were titrated in buffer at concentrations ranging from 50 pM to 50 aM and measured in the NGS HC assay. Each measurement point is a separate sample where all three SOMAmer analytes are sequenced together, and qSpike is added to all samples at the same concentration.

[0117] In graph (A) of Figure 9, qSpike exhibits a complementary shift due to the change in analyte dose-response signal. The NGS assay signal (read counts) is compared to the known spikes to generate a scale factor. In graph (B) of Figure 10, the NGS counts are scaled so that the spikes are equal for all samples, reconstructing the actual SOMAmer dose-response for the three SOMAmers measured in the assay.

[0118] The use of qSpike reporters to complement finite read counts can be incorporated into any of the next generation sequencing assays described above. For example, FIG. 11 shows some of the steps and by-products of an exemplary NGS assay, generally designated 1100, with four PCR groups and the addition of a qSpike reporter to each group. Thus, the steps of assay 1100 can be incorporated into any NGS assay that uses multiple PCR groups, such as assays 500 and 600 shown in FIGS. 5-6.

[0119] Step 1102 provides an eluate 1104 containing SOMAmers that have been hybridized to probes, captured on beads, washed, and eluted. Thus, eluate 1104 shall be considered to be generally similar to, for example, the eluate obtained from step 510 of assay 500 shown in Figure 5, or the eluate obtained from step 626 of assay 600 shown in Figure 6.

[0120] In step 1106, the eluate 1104 is divided into four equal portions or aliquots 1108, 1110, 1112, and 1114. Each aliquot may optionally be diluted to any desired extent at this stage in order to normalize the expected concentration of SOMAmer ID sequences that will be amplified in the next step.

[0121] In step 1116, the separate eluates resulting from step 1106 are prepared for next generation sequencing (NGS), including PCR amplification of the reporter regions. Similar to assays 500 and 600, a different set of primers and associated reporter regions are amplified in each separate eluate, resulting in amplification of a subset of SOMAmer ID sequences in each eluate. However, in this example, a different qSpike reporter is also added at a known concentration to each eluate prior to PCR amplification.

[0122] In step 1118, the separate solutions containing the amplified SOMAmer ID sequences and qSpike reporters in each aliquot are recombined into a single elution solution 1120. In some examples, the separate solutions can be recombined in different volumes to obtain a desired degree of dilution of the relatively abundant SOMAmer ID sequences and a normalized combined solution with less overall variation in SOMAmer ID concentrations, which can be analyzed with relatively fewer reads.

[0123] In step 1122, the solution 1120 is further prepared for NGS, sequenced, and the results written to a data file and analyzed. Preparation of the amplified eluate may include attaching adapter sequences and / or barcode sequences for demultiplexing to the amplified reporter sequences of the trimolecular compounds. The prepared solution may then be sequenced using NGS technology, and the data obtained from the sequencing may then be analyzed or otherwise processed to determine the concentration of the target analyte in the original biological sample. With the use of qSpike reporters, the analysis may include scaling or renormalizing the data to return the qSpike concentrations to known levels, thereby compensating for possible counting errors due to finite sequencing reads.

[0124] E. Determining the stability of SOMAmer probes As previously mentioned, in accordance with an embodiment of the present teachings, hybridization regions H1 and H2 are configured to bind to respective complementary portions of corresponding SOMAmers and have similar or purposefully different melting temperatures (T m ) As described in this section, in some instances, the melting temperature of the hybridization region can be computationally estimated using experimentally determined melting profiles of the SOMAmer-probe pair.

[0125] 1. Background technology The most widely used method for predicting the stability of nucleic acid duplexes is known as the nearest neighbor model. The nearest neighbor model assumes that the thermodynamic properties of helix formation depend mainly on the identity of adjacent base pairs in the duplex. This model has been widely applied for use in predicting the stability of duplex formation in primer design required for PCR and other applications where oligonucleotide duplex formation is important. For NGS assays according to the teachings of the present invention (i.e., involving SOMAmers), it is desirable to extend this method to accurately predict the stability of duplexes that are composed of one strand containing one type of modified DNA base and the second strand containing natural DNA bases.

[0126] Absorbance versus temperature profiles (melting curves) measured with a UV-vis spectrophotometer are traditionally used to examine the stability of DNA secondary structures. Hybridizations are typically performed in 1.0 M NaCl, 10 mM sodium cacodylate, and 0.5 mM Na2EDTA buffer, pH 7. Oligonucleotide concentrations are varied over a 100-fold range, and thermodynamic parameters are measured using the inverse melting temperature (T M -1 ) versus the natural logarithm of the total DNA concentration, and fitted as follows:

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[0127] According to an embodiment of the present teaching, an extension of the nearest neighbor model is developed for SOMAmers containing the three most common modified bases, Nap-dU, 2-Nap-dU, and Benzyl-dU. As discussed below, melting profiles were experimentally obtained using fluorescence measurements of over 400 SOMAmer-probe pairs. These data were used to define the nearest neighbor parameters required to predict the stability of SOMAmer probes under assay read conditions.

[0128] 2. Duplex formation to model SOMAmer-probe binding The formation of a SOMAmer-probe duplex follows the following process:

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[0129] 3.SOMAmer melting model A SOMAmer with an internal structure can also be viewed as a simple two-state model as follows:

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[0130] This is a unimolecular reaction, and under moderate dilution conditions, there is no entropic contribution from the concentration of SOMAmers, since all reactions are independent. The simplest model is to assume independent processes, where melting of the SOMAmer occurs after melting of the primer, or vice versa. Without additional experiments, such as melting of independent SOMAmers, there is no way to know a priori which of the two transitions is due to melting of the SOMAmer structure or melting of the hybridization primer. The primer melt corresponds better than the 16 base pair melt, and therefore presumably the higher free energy data.

[0131] 4. Experimental Determination of the Thermodynamics of Melting Fluorescence intensity versus temperature profiles (melting curves) were measured using the fluorescent dye SYBR Green I. The fluorescence intensity of SYBR Green I increases 100-fold upon binding to duplex DNA compared to single-stranded DNA, so that as the SOMAmer-probe duplex structure melts, the fluorescence intensity decreases.

[0132] Thermal melts were performed in a buffer containing components defined by the SOMAscan assay eluate (in this case 100 mM Tris pH 8.0, 200 mM NaCl, and 0.9 M perchlorate). Perchlorate is known to reduce the stability of DNA duplexes. All thermal melts were performed in 120 μL (8.3×10 -7 The melting profiles were obtained at a single concentration of 100 pM in 100 mM. Thus, all thermodynamic parameters were obtained from a single fit to the individual thermal melting profiles. Melting profiles exhibiting more complex behavior than predicted by the two-state model were excluded from the analysis. A total of four plates were measured for each of the H1 and H2 probes. These 800 melting profiles were evaluated to generate data consistent with the assumed two-state model of duplex formation. Of these 800 profiles, a total of 408 melting profiles of SOMAmers containing three different modified nucleotides (Nap-dU, 2-Nap-dU, and Benzyl-dU) were used in this analysis.

[0133] a. Single-phase model fit Figure 12 shows typical thermal melting data for a SOMAmer-probe duplex. The vertical axis is fluorescence measured in RFU, and the horizontal axis is temperature, with the following two-state model fit overlaid: First, a high-temperature baseline and a low-temperature baseline are fitted to the data using the first and last 15 data points. The low-temperature baseline corresponds to double-stranded material, and the high-temperature baseline corresponds to single-stranded material, and is shown as follows:

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[0134] Using nonlinear regression, six free parameters

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[0135] b. Fit of the biphasic model In many cases, the data exhibit more complex melting behavior, presumably due to the internal SOMAmer structure melting first, followed by the SOMAmer-probe duplex. Figure 13 shows data that exhibit typical biphasic behavior, again overlaid with a theoretical model fit indicated by the solid red line. Two clear transitions occur in the data, presumably the first being the melting of the internal SOMAmer structure, followed by the melting of the SOMAmer-probe duplex. The two transitions are assumed to be independent. Three baselines are required to fit the biphasic model. The first corresponds to the temperature dependence of the internal SOMAmer structure, the second to the temperature dependence of the double-stranded SOMAmer-probe, and the third to the temperature dependence of the combined single-stranded material. The latter two are the same as the baselines above. The former is denoted as follows:

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[0136] 5. Nearest Neighbor Model Once the experimental data has been fitted to an appropriate model (e.g., single-phase or two-phase as discussed above) and the individual thermodynamic parameters have been compiled, the parameters of the nearest neighbor model can be obtained from the data. The change in free energy is approximated as:

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[0137] Parameters

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[0138] 6. Illustrative Results In an exemplary process following the above teachings, the thermal melts of duplexes containing three different modified nucleotides, Nap-dU, 2-Nap-dU, and Benzyl-dU, were used to extend the nearest neighbor model parameters. Based on these extended nearest neighbor parameters, a Python code has been developed to calculate the thermodynamics and melting temperatures.

[0139] The following table summarizes the 31 parameters required for the nearest neighbor model, including 10 parameters for the four standard bases and 7 additional parameters for each modified base.

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[0140] Using the parameters in the table above, we estimate the melting temperatures, T, of duplexes containing the modified nucleotide bases Nap-dU, 2-Nap-dU, and benzyl-dU. m may be computationally determined. Similar procedures may be used to calculate the estimated melting temperatures of any other SOMAmer-containing duplexes. In accordance with an embodiment of the present teachings, these melting temperatures may be used to determine where to split the hybridization complement to the SOMAmer, i.e., the split point between the first and second hybridization regions H1 and H2 (and thus between the first probe 104 and the second probe 106) of a trimolecular compound such as compound 100 shown in FIG. 1.

[0141] F. Illustrative Combinations and Additional Examples This section describes additional aspects and features of systems and methods for detecting and quantifying target molecules in biological samples in accordance with embodiments of the present teachings, and is presented without limitation as a series of paragraphs, some or all of which may be designated alphanumeric for clarity and efficiency. Each of these paragraphs may be combined in any suitable manner with one or more of the other paragraphs, and / or with disclosure from elsewhere in this application, including material incorporated by reference in cross-references. Some of the following paragraphs explicitly refer to and further limit other paragraphs, and provide, without limitation, some examples of suitable combinations.

[0142] A. A system for quantifying the abundance of a target protein in a biological sample, the system comprising: a plurality of aptamers, each configured to bind to a particular target protein when a biological sample containing the target protein is exposed to the aptamer, thereby forming an aptamer-containing eluate; a plurality of capture probes, each configured to hybridize to a particular aptamer, wherein the plurality of capture probes includes a first capture probe having a portion hybridized to a first portion of the respective aptamer, a second capture probe having a portion hybridized to a second portion of the respective aptamer, a DNA primer region, and an aptamer ID sequence corresponding to the aptamer; means for forming a plurality of trimolecular complexes by exposing the aptamers in the eluate to the capture probes, and means for sequencing the aptamer ID sequence, thereby determining the abundance of the target protein in the biological sample.

[0143] B. A system for quantifying the abundance of two or more target proteins in a biological sample, comprising: a plurality of aptamers, each configured to capture a specific protein in the sample, thereby forming an aptamer-containing eluate upon isolation of an aptamer that captured one of the target proteins in the biological sample; a plurality of first probes, each hybridizing to a corresponding first portion of the specific aptamer; a plurality of second probes, each hybridizing to a corresponding second portion of the specific aptamer, each second probe comprising at least one DNA primer region and an aptamer ID sequence corresponding to the specific aptamer; a means for sequencing the aptamer ID sequences; and a means for quantifying the abundance of the target proteins based on the sequenced aptamer ID sequences.

[0144] C. A system for detecting a target protein in a biological sample, comprising: a plurality of aptamers, each configured to capture a specific protein; a plurality of first probes, each comprising a portion hybridized to a corresponding first portion of one of the aptamers that captured the target protein; a plurality of second probes, each comprising a portion hybridized to a corresponding second portion of one of the aptamers that captured the target protein, at least one DNA primer region, and an aptamer ID sequence corresponding to the aptamer that captured the target protein; a means for amplifying the aptamer ID sequence; and a means for sequencing the aptamer ID sequence to identify the aptamer ID sequence, thereby identifying the aptamer that captured the target protein and the target protein.

[0145] D. The system of any of the preceding paragraphs, further comprising means for normalizing complementary read counts of all samples.

[0146] E. The system of any of the preceding paragraphs, further comprising means for performing dynamic range compression of the abundance of aptamers and / or aptamer ID sequences prior to sequencing and counting.

[0147] Benefits, Features, and Profits Various embodiments and examples of methods and systems for detecting and quantifying the presence of target molecules in biological samples described herein offer several advantages over previously known solutions. For example, the exemplary embodiments and examples described herein enable aptamer-based protein detection to be quantified using next generation sequencing by simplifying the sequencing target from the aptamer to an aptamer-identifying sequence.

[0148] Additionally, among other benefits, the exemplary embodiments and examples described herein enable accurate detection of target molecules spanning several orders of magnitude in abundance by splitting the assay eluate into multiple dilution groups at one or more stages of the assay and recombining prior to next generation sequencing.

[0149] Additionally, among other advantages, the exemplary embodiments and examples described herein allow for the addition of a quantitative spike reporter to the assay eluate to normalize complementary read counts across samples, thereby correcting for errors arising from finite sequencing reads.

[0150] No known system or device is capable of performing these functions. However, not all embodiments and examples described herein provide the same advantages or to the same degree of advantages.

[0151] conclusion The above disclosure may encompass multiple separate examples with independent utility. Each of these is disclosed in its preferred form(s), but the specific embodiments disclosed and illustrated herein should not be considered in a limiting sense, as numerous variations are possible. Where section headings are used within this disclosure, such headings are for organizational purposes only. The subject matter of the present disclosure includes all novel and unobvious combinations and subcombinations of the various elements, features, functions, and / or properties disclosed herein. The following claims specifically point out certain combinations and subcombinations that are deemed novel and unobvious. Other combinations and subcombinations of features, functions, elements, and / or properties may be claimed in applications claiming priority from this or related applications. Such claims are deemed to be within the subject matter of the present disclosure, whether broader, narrower, equal, or different than the scope of the original claims.

Claims

1. 1. A method for quantifying the abundance of a target protein in a biological sample, comprising: capturing the target protein by exposing the biological sample to a plurality of aptamers, each aptamer configured to bind to a specific protein; isolating the aptamer that captured one of the target proteins in the aptamer-containing eluate; forming a plurality of trimolecular complexes by exposing the aptamers in the eluate to a plurality of capture probes, each capture probe being configured to hybridize to a particular aptamer, each trimolecular complex comprising: one of the aptamers from the eluate; a first capture probe comprising a portion hybridized to a first portion of the aptamer; a second capture probe comprising a portion hybridized to a second portion of the aptamer, a DNA primer region, and an aptamer ID sequence corresponding to the aptamer; separating the trimolecular complex from capture probes that are not bound to the aptamer; dissociating the capture probe from the corresponding aptamer in the trimolecular complex; amplifying the aptamer ID sequence in the released capture probe; determining the aptamer ID sequence by next generation sequencing; and determining the abundance of the target protein in the biological sample based on data obtained by sequencing the aptamer ID sequence.

2. The method of claim 1 , wherein the aptamer is a SOMAmer.

3. 2. The method of claim 1, further comprising splitting the aptamer-containing eluate into two or more groups prior to forming the trimolecular complex, and wherein the plurality of capture probes comprises two or more separate sets of capture probes corresponding to the two or more groups.

4. 2. The method of claim 1, further comprising splitting the dissociated capture probe into multiple aliquots prior to amplifying the aptamer ID sequences, and wherein amplifying the aptamer ID sequences comprises amplifying a different subset of aptamer ID sequences in each aliquot.

5. The method of claim 4, further comprising diluting the at least one aliquot prior to amplifying the aptamer ID sequence.

6. 5. The method of claim 4, further comprising adding a quantitative spike reporter to each aliquot prior to amplifying the aptamer ID sequence.

7. The method of claim 6, further comprising recombining the aliquots prior to sequencing the aptamer ID sequence.

8. 1. A method for quantifying the abundance of two or more target proteins in a biological sample, the method comprising: capturing the target protein by exposing the biological sample to a plurality of aptamers, each aptamer configured to capture a particular protein; isolating the aptamers that captured one of the target proteins in the biological sample, thereby forming an aptamer-containing eluate; forming a plurality of trimolecular complexes, each trimolecular complex comprising: A specific aptamer present in the aptamer-containing eluate; a first probe hybridized to a corresponding first portion of the specific aptamer; a second probe comprising a portion hybridized to a corresponding second portion of the specific aptamer, at least one DNA primer region, and an aptamer ID sequence corresponding to the specific aptamer; amplifying the aptamer ID sequence; Sequencing the aptamer ID sequence; and quantifying the abundance of the target protein based on the sequenced aptamer ID sequence.

9. The method of claim 8 , wherein the aptamer is a SOMAmer.

10. 9. The method of claim 8, further comprising splitting the aptamer-containing eluate into two or more dilution groups prior to forming the trimolecular complex, and wherein the plurality of capture probes comprises two or more separate sets of capture probes corresponding to the two or more dilution groups.

11. 9. The method of claim 8, further comprising splitting the dissociated capture probes into multiple aliquots prior to amplifying the aptamer ID sequences, wherein diluting at least one of the aliquots and amplifying the aptamer ID sequences comprises amplifying a different subset of aptamer ID sequences in each aliquot.

12. The method of claim 11, further comprising adding a quantitative spike reporter to each aliquot prior to amplifying the aptamer ID sequence.

13. The method of claim 12, further comprising recombining the aliquots prior to sequencing the aptamer ID sequence.

14. The method of claim 8, wherein sequencing the aptamer ID sequence is accomplished by next generation sequencing.

15. 1. A method for detecting a target protein in a biological sample, comprising: capturing target proteins with aptamers by combining the biological sample with a plurality of aptamers, each aptamer configured to capture a specific protein; forming a trimolecular complex, An aptamer that captures the target protein; a first probe comprising a portion hybridized to a corresponding first portion of the aptamer that has captured the target protein; forming a trimolecular complex comprising a portion hybridized to a corresponding second portion of the aptamer that captured the target protein, at least one DNA primer region, and a second probe comprising an aptamer ID sequence corresponding to the aptamer that captured the target protein; amplifying the aptamer ID sequence; and sequencing the aptamer ID sequence to identify the aptamer ID sequence, thereby identifying the aptamer that captured the target protein and the target protein.

16. 16. The method of claim 15, further comprising forming the first probe and the second probe by determining a target region on the aptamer and then determining a boundary to divide the complement of the target region into the first probe and the second probe.

17. 17. The method of claim 16, wherein determining the boundary is based on achieving a desired melting temperature of the hybridized portions of the first and second probes.

18. 18. The method of claim 17, further comprising: determining by a computation the melting temperature of the complement of the target region; and then incrementally modifying the boundary between the hybridized portions of the first and second probes until a desired melting temperature of the hybridized portion is achieved.

19. The method of claim 17, wherein sequencing the aptamer ID sequence is accomplished by next generation sequencing.

20. 16. The method of claim 15, further comprising dissociating the second probe from the aptamer prior to amplifying the aptamer ID sequence.

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