New generation of sequencing for protein measurement

Abstract

Methods for the detection and quantification of target molecules, such as proteins, in biological samples are provided. The disclosed method includes capturing a target molecule by an aptamer, replacing the aptamer with an aptamer identification sequence, and then sequencing the aptamer identification sequence by a new generation sequencing technique.

Description

[0001] Cross-referencing This application is a divisional application of Chinese Patent Application No. 202280089749.0. Chinese Patent Application No. 202280089749.0 is the Chinese national phase application of international application PCT / US2022 / 082594, which claims priority to U.S. Provisional Application 63 / 294,964, filed on December 30, 2021, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to systems and methods for the quantitative measurement of proteins in biological samples. More specifically, the disclosed embodiments involve capturing a target protein with a specially designed aptamer, forming an eluent containing the aptamer containing the captured target protein, and then replacing the aptamer in the eluent with a “reporter” DNA molecule that is easier to sequence than the aptamer itself. Background Technology

[0003] Typically, various attempts to assess gene activity and / or decode biological processes (including disease processes or pharmacological processes) focus on genomics. However, proteomics can provide further information about the biological functions of cells and organisms. Proteomics involves detecting and quantifying expression at the protein level, rather than the gene level, to achieve qualitative and quantitative measurements of gene activity. Proteomics also includes the study of non-gene-coding events, such as post-translational protein modifications and protein-protein interactions.

[0004] Currently, obtaining vast amounts of genomic information is possible. DNA microarrays, as molecular arrays, have been put into practical use, and the price of direct DNA sequencing continues to decline significantly. Similarly, the demand for high-throughput proteomics is constantly increasing. In health monitoring, proteomics is preferable to genomics because the genome is static, only indicating medical possibilities, while the proteome dynamically changes with a patient's medical status and can even be considered to define that status. However, detecting and quantifying proteins is difficult, while detecting and quantifying nucleic acids is relatively easy, at least in part because proteins are more complex and variable than DNA in terms of biological function. This has spurred many attempts to represent protein concentration by measuring messenger RNA (mRNA) concentration. However, it has been shown that mRNA concentration does not correlate well with protein concentration. Proteomics appears to rely on the ability to directly detect proteins.

[0005] One method for detecting and quantifying the presence of specific proteins in biological samples is using Protein Capture Sustained-Release Modified Aptamer (SOMAmer®) reagents. SOMAmer reagents are composed of chemically modified nucleotides that greatly expand the physicochemical diversity of large random nucleic acid libraries from which SOMAmer reagents are selected. Assays using SOMAmer reagents measure native proteins in a composite matrix by converting each individual protein concentration into a corresponding SOMAmer reagent concentration, which is then quantified using standard DNA techniques such as microarrays or qPCR.

[0006] SOMAmer reagents are DNA-based single-stranded protein affinity reagents that include chemically modified nucleotides that mimic the side chains of amino acids, thereby increasing 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 for an iterative selection and amplification process called System Evolution of Ligands by Exponential Enrichment (SELEX), in which SOMAmer reagents are selected. SOMAmer reagents can be generated using a SELEX-type process to capture proteins resistant to selection of unmodified nucleic acids (ACTG conventional aptamers). SOMAmer reagents can be customized to select the desired specificity and sustained-release properties, as well as mimicking the assay conditions to be used with the reagent.

[0007] In SOMAmer-based assays, the presence of protein in a sample is translated into a specific SOMAmer-based DNA signal. Following the SOMAmer-protein binding step is a series of separation and washing steps to convert the relative protein concentration into a measurable nucleic acid signal, which is quantified using DNA detection techniques, such as hybridizing fluorescently labeled SOMAmers with a custom-designed DNA microarray. After laser scanning of the microarray, the reading, expressed in relative fluorescence units (RFU), is proportional to the amount of the target protein in the initial sample.

[0008] The use of microarray hybridization for protein quantification has several drawbacks, including limited scalability, fixed assay costs, and limited commercial availability of microarrays. Therefore, there is a need to develop alternative quantification methods for SOMAmer molecules in the post-capture eluent. Summary of the Invention

[0009] This disclosure provides systems, apparatus, and methods related to the detection and quantification of proteins using parallel sequencing technologies known as next-generation sequencing or NGS. More specifically, this disclosure relates to hybridization capture (HC) technology, in which SOMAmer elution molecules characterizing protein capture are replaced with “reporter” DNA molecules containing SOMAmer-specific recognition markers or “SOMA IDs,” and then sequenced using NGS technology.

[0010] In some embodiments, this disclosure relates to a system and method for quantifying the abundance of a target protein in a biological sample, comprising: exposing the biological sample to a plurality of aptamers configured to capture a target protein, each of the plurality of aptamers being configured to bind to a specific protein; isolating the aptamer containing the aptamer from an elution buffer containing the aptamer from the target protein; and forming a plurality of trimolecular complexes by exposing the aptamer in the elution buffer to a plurality of capture probes, each of the plurality of capture probes being configured to hybridize to a specific aptamer, each trimolecular complex comprising: one of the plurality of aptamers from the elution buffer, the second aptamer being a third aptamer being a fourth aptamer being a fifth aptamer being a fifth aptamer being a sixth ... A capture probe, the first capture probe including a portion hybridizing with a first part of an aptamer, and a second capture probe including a portion hybridizing with a second part of an aptamer, a DNA primer region, and an aptamer ID sequence corresponding to the aptamer; separating a three-molecule complex from the capture probes that are not bound to the aptamers; dissociating the capture probes in the three-molecule complex from the corresponding aptamers; amplifying the aptamer ID sequence in the dissociated capture probes; sequencing the aptamer ID sequence using next-generation sequencing; and determining the abundance of the target protein in the biological sample based on the data obtained by sequencing the aptamer ID sequence.

[0011] In some embodiments, this disclosure relates to a system and method for quantifying the abundance of two or more target proteins in a biological sample, comprising: capturing target proteins by exposing the biological sample to a plurality of aptamers, each of the plurality of aptamers being configured to capture a specific protein; forming an aptamer-containing eluent by isolating an aptamer containing one of the target proteins captured in the biological sample; forming a plurality of tripoleons, each tripoleon comprising: a specific aptamer present in the aptamer-containing eluent; a first probe that hybridizes to a corresponding first portion of the specific aptamer; and a second probe comprising a portion that hybridizes 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.

[0012] In some embodiments, this disclosure relates to a system and method for detecting a target protein in a biological sample, comprising: capturing a target protein by binding the biological sample to a plurality of aptamers, each of the plurality of aptamers being configured to bind to a specific protein; forming a three-molecule complex comprising: an aptamer that has captured the target protein; a first probe including a portion that hybridizes to a corresponding first portion of the aptamer that has captured the target protein; and a second probe including a portion that hybridizes to a corresponding second portion of the aptamer that has captured the target protein, at least one DNA primer region, and an aptamer ID sequence corresponding to the aptamer that has 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 has captured the target protein and the target protein.

[0013] In some embodiments according to various aspects of this disclosure, aptamers used to capture target proteins can be sequenced directly, for example, using next-generation sequencing technology, without the need to use three-molecule complexes to convert the aptamers into simpler sequences.

[0014] In some embodiments according to various aspects of this disclosure, the aptamers used to capture the target protein may be SOMAmers.

[0015] In some embodiments according to various aspects of this disclosure, the aptamer-containing eluent can be divided into multiple groups before and / or after exposure to the hybridization probe region. In some cases, some or all of the eluent groups can be diluted to the desired extent.

[0016] In some embodiments according to various aspects of this disclosure, a quantitative peak reporter factor may be added during the desired assay stage to correct for compensatory changes in the analyte count ratio.

[0017] The features, functions, and advantages may be implemented independently in the various embodiments of this disclosure or may be combined with other embodiments, further details of which can be found in the following description and drawings. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of a tripoleon comprising an aptamer and two probes hybridizing with the aptamer, according to various aspects of this disclosure.

[0019] Figure 2A Manufactured in accordance with all aspects of this instruction manual Figure 1 A flowchart of the steps of an exemplary method for preparing hybridization probes required for trimolecular complexes.

[0020] Figure 2B This is based on all aspects of this instruction manual. Figure 1A flowchart of the steps of an exemplary method for preparing a three-molecule complex.

[0021] Figure 3A This is based on the use of the three-molecule complex in various aspects according to this specification, such as... Figure 1 A flowchart of the steps of an exemplary method for performing next-generation sequencing content determination using a complex of [a specific compound].

[0022] Figure 3B This is a flowchart illustrating the steps of an exemplary method for performing next-generation sequencing content determination according to various aspects of this specification, the method including capturing a target protein and then forming a three-molecule complex, such as... Figure 1 Complexes.

[0023] Figure 4 This is a flowchart of the steps and byproducts for exemplary next-generation sequencing content determination involving four hybrid groups, based on various aspects of this specification.

[0024] Figure 5 This is a flowchart illustrating the steps and byproducts of exemplary next-generation sequencing assays involving one hybridization group and four PCR groups, based on various aspects of this specification.

[0025] Figure 6 This is a flowchart of the steps and byproducts for exemplary next-generation sequencing content determination involving four hybridization groups and four PCR groups, according to various aspects of this specification.

[0026] Figure 7 It is a histogram depicting the hypothetical, simplified results of two-analyte assays for two different samples.

[0027] Figure 8 This is based on all aspects of this instruction manual. Figure 7 The graph shows the content determination results, where quantitative spike (qSpike) control reporter factors were added to both samples at the same concentration.

[0028] Figure 9 This is a histogram of the raw results of the determination of SOMAmer content in three analytes in eight different samples with different analyte concentrations according to various aspects of this specification, with four different qSpike reporter factors added to each sample.

[0029] Figure 10 This is based on all aspects of this instruction manual. Figure 9 Histogram of normalized results for content determination.

[0030] Figure 11 This is a flowchart of some steps and byproducts of an exemplary next-generation sequencing assay involving four PCR groups, each with the addition of a qSpike control reporter factor, according to various aspects of this specification.

[0031] Figure 12 The graph shows the relative fluorescence units (RFU) versus temperature according to various aspects of this specification. It illustrates the experimentally obtained single-phase thermal melting data of the SOMAmer-probe duplex and is covered by curve fitting of the two-state theoretical model.

[0032] Figure 13 The graphs are relative fluorescence units (RFU) versus temperature based on various aspects of this specification. They show the experimentally obtained biphase thermal melting data of the SOMAmer-probe duplex and are covered by curve fitting of the biphase theoretical model. Detailed Implementation

[0033] The following describes and illustrates, in the accompanying drawings, various aspects and examples of hybridization capture, next-generation sequencing assay systems, and related methods for protein detection and quantification. Unless otherwise stated, protein assays and / or their various components according to this specification may include at least one of the structures, components, functions, and / or variations described, illustrated, and / or incorporated herein. Furthermore, unless specifically excluded, process steps, structures, components, functions, and / or variations incorporated into the present specification may be included in other similar apparatuses and methods, including those interchangeable between the disclosed embodiments. The following descriptions of the examples are merely illustrative in nature and are not intended to limit this disclosure, its application, or use. Additionally, the advantageous effects achieved by the examples and embodiments described below are exemplary in nature, and not all examples and embodiments have the same or the same degree of advantage.

[0034] The detailed description includes the following sections immediately following: (1) Definition; (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 several subsections, each of which is marked accordingly.

[0035] definition Unless otherwise stated, the following definitions apply in this document.

[0036] The terms “include,” “contain,” and “have” are used interchangeably to indicate that something includes but is not limited to, and are open-ended terms not intended to exclude other undescribed elements or methodological steps.

[0037] Terms such as “first,” “second,” and “third” are used to distinguish or identify multiple members in a group, etc., and are not intended to indicate sequence or number restrictions.

[0038] “AKA” means “also known as” and can be used to indicate an alternative name or corresponding term for one or more given elements.

[0039] Directional terms such as “up,” “down,” “vertical,” and “horizontal” should be understood within the context of the specific object being discussed. For example, an object can be oriented around defined X, Y, and Z axes. In these examples, the XY plane is defined as horizontal, upward is defined as the positive direction of the Z-axis, and downward is defined as the negative direction of the Z-axis.

[0040] In the context of a method, “providing” can include receiving, obtaining, purchasing, manufacturing, generating, processing, preprocessing, and / or similar operations to place the provided object or material in a state and configuration that can be used for other steps to be performed.

[0041] "NGS" stands for "next-generation sequencing".

[0042] “HC” means “hybrid capture”.

[0043] "SOMAmer" refers to a "slow-release modified aptamer" reagent developed and manufactured by SomaLogic Operating Co, Inc. ("SomaLogic") in Boulder, Colorado.

[0044] "SOMAmer ID sequence" or "SOMA ID" or "reporter factor" refers to a portion of a three-molecule complex that includes a SOMAmer-specific DNA strand that can be sequenced using NGS technology.

[0045] "Quantitative spike," "report spike," or "qSpike" refers to an amplified reporting factor used to normalize compensating readings across samples, thereby allowing the identification of true signal variations.

[0046] In this disclosure, one or more publications, patents, and / or patent applications may be incorporated by reference. However, such materials are incorporated only where there is no conflict with the statements and drawings set forth herein. In the event of any such conflict (including any conflict in terminology), this disclosure shall prevail.

[0047] Overview In general, this disclosure relates to methods for the detection and quantification of target molecules (such as proteins) in biological samples. The disclosed methods may include capturing the target molecule via an aptamer, replacing the aptamer with an aptamer identifier sequence, and then sequencing the aptamer identifier sequence using next-generation sequencing technology. Alternatively, the disclosed methods may include capturing the target molecule via an aptamer and then directly sequencing the aptamer.

[0048] Examples, components and alternatives The following sections describe some aspects of protein detection and quantification using aptamers (such as SOMAmer reagents), and related systems and / or methods, wherein SOMAmers are replaced by reporter DNA molecules containing SOMAmer-specific fragments via hybridization capture, which can be sequenced using next-generation sequencing technologies. The examples in these sections are intended to be illustrative and should not be construed as limiting the scope of this disclosure. Each section may include one or more different embodiments or examples, and / or contextual or related information, functionality, and / or structure.

[0049] A. Exemplary Adapter This section describes sustained-release modified aptamers (SOMAmers), which are illustrative examples of aptamers suitable for use in conjunction with the example systems and methods described herein.

[0050] A method called "evolution through exponentially enriched ligand systems" (sometimes also known as the SELEX process) has clearly demonstrated that nucleic acids, like proteins, possess three-dimensional structural diversity. The SELEX process is a method for the in vitro evolution of nucleic acid molecules to achieve a desired activity. Here, SELEX for generating nucleic acid molecules that bind with high specificity to target molecules is described. The SELEX process provides a 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 recognized by SELEX is a specific ligand for a given target compound or molecule. This SELEX process is based on the unique perspective that nucleic acids have sufficient capacity to form a wide variety of two-dimensional and three-dimensional structures, and that their monomers possess sufficient chemical versatility to act as ligands (forming specific binding pairs) for virtually any compound, whether monomer or polymer. Molecules of any size or composition can serve as targets.

[0051] The SELEX method for high-affinity binding involves selecting from a mixture of candidate oligonucleotides and using the same universal selection scheme for stepwise iterative binding, dissociation, and amplification to achieve virtually any desired binding affinity and selectivity criterion. The SELEX method begins with a mixture of nucleic acids (preferably comprising a random sequence) and includes the following steps: contacting the mixture with the target under binding-favorable conditions; separating unbound nucleic acids from those specifically bound to the target molecule; dissociating the nucleic acid-target complex; amplifying the dissociated nucleic acids from the nucleic acid-target complex to produce a ligand-rich nucleic acid mixture; and then repeating the binding, dissociation, dissociation, and amplification steps until the desired number of cycles is reached to produce a nucleic acid ligand with high specificity and high affinity for the target molecule. In this way, aptamers suitable for binding to virtually any target protein can be discovered.

[0052] More specifically, SOMAmers are protein-binding aptamers discovered through modification using the SELEX process, with a dissociation rate (t... 1 / 2 The average time required for half of the protein-aptamer complex to dissociate is typically between 30 and 240 minutes. Furthermore, SOMAmers contain modified nucleosides that provide various intrinsic functions. These functions can include tags for immobilization, tags for detection, means to promote or control separation, amino acid-like side chains that provide better affinity for proteins, etc. Modifications that improve protein affinity are typically chemical groups attached to the 5-position of a pyrimidine base. By functionalizing this 5-position with protein-like groups (such as phenyl or 2-naphthyl), the chemical diversity of SOMAmers is expanded, allowing for high-affinity binding to a wider range of target molecules. Additionally, some polymerases are still capable of transcribing DNA modified at these positions, thus allowing the amplification required by the SELEX process.

[0053] It should be noted that while binding aptamers (including SOMAmers) are typically discovered via the SELEX process, other methods may exist for selecting them. For example, with advancements in computer modeling of molecular interactions, it may become possible to directly calculate the ideal nucleic acid sequences of aptamers and the relevant chemical modifications to SOMAmers, thereby generating capture agents specific to a given target molecule. Besides SELEX, other chemical techniques for screening aptamers and SOMAmers are also possible.

[0054] Determination of the content of physiologically significant molecules in biological and other samples is an important tool in scientific research and healthcare. Each SOMAmer is capable of binding to target molecules in a sample with high specificity and very high affinity. After appropriate washing and separation steps, unbound proteins are first removed, followed by the removal of unbound SOMAmers, eluting the SOMAmers from the resulting SOMAmer-protein complex. The SOMAmer eluent is then contacted with a microarray containing complement for SOMAmers, enabling the determination of the absence, presence, quantity, and / or concentration of target molecules in the sample.

[0055] B. Exemplary method for determining hybridization capture content This section describes a targeted hybridization capture (HC) assay in which the SOMAmer signal from the assay eluent is replaced by a “reporter” DNA molecule containing a SOMAmer-specific identifier sequence or “SOMA ID” to enable sequencing.

[0056] Prior to the HC content determination described in this section, a SOMAmer binding step has been performed to produce an eluent containing SOMAmer reagent, indicating the presence of the corresponding target protein in the sample. For example, but not limited to, the following steps can be performed to obtain an eluent containing SOMAmer: (1) Protein-specific SOMAmer reagent labeled with 5' fluorescent group, photodegradable linker and biotin was immobilized on streptavidin (SA) coated beads and incubated with one or more samples of a protein-containing complex mixture; (2) A SOMAmer-target protein complex is formed on the bead; (3) Wash the bead to remove unbound proteins and label the bound proteins with biotin; (4) The SOMAmer-protein complex is released from the bead by photolysis of the linker under ultraviolet light; (5) Incubation in a buffer containing multiple anion competitors prevents the rebinding of dissociated proteins, thereby increasing the dynamics of complexes that specifically bind to the target protein and have a slow dissociation rate compared to interactions that indicate the binding of the target protein to the corresponding SOMAmer with a rapid dissociation rate. (6) SOMAmer-protein complexes were recaptured on the second group of streptavidin-coated beads by biotin-labeled proteins, followed by an additional washing step to facilitate further removal of non-specifically bound SOMAmer reagents; and (7) Release the SOMAmer reagent from the beads in the denaturing buffer to form an eluent containing SOMAmer suitable for quantitative analysis.

[0057] Now let's turn to the focus of this section: hybrid capture methods. Figure 1 The diagram schematically illustrates a three-molecule complex, collectively denoted as 100, that can be used for next-generation sequencing assays to identify target proteins. Complex 100 includes SOMAmer 102, which is one of the SOMAmers retained in the eluent after assaying, i.e., after exposure to the biological sample and (e.g.) the other steps described above. In other words, the presence of SOMAmer 102 in the eluent after assaying indicates the presence of the corresponding target protein (or other target molecule) in the sample.

[0058] Complex 100 further includes a first probe 104 and a second probe 106. The first probe 104 includes a... Figure 1 The left-hand complementary hybridization region H1 of SOMAmer 102. The second probe 106 includes... Figure 1The right-hand complementary hybridization region H2 of SOMAmer 102 also includes universal primer regions P1 and P2, as well as the unique SOMAmer recognition sequence I corresponding to SOMAmer 102. S Or “SOMA ID”, as described in more detail below. The positions of H1 and H2 can also be reversed, i.e., accompanied by the unique SOMAmer recognition sequence I in the universal primer regions P1 and P2 and H2. S With proper inversion, H1 hybridizes to the right side of SOMAmer 102, and H2 hybridizes to the left side of SOMAmer 102.

[0059] Hybrid regions H1 and H2 are configured to specifically bind different complementary portions of the corresponding SOMAmers and can be designed to have similar melting temperatures (T0). m To achieve simultaneous hybridization under a given set of content determination conditions. For example, the hybridization region of complex 100 can be designed and formed according to the following steps.

[0060] First, the target region to be used as a probe is determined on the SOMAmer. For truncated SOMAmers, the entire SOMAmer sequence can be used as the target region, including the five bases of the fixed region used for amplification from each end of the random region in SELEX. For full-length SOMAmers, the SOMAmer can be truncated to any desired length, such as 50-mer, via computer simulation (i.e., computationally), and then the hybridization complement is determined.

[0061] Next, the boundary used to divide the target region into two parts is determined. In some examples, to ensure that the two hybridization regions have similar melting temperatures, the melting temperature of the 25-mer duplex (e.g., between SOMAmer and H1 and H2) can be calculated and then the boundary variation between the two regions is gradually adjusted until the melting temperatures between the H1- and H2-SOMAmer duplexes reach maximum equilibrium. In other examples, different melting temperatures can be intentionally chosen, for example, a first melting temperature for the H1 probe (e.g., 45ºC) and a second melting temperature for the H2 probe (e.g., 35ºC).

[0062] Length constraints can also be imposed on the hybridization regions. For example, the minimum length of H1 and H2 can be set to 18-mer. Similarly, the maximum length of H2 can be set to 30-mer, to ensure that H2 plus the remaining reporter portion of the second probe is still shorter than the maximum length required for subsequent synthesis, such as 100 bases. Under these constraints, hybridization regions H1 and H2 can be generated by calculation.

[0063] Generate universal primer regions P1 and P2 and SOMAmer ID sequence I for the second probe 106. S Various factors can be considered. For example, sequencing amplification designs for counting applications must strike a good balance between the need for short and inexpensive reads and the need for sequences with sufficient length and information content to serve as identifier sequences such as SOMAmer IDs for counting and barcode sequences for reuse. For these reasons, the actual area of ​​the reporter region, which ultimately becomes the largest part of the sequencing template, may be limited in length when scaling up the content. For example, the length of primer regions P1 and P2 may be limited to 24-mer, and the SOMAmer ID sequence I... S The length can be limited to 15-mer, the edit distance length must be at least 5, and the homopolymer length must not exceed 2-mer. Other length limitations and options may also apply to the primer region and SOMAmer ID sequence.

[0064] Figure 2A It generates hybridization probes: H1 (104) and H2 (106) (used to form Figure 1 A flowchart of the steps of an exemplary method 200 (a three-molecule complex). In step 202, a set of SOMAmer sequences for content determination is provided.

[0065] In step 204, hybridization probe regions H1 and H2 are generated. These hybridization probe regions can be calculated and determined, for example, under various lengths and / or other constraints, as described above. Also, as previously mentioned, in some cases, hybridization regions can be isolated from a single SOMAmer complement structure based on factors such as balancing the melting temperature of each region.

[0066] In step 206, a SOMAmer ID (I) uniquely corresponding to each SOMAmer is generated. S The SOMAmer ID area can be designed using various methods. For example, I... S Regions can be designed "by eye" (e.g., with maximum edit distance), or they can be computationally generated along with the computational generation of hybrid regions H1 and H2. After generating the library of SOMAmer ID regions, SOMAmer IDs can be randomly or in any other suitable manner assigned to SOMAmers to produce a unique reporting factor corresponding to each SOMAmer.

[0067] In step 208, universal primer regions P1 and P2 are generated. Universal primers may be designed for stability and to reduce the risk of downstream bias. For example, in some cases, primer lengths may be 24 or 25-mers, with an estimated melting temperature of approximately 70°C. In some cases, primers may terminate at the 3' end of guanine (G) to maintain stability. In some cases, primers may be evaluated using an oligonucleotide analyzer to reduce the potential risk of dimer formation. In some cases, primers may be further customized to avoid non-specific interactions with functional oligonucleotides used in known sequencing technologies.

[0068] In step 210, first and second SOMAmer-specific probes (sometimes referred to as “capture probes”) are generated. Each first probe includes a hybridization region H1 and one or more elements suitable for binding to assay beads, such as biotin for binding to streptavidin-coated beads. The first probe may also include other elements such as a photolyzable linker. Each second probe includes a hybridization region H2, universal primer regions P1 and P2, and the SOMAmer ID sequence I. S As part of the generation of the second probe, the SOMAmer ID region can be attached to a universal primer to generate an amplifiable reporter factor.

[0069] Figure 2B It is used to generate trimolecular complexes (e.g. Figure 1 The flowchart illustrates the steps of an exemplary method 250 (shown as a three-molecule complex). In step 252, an eluent containing SOMAmers is provided, the SOMAmers in the eluent indicating the presence of one or more target proteins or other target molecules in one or more biological samples exposed to the SOMAmer library, as previously described.

[0070] In step 254, a set of SOMAmer-specific capture probes or a library of SOMAmer-specific capture probes generated by method 200 is bound to the SOMAmer-containing eluent after assay. In one example, 25 µl of SOMAmer-containing eluent is bound to 25 µl of probe-containing solution to form a 50 µl hybridization solution. To promote hybridization, the concentration of capture probes can be comparable to or higher than the concentration of SOMAmers in the eluent. For example, a suitable probe concentration may be in the range of 0.05 nM to 5.0 nM, such as 0.5 nM (where nM is nanomoles per liter).

[0071] In some cases, in optional step 253 of method 250, the eluent containing SOMAmers can be separated and selectively diluted before hybridization, and then re-pooled before sequencing. More specifically, the eluent after assay can be divided into two or more dilution groups (e.g., four dilution groups) based on the expected relative abundance of SOMAmers in each group. The sample with the lowest concentration (highest dilution) can contain the largest amount of SOMAmers in the eluent. Conversely, the sample with the highest concentration (lowest dilution) can contain the smallest amount of SOMAmers in the eluent. In this way, SOMAmer counts can be "leveled" to improve the accuracy and precision of detecting SOMAmers with lower abundance. Each dilution group can then be hybridized individually by exposure to the corresponding subset of probes. Further details regarding the use of dilution groups will be given in subsequent sections of this disclosure.

[0072] Furthermore, leveling can be achieved by introducing a fixed proportion of H1 probes, which may or may not carry capture tags for certain high-abundance SOMAmers in the eluent. SOMAmers that form trimolecular complexes with H1 probes lacking capture tags will be removed in the washing step of method 300, as described in detail later.

[0073] In step 256, the first and second probes are hybridized with SOMAmers to form a three-molecule complex, each three-molecule complex comprising (i) a SOMAmer, (ii) a first probe that binds to the SOMAmer via hybridization region H1, and (iii) a second probe that binds to the SOMAmer via hybridization region H2. Each second probe includes the SOMAmer ID sequence I. S The probe can be sequenced to indicate the presence of the corresponding SOMAmer in the elution buffer, thereby indicating the presence of the corresponding protein captured by that SOMAmer in the original biological sample. Hybridization of the capture probe with SOMAmers can be accomplished using any suitable technique, such as appropriate thermal cycling, and additives can be included to enhance hybridization kinetics.

[0074] Figure 3A This is a flowchart of the steps of an exemplary method 300 for performing next-generation sequencing content determination using a three-molecule complex, such as... Figure 1 As shown and through, for example Figure 2B The method generates the content. In step 302, a hybridized three-molecule complex corresponding to the desired SOMAmers library after content determination (e.g., each SOMAmer has a structure like complex 100 and is generated by, for example, method 250) is provided for sequencing content determination.

[0075] In step 304, the ternary complex is captured on the magnetic beads. For example, the complex can be captured by binding biotin attached to the hybridization region H1 of the first probe to streptavidin on the bead. Capture can be accomplished using any suitable technique. For example, in one instance, a 30 μl hybridization volume solution containing the bead at a concentration of 20 mg / ml can be prepared and then mixed at 45ºC for 30 minutes using a thermal mixer at 1200 rpm.

[0076] In step 306, the solution containing the bead-capturing probe is washed once or multiple times to remove unbound H2 probe reporter factors, i.e., probes that have not hybridized with the corresponding SOMAmer. For example, washing can be performed with a suitable buffer solution, such as a 20 mM phosphate buffer solution containing 1 mM EDTA and 0.05% sodium dodecyl sulfate (SDS). The washing phase can be performed statically, dynamically using a hot mixer, or a combination of both. In one example, there may be two 5-minute static washing phases and two 10-minute dynamic washing phases, with the dynamic washing phases rotating at 1200 rpm. In any case, the resulting solution after washing should contain a three-molecule complex bound to the bead, with at most a small amount of unbound H2 probe / reporter factor remaining.

[0077] In some SOMAmer elution buffer leveling or dynamic range compression embodiments, the three-molecule complex lacking the bead-capturing tag on H1 will be removed in step 306 along with the unbound H2 probe reporter factor. These complexes will result in a reduction in the replication number of those SOMAmers in the final NGS sequencing, thereby reducing the number of these abundant SOMAmers.

[0078] In step 308, the bound ternary complex is eluted from its attached beads, for example, by exposure to a solvent, heat, or by any other suitable elution method. As part of this step, the components of the complex may also dissociate, resulting in separated SOMAmers and probes. In one example, the complex is eluted by adding 85 µl of 20 mM NaOH to the eluent containing the bound complex, followed by mixing with a hot mixer at 1200 rpm for 3 minutes, and then dissociating the complex for 5 minutes. The solution containing the eluted complex is then mixed with 20 µl of hydrochloric acid.

[0079] In some examples, in optional step 309 of method 300, the eluted solution (i.e., the eluent) produced in step 308 can be partitioned and / or diluted into two or more groups, such as four dilution groups or primer amplification groups. As described in the context of the preceding method 250, using multiple dilution groups or primer amplification groups (which in some cases may not be diluted) corresponding to subsets of SOMAmers with different expected abundances levels to level the relative abundance of the entire SOMAmers set or compress its wide distribution can result in greater accuracy and precision when detecting relatively scarce target molecules. The partitioning into these groups can be performed before hybridization (as in step 253 of method 20) or after hybridization (as in step 309 described here), or both. Further details of possible dilution and recombination techniques will be described in subsequent sections of this disclosure.

[0080] In step 310, the solution produced by steps 308 and optionally 309 is prepared for next-generation sequencing (NGS). This may include solutions containing universal primers ( Figure 1 (P1 and P2 in the sequence) and the associated SOMAmer ID sequence I S PCR amplification of the reporter region. If the elution buffers from multiple samples are to be combined before sequencing, the NGS preparation process may also include ligating aptamer sequences and / or barcode sequences for demultiplexing. The generation and ligation of aptamer and barcode sequences in the reporter region can be performed in any suitable manner known in the art, which is common practice in preparing samples for next-generation sequencing. In some cases, barcode sequences may be added as part of a first preparation step, and NGS aptamers may be added as part of a second preparation step.

[0081] In optional step 311, the groups that remain separated after step 310 can be recombined to prepare for sequencing.

[0082] In step 312, the prepared sample is 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. of San Diego, California. However, the method disclosed herein is also suitable for use with other NGS sequencing platforms.

[0083] After NGS, in optional step 314, the data obtained from sequencing can be analyzed or otherwise processed to determine the concentration of an analyte (e.g., a target protein) in the original biological sample. Generally, such analysis includes demultiplexing the sequencing data using barcodes corresponding to each original sample (if multiple samples are multiplexed), counting the reporter factor sequences, and scaling and / or normalizing the data to extract accurate results. For analysis, the sequencing data can be written to a data file in a standard format, such as the ADAT format developed by SomaLogic. Possible quantitative analysis methods will be discussed in more detail below.

[0084] Figure 3B FIG. 4 is a flowchart of steps of an exemplary method 350 for performing a next-generation sequencing assay, which includes capturing a target protein with an aptamer, forming a trimolecular complex from the aptamer, and then using the trimolecular complex as a basis for identifying the captured target protein. It should be understood that any step of method 350 may be similar to the corresponding steps of the methods described previously (i.e., methods 200 and 300), and thus the same details will not be described again.

[0085] In step 352 of method 350, the target protein is captured by exposing the biological sample to a plurality of aptamers, such as SOMAmers, each aptamer being configured to bind to a specific protein. By exposing the sample to a library containing many such SOMAmers, a large number of target protein species can be detected in a single assay.

[0086] In step 354, the aptamer that has captured one of the target proteins is separated in an aptamer-containing eluate. For example, during an assay called SomaScan performed by SomaLogic, an aptamer-containing eluate can be formed, which includes binding the aptamer to assay beads, capturing proteins with the aptamer, washing away unbound proteins, labeling the bound proteins with biotin, releasing the aptamer from the beads, capturing the labeled proteins on new beads, removing unbound aptamers, denaturing the aptamers from the captured proteins, and then separating the aptamers into the eluate.

[0087] In optional step 356, the aptamer-containing eluate can be divided into multiple groups, which may be dilution groups, with more details as shown below in Figure 4-6 and Figure 11 shown.

[0088] In step 358, multiple trimolecular complexes are formed by exposing the aptamers in the eluate (or each eluate dilution group) to a plurality of capture probes, each capture probe being configured to hybridize with a specific aptamer. For example, each complex can have a structure similar to Figure 1The structure of complex 100 is shown. Therefore, each tripolemic complex includes (i) a specific aptamer from the elution buffer; (ii) a first capture probe including a portion that hybridizes to a first portion of the aptamer; and (iii) a second capture probe including a portion that hybridizes to a second portion of the aptamer, further including one or more DNA primer regions and an aptamer ID sequence corresponding to the specific aptamer. If multiple separate dilutions are formed, each dilution will be exposed to a specific set of capture probes corresponding to a specific subset of the aptamer. Optionally, some H1 probes may lack bead capture tags for additional leveling of SOMAmer counts.

[0089] In step 360, the groups formed in step 356 (if any) can be recombinated.

[0090] In step 362, the ternary complex is separated from the capture probes that have not bound to the aptamer. For example, the hybridized complex can be captured onto magnetic beads, and then the unbound probes can be removed by washing.

[0091] In step 364, the capture probe in the three-molecule complex is dissociated from the corresponding aptamer. This may include eluting the complex from the bead, but in any case, the result of step 362 is that the capture probe no longer binds to the aptamer.

[0092] In step 366, the eluent containing unbound capture probes may be selectively separated and / or diluted (a second dilution may be performed, as follows). Figure 6 (as described above) to form multiple PCR groups.

[0093] In step 368, the aptamer ID sequence in the dissociated capture probe in the elution buffer is amplified, for example by PCR amplification of the DNA primer region and the associated ID sequence. Other NGS preparations may also be performed at this stage, such as ligating the aptamer sequence and / or demultiplexing the barcode sequence.

[0094] In step 370, different PCR groups (if any) can be recombinated.

[0095] In step 372, the aptamer ID sequence is sequenced using next-generation sequencing technology. In some examples, this can be done using a next-generation sequencing platform developed by Immena, Inc., San Diego, California.

[0096] In step 374, the data obtained by sequencing the aptamer ID sequence can be used to determine the abundance of the target protein in the original biological sample.

[0097] C. Exemplary dilution groups or dynamic range compression for next-generation sequencing This section describes possible methods, according to various aspects of this specification, for achieving higher assay efficiency, reproducibility, performance, and / or manufacturing feasibility in next-generation sequencing systems. See also: Figure 4-6 .

[0098] First, it should be understood that NGS can be performed on SOMAmer-containing elutions without dividing the elution into multiple dilution groups; that is, on a single elution solution that has never been grouped, diluted, or recombined. This assay is within the scope of this disclosure and has the advantage of requiring less elution and only one hybridization plate per 96 samples. However, this assay faces challenges in terms of sensitivity and accuracy, for example, because the maximum range of SOMAmer abundance in the original elution can span multiple orders of magnitude. For example, the target protein in the sample may have a concentration in the fM-µM range (i.e., spanning approximately nine orders of magnitude), resulting in eluted SOMAmer concentrations spanning five or more orders of magnitude. Assaying such elutions may result in over-counting of more SOMAmers and correspondingly under-counting of fewer SOMAmers. Therefore, it may be necessary to level or compress the abundance range of SOMAmers before sequencing and counting.

[0099] The system and method disclosed herein address this problem by incorporating dilution to subgroup SOMAmers before counting, thereby achieving leveling or dynamic range compression of counts across subgroups when they are grouped together for sequencing and counting. In some examples, the SOMAmer probe set is subdivided into multiple subgroups based on SOMAmer elution abundance (a first subgroup of very few SOMAmers, a second subgroup of few SOMAmers, a third subgroup of abundant SOMAmers, etc.). The dynamic range of each subgroup is smaller than that of the original (undivided) elution buffer, and in some examples, even much smaller. As described below, dilution groups can be formed before and / or after SOMAmers hybridize with probes, i.e., before and / or after the formation of a three-molecule complex suitable for NGS.

[0100] Besides leveling through dilution, high-abundance SOMAmers can also be "leveled" by introducing H1 probes lacking bead-capturing tags as well as probes containing these tags. The ratio of H1 probes containing to those without bead-capturing tags will reduce the amount of trimolecular complexes captured in steps 304 of method 300 and step 358 of method 350 by a corresponding amount. For example, if the ratio of unlabeled probes to labeled probes is 10:1, only 10% of the trimolecular complexes are captured, resulting in an order-of-magnitude reduction in the count in the NGS output compared to a assay without the introduction of unlabeled probes. The ratio of unlabeled to labeled probes may vary for different SOMAmers, depending on the expected count for each SOMAmer.

[0101] 1. Four hybrid groups Figure 4 The steps and byproducts of an exemplary NGS assay involving four hybridization groups (indicated by 400 in total) are shown. In step 402, an eluent 404 containing SOMAmers is provided. As previously described, eluent 404 contains SOMAmers generated through prior exposure to the biological sample and separation from the target molecule.

[0102] In step 406, the eluent 404 is divided into four equal portions or samples 408, 410, 412, and 414. Figure 4 In the content determination, sample 408 is diluted at a ratio of 1:16, i.e., one part eluent to 16 parts buffer solution; sample 410 is diluted at a ratio of 1:4; and samples 412 and 414 are each diluted at a ratio of 1:2. In some cases, the sample may not be diluted.

[0103] In step 416, the four samples are each bound to a set of hybridization capture probes, labeled “Group 1”, “Group 2”, “Group 3”, and “Group 4”, respectively. In this example, capture probe group 418 binds to the elution buffer with the highest dilution, and therefore contains capture probes configured to bind to the most common SOMAmers in the elution buffer. Similarly, capture probe group 420 contains probes configured to bind to the next most common SOMAmers, and capture probe groups 422 and 424 each contain probes configured to bind to different subsets of relatively fewer SOMAmers. Thus, the result of step 416 is four different solutions, each configured to produce a set of three-molecule compounds after hybridization, each compound including an SOMAmer and a corresponding probe hybridizing to the SOMAmer. Each compound may be substantially similar to Figure 1 Compound 100 in the compound.

[0104] Any of the four sets of hybridization capture probes may contain a fixed proportion of H1 probes that include and exclude some subset of bead-capturing markers for SOMAmers within each set, for further count leveling.

[0105] In step 426, the four solutions generated in step 416 are respectively hybridized, captured onto beads, washed, and eluted. This can be done according to the previously described... Figure 3A Steps 304, 306, and 308 of method 300 shown are implemented.

[0106] In step 428, the different eluents generated in step 426 are recombine into a single eluent 430. In some cases, the different solutions may be recombinated in different volumes, thereby further diluting the relatively abundant capture groups (i.e., those corresponding to abundant SOMAmers, and thus to the abundant target molecule species in the original biological sample). This produces normalized combined solutions with smaller overall variations in the concentrations of different trimolecular compounds, which can be analyzed with relatively fewer sequencing “reads.” For example, the combination of dilution and normalization may reduce the number of reads required per sample from approximately 200 million to less than 5 million, allowing multiple samples to be reused in each sequencing cycle and reducing the cost per sample while still achieving acceptable accuracy (measured by the coefficient of variation (CV) in the results).

[0107] In step 432 (which can be considered a combination of steps 310, 312, and 314 of the aforementioned method 300), the solution 430 generated in step 428 is prepared for next-generation sequencing (NGS), sequenced, and the results are written to a data file and analyzed as needed. Preparation may include the use of universal primers ( Figure 1 P1 and P2) and related SOMAmer ID sequence I S PCR amplification is performed on the reporter region. As previously mentioned, preparation may also include ligating the aptamer sequence and / or the barcode sequence for demultiplexing to the three-molecule compound. The prepared solution is then sequenced using NGS technology, such as a next-generation sequencing platform developed by Immena, San Diego, California, or any other NGS sequencing platform. Following NGS, the sequencing data is analyzed or otherwise processed to determine the concentration of the analyte, such as the target protein, in the original biological sample. This may include demultiplexing the sequencing data using barcodes corresponding to each original sample (if multiple samples are pooled), counting reporter factor sequences, and scaling and / or normalizing the data to extract accurate results. Sequencing data may be written to a data file in a standard format, such as the ADAT format developed by SomaLogic.

[0108] 2. One hybridization group and four PCR groups Figure 5 The steps and byproducts of an exemplary NGS assay (typically indicated as 500) involving one hybridization group and four PCR groups are shown. In step 502, an eluent 504 containing SOMAmers is provided. As previously described, eluent 504 contains SOMAmers generated from prior exposure to the biological sample and separation from the target molecule.

[0109] In step 506, the eluent 504 is bound to a set of hybridization capture probes 508 (i.e., a set of probes configured to bind to all SOMAmers in the eluent). As described above, this set of probes may also contain a fixed proportion of labeled and unlabeled H1 probes.

[0110] In step 510, the solution produced in step 506 is hybridized, captured onto beads, washed, and eluted. This can be done according to... Figure 3A Steps 304, 306, and 308 of method 300 described herein are implemented. However, in this case, instead of a single universal primer set, four sets of primers can be used, each set associated with a specific subset of SOMAmer IDs, corresponding to a set of SOMAmers falling within a specific expected concentration range. In other words, step 510 produces four distinct sets of three-molecule compounds corresponding to different abundance groups of SOMAmers and thus to different abundance groups of target molecules in the SOMAmer eluent from the original biological sample, each set of three-molecule compounds containing different PCR primers, thus allowing for individual amplification.

[0111] In step 512, the eluent produced in step 510 is divided into four equal fractions, or samples 514, 516, 518, and 520. At this stage, each sample can be selectively diluted to any desired extent to normalize the expected concentration of the SOMAmer ID sequence to be amplified in the next step. However, Figure 5 No dilutions in step 512 are described.

[0112] In step 522, different elution buffers generated from step 512 are prepared for next-generation sequencing (NGS), including PCR amplification of the reporter region. However, in this case, different primer sets and associated reporter regions are amplified in each isolated elution buffer, resulting in only a subset of known SOMAmer ID sequences being amplified in each elution buffer.

[0113] In step 524, the different solutions containing the amplified SOMAmer ID sequence in each sample are recombine into a single eluent 526. In some cases, the separated solutions can be recombine at different volumes to achieve the dilution required for relatively rich SOMAmer ID sequences and to obtain a normalized combined solution with a smaller overall variation in SOMAmer ID concentration, which can be analyzed with relatively fewer reads.

[0114] In step 528, solution 526 is further prepared for next-generation sequencing (NGS), sequencing is performed, and the results are written to a data file and analyzed as needed. Preparation of the amplification eluent may include ligating the aptamer sequence and / or the barcode sequence for demultiplexing to the reporter region of the three-molecule compound. The prepared solution is then sequenced using NGS technology, and the sequencing data can then be analyzed or otherwise processed to determine the concentration of the target analyte in the original biological sample, as previously described.

[0115] 3. Four hybridization groups and four PCR groups Figure 6 The steps and byproducts of an exemplary NGS assay involving four hybridization groups and four PCR groups (total expressed as 600) are shown, thus merging the results. Figure 4-5 The content determination of 400 and 500 involves multiple aspects. In step 602, an eluent 604 containing SOMAmers is provided, which contains SOMAmers generated from prior exposure to the biological sample and separation from the target molecule.

[0116] In step 606, the eluent 604 is divided into four equal portions or samples 608, 610, 612, and 614. These samples may optionally be diluted to different degrees, or in some cases, the samples may not be diluted.

[0117] In step 616, the four samples are each bound to a set of hybridization capture probes, with sets 618, 620, 622, and 624 labeled "Group 1," "Group 2," "Group 3," and "Group 4," respectively. Each set of probes is configured to bind to a specific subset of SOMAmers in the elution buffer, and the samples are then hybridized separately. Therefore, the result of step 616 is four distinct solutions, each containing a set of three-molecule compounds, including the SOMAmer and the corresponding probe that hybridizes with it. Each compound may be substantially similar to... Figure 1 Compound 100 in the compound.

[0118] Any one of the four sets of hybridization capture probes may selectively include a fixed proportion of unlabeled and labeled H1 probes for further count leveling.

[0119] In step 626, the four hybridization solutions generated in step 616 are combined into a single eluent 628, which is then sequentially captured onto the beads, washed, and eluted. This can be done according to the previously described... Figure 3A Steps 304, 306, and 308 of method 300 are performed. As previously stated, each hybridization solution may be diluted or undiluted before recombination, and the differential volume of each group may also be used to compress the analyte changes before bead capture and washing.

[0120] In step 630, the eluent produced in step 626 is divided into four equal fractions, or samples 632, 634, 636, and 638. At this stage, each sample can be selectively diluted to any desired extent to normalize the expected concentration of the SOMAmer ID sequence to be amplified in the next step. However, Figure 6 No dilutions in step 630 are described.

[0121] In step 640, the different elution buffers generated in step 630 are used for next-generation sequencing (NGS), a process that includes PCR amplification of the reporter region. Figure 5 As shown in Figure 500, different primer sets and associated reporter regions were amplified in each separated eluent, thereby amplifying a subset of the SOMAmer ID sequence in each eluent.

[0122] In step 642, different solutions containing the amplified SOMAmer ID sequence in each sample are recombine into a single eluent 644. In some cases, the different solutions can be recombine at different volumes to achieve the dilution required for relatively rich SOMAmer ID sequences, and to obtain a normalized combined solution with a smaller overall variation in SOMAmer ID concentration, which can be analyzed with relatively fewer reads.

[0123] In step 646, solution 644 is further prepared for NGS and sequencing, and the results are written to a data file and analyzed as needed. Preparation of the amplification eluent may include ligating the aptamer sequence and / or the barcode sequence for demultiplexing to the reporter region of the three-molecule compound. The prepared solution is then sequenced using NGS technology, and the sequencing data can then be analyzed or otherwise processed to determine the concentration of the target analyte in the original biological sample.

[0124] D. PCR group quantitative peak normalization In NGS-based systems, signals are measured as sequence read counts. Reads for all analytes measured in the same sequencing run for a given sample are mixed together as a fixed or finite set of total read counts. As parts of the same mixture, the NGS read counts of all analytes measured in a given sample influence each other; therefore, the signal count observed from each analyte is the "net result" of increases and decreases in all analyte counts measured in a "zero-sum game" for each sample with a fixed total read count. More specifically, in an NGS system with a fixed total read count, any increase in the count of one analyte will result in a corresponding decrease in the counts of other analytes, distributed proportionally to each analyte's share of the total read count.

[0125] Figure 7 This "zero-sum game" is graphically illustrated by showing the results of a simplified dual-analyte assay for two different samples in the form of a histogram, where the vertical axis represents the total number of readings for each analyte, and this total number of readings is fixed at 2 million. In Sample 1, the counts for analytes A and B are equal. In Sample 2, analyte A increases by 500,000 counts, while analyte B decreases by a corresponding 500,000 counts. However, due to the finite number of total reads, it is impossible to obtain a complete analysis of the results from the two analyte assays. Figure 7 The difference in the counts of analytes A and B in sample 2 was determined to be due to an increase in analyte A, a decrease in analyte B, or a combination of both.

[0126] Figure 8 This paper describes how to normalize cross-sample compensated readings by introducing a reference reporter factor, or a quantitative spike control reporter factor (“qSpike”), thereby allowing the identification of true signal variations. Figure 8 In the NGS assay shown, the qSpike reporter factor was physically added (spiked) to a dual analyte system containing analytes A and B, where all samples had the same known concentration. In this case, the qSpike reporter factor was exactly the same as the concentrations of analytes A and B in sample 1. In sample 2, as before, an increase in analyte A and a decrease in analyte B were observed. However, it can now be observed that the qSpike in sample 2 is reduced relative to sample 1, even though the same concentration of qSpike was added to sample 1. Figure 8 As shown in the "Sample 2 qSpike Adjustment" histogram, scaling / adjusting all analytes in Sample 2 to push the spike back to its expected concentration will result in a noticeable increase in analyte A relative to analyte B.

[0127] In more practical NGS assays, a mixture of multiple qSpike reference reporter factors can be used to correct for compensatory variations in analyte count ratios. For example, the assay according to this specification can use a mixture of four unique H2 reporter factors, i.e., four unique amplifiable reporter factors forming part of a second probe, which is, for example... Figure 3A The assay 300 shown is introduced after eluting the three-molecule complex in step 308. Alternatively, specific qSpike SOMAmers can be introduced into the eluent, and a suitable qSpike reporter factor can be included in the SOMAmer-specific probe library. qSpike reporter factors or SOMAmers may be provided at different relative concentrations.

[0128] Figure 9-10 This is a histogram, depicting both the raw results and the qSpike-adjusted results of this content determination. The legend has the following meanings: QSpike-H is a high-concentration qSpike reporter factor. • QSpike-MH is a medium-to-high concentration qSpike reporter factor. QSpike-ML is a low to medium concentration qSpike reporter factor. QSpike-L is a low-concentration qSpike reporter factor. • Apolipoprotein E2, transferrin, and kininogen HMW were SOMAmer analytes.

[0129] exist Figure 9-10 In the assays shown, three SOMAmers—apolipoprotein E2, transferrin, and kininogen HMW—were titrated in buffer at concentrations ranging from 50 pM to 50 aM and measured in an NGS HC- assay. Each measurement point was a separate sample in which the three SOMAmer analytes were sequenced together, and qSpike was added to all samples at the same concentration.

[0130] exist Figure 9 In this study, qSpike exhibited compensatory changes due to variations in the analyte dose response signal. The NGS content determination signal (reading) was compared to a known peak, and a scaling factor was generated. Figure 10 In this study, the NGS counts have been scaled so that the peaks of all samples are uniform, which restores the actual SOMAmer dose to the response of the three SOMAmers measured in the assay.

[0131] Using the qSpike reporter factor to compensate for limited readings can be incorporated into any of the next-generation sequencing assays described above. For example, Figure 11Some steps and byproducts of an exemplary NGS assay (overall denoted as 1100) are described, involving four PCR groups and the addition of a qSpike reporter factor in each group. Therefore, the steps of assay 1100 can be incorporated into any NGS assay using multiple PCR groups, such as... Figure 5-6 The content determinations shown are 500 and 600.

[0132] In step 1102, an eluent 1104 containing SOMAmers, which have been hybridized with the probe, captured onto the bead, washed, and eluted, is provided. Therefore, eluent 1104 should be considered substantially similar to, for example... Figure 5 The content determination step 510 shown in step 500 is as follows: Figure 6 The eluent produced in step 626 of the content determination 600 shown.

[0133] In step 1106, the eluent 1104 is divided into four equal fractions or samples 1108, 1110, 1112, and 1114. At this stage, each sample can be selectively diluted to any desired extent to normalize the expected concentration of the SOMAmer ID sequence to be amplified in the next step.

[0134] In step 1116, the different elution buffers generated from step 1106 are prepared for next-generation sequencing (NGS), including PCR amplification of the reporter region. As in assays 500 and 600, different primer sets and associated reporter regions are amplified in each individual elution buffer, thereby amplifying a subset of the SOMAmer ID sequence in each elution buffer. However, in this case, different qSpike reporter factors are added to each elution buffer at known concentrations prior to PCR amplification.

[0135] In step 1118, different solutions containing the amplified SOMAmer ID sequence and qSpike reporter factor in each sample are recombine into a single elution buffer 1120. In some cases, the different solutions may be recombinated in different volumes to achieve the dilution required for relatively rich SOMAmer ID sequences and to obtain a normalized combined solution with a smaller overall variation in SOMAmer ID concentration, which can be analyzed with relatively fewer reads.

[0136] In step 1122, a solution 1120 for NGS is further prepared, sequenced, and the results are written to a data file and analyzed as needed. Preparation of the amplification eluent may include ligating the aptamer sequence and / or the barcode sequence for demultiplexing to the amplified reporter region of the three-molecule compound. The prepared solution is then sequenced using NGS technology, after which the sequencing data can be analyzed or otherwise processed to determine the concentration of the target analyte in the original biological sample. Due to the use of the qSpike reporter factor, the analysis may include scaling or renormalizing the data to restore the qSpike concentration to a known level, thereby compensating for counting errors that may occur due to limited sequencing reads.

[0137] E. Determining the stability of SOMAmer probes As previously stated, according to various aspects of this specification, hybridization regions H1 and H2 are configured to combine with the respective compensating portions of the corresponding SOMAmer, and can be designed to have similar or intentionally different melting temperatures (T0). m This allows for simultaneous hybridization under a given set of assay conditions. As discussed in this section, in some cases, the melting temperature of the estimated hybridization region can be calculated using experimentally determined melting profiles of the SOMAmer-probe pair.

[0138] 1. background The most widely used method for predicting the stability of nucleic acid double strands is known as the nearest neighbor model. The nearest neighbor model assumes that the thermodynamic properties of helix formation depend primarily on the identity of adjacent base pairs in the double strand. This model has been widely used to predict the stability of double strand formation required for primer design in PCR, as well as other applications where double strand formation is critical. For NGS assays according to this specification (i.e., involving SOMAmers), this method needs to be extended to accurately predict the stability of double strands consisting of one strand containing a modified DNA base and another strand containing native DNA bases.

[0139] Traditionally, absorbance curves as a function of temperature (melting curves), measured using a UV-Vis spectrophotometer, have been used to study the stability of DNA secondary structures. Hybridization is typically performed in a buffer solution of 1.0 M NaCl, 10 mM sodium carboxylate, and 0.5 mM Na₂EDTA at pH 7. The oligonucleotide concentration varies within a 100-fold range, and the thermodynamic parameters are determined by the remelting temperature (Tremelting). M -1 The curve was obtained by plotting the natural logarithm of the total DNA concentration against the total DNA concentration, and then fitted.

[0140] in addition, and Alternatively, the data can be obtained by separately fitting the melt curves and averaging them across different concentrations. Both methods are essentially van 'Hoff' analyses of the data. Thermodynamic data obtained from these two methods are typically within 10% agreement. This section will specifically use the latter method—separately fitting the melt curves—to extract thermodynamic parameters for predicting the stability of the mixed double strands. Furthermore, the buffer composition from which the thermal melt curves are obtained will be matched to the composition read from a typical SOMAmer content assay.

[0141] Based on various aspects of this specification, an extension of the nearest neighbor model was developed for SOMAmers containing the three most common modified bases: Nap-dU, 2-Nap-dU, and benzyl-dU. Melting curves were experimentally obtained by fluorescence measurements of over 400 SOMAmer-probe pairs, as described below. These data were used to define the nearest neighbor parameters required to predict the stability of SOMAmer-probes under assay readout conditions.

[0142] 2. Model SOMAmer-probe binding double strand formation SOMAmer-probe double strand formation follows the process:

[0143] in, It's SOMAmer. It is a hybridization probe. It is a double strand. Defined as the initial total concentration of DNA: , Assuming the initial concentrations of SOMAmer and probe are equal, the following equation can be obtained according to stoichiometry:

[0144]

[0145] in, This represents the mole fraction of the double strand. The equilibrium constant for double strand formation is...

[0146] in, and The enthalpy and entropy generated by the double strand. The absolute temperature is K. Let be the gas constant (1.9872 cal / K mol). Substituting the concentration into the equation, we get:

[0147] According to the definition, The temperature corresponding to the equal amounts of double-stranded and non-double-stranded molecules, i.e. The expression for the melting temperature is given below:

[0148] 3. SOMAmer melt model SOMAmers with internal structures can also be viewed as a simple two-state model as follows:

[0149] in, and These are structured and unstructured SOMAmers, respectively. Defined as the initial total concentration of DNA.

[0150] The following equations are derived based on chemometrics.

[0151]

[0152] in, This represents the mole fraction of the structured SOMAmer. The equilibrium constant of the structured SOMAmer is...

[0153] in, and The enthalpy and entropy of the structure formation, The absolute temperature is K. The gas constant is 1.9872 cal / K mol. Substituting the concentration equation into the mole fraction of the structured SOMAmer yields...

[0154] Similarly, according to the definition, The temperature corresponding to the equivalent amount of structured and unstructured SOMAmer, i.e. ,as follows:

[0155] The concentration of the SOMAmer does not contribute to the entropy because it is a unimolecular reaction, and all reactions are independent under appropriate dilution conditions. The simplest model assumes that SOMAmer melting and subsequent primer melting are independent processes, and vice versa. Without additional experiments, such as a standalone SOMAmer melt, it is impossible to know a priori which of the two transitions is caused by SOMAmer structural melting or hybridization primer melting. Primer melting is most likely due to higher free energy data, as it corresponds to melting of more than 16 base pairs.

[0156] 4. Experimental determination of melting thermodynamics The fluorescence intensity as a function of temperature (melting curve) was measured using the fluorescent dye SYBR Green I. Compared to single-stranded DNA, SYBR Green I exhibits a 100-fold increase in fluorescence intensity when binding to double-stranded DNA; therefore, the fluorescence intensity decreases as the SOMAmer-probe double-stranded structure melts.

[0157] The components defined by the SOMAscan eluent were thermally melted in a buffer solution: 100 mM tris(hydroxymethyl)aminomethane, pH 8.0, 200 mM NaCl, and 0.9 M perchlorate. Perchlorate is known to decrease the stability of DNA double strands. When both the SOMAmer and probe concentrations were at 120 µL (8.3 × 10⁻⁶), the eluent was determined to be... -7 At 100 pM, all thermal melting was achievable. Therefore, all thermodynamic parameters were obtained through a single-step fitting of individual thermal melting curves. Melting curves exhibiting more complex behavior than those expected by the two-state model were excluded from the analysis. Four plates were measured each for the H1 and H2 probes. These 800 melting curves were evaluated to obtain data consistent with the assumed two-state model of double-strand formation. Of these 800 curves, 408 SOMAmers containing three different modified nucleotides: Nap-dU, 2-Nap-dU, and benzyl-dU were used in this analysis.

[0158] a. Single-phase model fitting Figure 12 Data for typical thermal melting of SOMAmer-probe duplexes are shown, where the vertical axis represents fluorescence in RFU (reactive power of fluorescence), the horizontal axis represents temperature, and the data is covered by a two-state model fitting as follows. First, the high-temperature and low-temperature baselines are fitted using the first 15 and last 15 data points, respectively. The low-temperature baseline corresponds to the duplex material, and the high-temperature baseline corresponds to the single-chain material, denoted as […].

[0159] .

[0160] For a given and The value is calculated first. Then calculate The function relating the fraction of the double strand to temperature was obtained.

[0161] in .

[0162] The curve of the hot melt, According to the following calculations: .

[0163] Six free parameters were found using nonlinear regression. , , , , and The optimal value is obtained. The initial estimates for the single-chain and double-chain baselines are as described above. and The initial values ​​are -200 kcal / mol and -0.6 kcal / mol K, respectively. Figure 12 The model fit for the data is shown as follows Figure 12 The solid red line in the diagram. The subscript 'p' indicates SOMAmer-probe thermodynamics. This model fits the data very well.

[0164] b. Biphasic model fitting Typically, the data exhibit more complex melting behavior, which is likely due to the initial melting of the SOMAmer internal structure, followed by the melting of the SOMAmer-probe duplex. Figure 13 Data representing typical biphasic behavior are shown, again covered by the theoretical model fit represented by the red solid line. Two distinct transitions emerge in the data: the first likely is the melting of the internal SOMAmer structure, followed by the double-chain melting of the SOMAmer-probe. These two transitions are assumed to be independent. To fit the biphasic model, three baselines are needed. The first corresponds to the temperature dependence on the internal SOMAmer structure, the second to the temperature dependence on the double-chain SOMAmer-probe, and the third to the temperature dependence on the combined single-chain material. The latter two are the same as the baselines described above. The former is denoted as...

[0165] Here, fluorescence is assumed to be an additive, thus indicating that the fluorescence of the double-stranded SOMAmer-probe complex increases with increasing fluorescence. At low temperatures, the internal structure of SOMAmer and the double-stranded structure of SOMAmer-probe coexist; therefore, net fluorescence is assumed to be the sum of the individual structures. The mole fraction of the internal structure of SOMAmer is denoted as... The mole fraction of the SOMAmer-probe double strand is denoted as The thermal melting curve is .

[0166] At low temperatures, and Both are 1, and the temperature dependence is... As the SOMAmer structure melts, the fluorescence reaches the baseline of the duplex completely. Similar to the single-phase case, the two-phase model fits the data very well.

[0167] 5. Nearest neighbor model Once the experimental data fits a suitable model (e.g., single-phase or two-phase as described above) and conforms to the various thermodynamic parameters, the parameters of the nearest-neighbor model can be obtained from the data. The change in free energy is approximated as:

[0168] in, The subscript indicates that the SOMAmer-probe is double-stranded. It is the free energy of the nearest neighbor stacking interaction. It is the nearest neighbor. In double strand The number of times it appears in This is the initial free energy resulting from entropic considerations. Nearest neighbor interactions include the ten standard Watson-Crick nearest neighbor stacking interactions (such as...). , (e.g., symbols) express Watson-Crick bases (Pairing). Furthermore, each modified base introduces additional 7 modified nucleotide stacking interactions (e.g., , (etc.), among which Indicates modification Nucleotides, whose bases are similar to standard... Nucleotide pairing. and They have similar expressions.

[0169] For a group Double strand and Nearest neighbor interactions, calculated by factors Construct a dimensionless array from sequence data. "Stacked matrix" Thermodynamic values ​​observed in the experiment Represented as length The column vectors. Unknown nearest neighbor stacking interactions. Represented as length The column vectors are obtained by solving the following overdetermined linear equations using ordinary least squares regression: .

[0170] parameter Take Ou's The minimum value of the norm, .

[0171] 6. Exemplary results In the exemplary procedure according to the above specification, the nearest neighbor model parameters were extended using double-stranded thermal melting of three different modified nucleotides: Nap-dU, 2-Nap-dU, and benzyl-dU. Python code has been developed to calculate the thermodynamic and melting temperatures based on these extended nearest neighbor parameters.

[0172] The table below summarizes the 31 parameters required for the nearest neighbor model, including 10 parameters for the standard 4 bases and 7 additional parameters for each modified base. This includes the parameters for each neural network pair. and The values, and the total number of occurrences of each nearest neighbor parameter in the dataset. (AT / TA) and (TA / AT) appear relatively infrequently in the data because they only need to appear in fixed regions.

[0173]

[0174] The parameters in the table above can be used to calculate and determine the estimated melting temperature T for double strands containing the modified nucleotide bases Nap-dU, 2-Nap-dU, and benzyl-dU. m A similar process can be used to calculate the estimated melting temperature of any other SOMAmer-containing double strand. According to various aspects of this specification, these melting temperatures can then be used to determine where the hybrid complement is divided into SOMAmers, i.e., tripoleonous compounds (e.g., SOMAmers). Figure 1 The dividing point between the first and second hybridization regions H1 and H2 of compound 100 shown (and therefore between the first and second probes 104 and 106).

[0175] F. Illustrative combinations and additional examples This section describes other aspects and features of the systems and methods for the detection and quantification of target molecules in biological samples, based on various aspects of this specification. These are presented as a series of paragraphs, which, for clarity and efficiency, may be indicated by alphanumeric symbols in some or all of them. Each of these paragraphs may be combined in any suitable manner with one or more other paragraphs, and / or with disclosures elsewhere in this application (including material incorporated by cross-reference). Certain paragraphs below explicitly refer to and further limit the other paragraphs, and provide examples of suitable combinations, but are not limited thereto.

[0176] A quantitative system for the abundance of a target protein in a biological sample includes a plurality of aptamers, each aptamer configured to bind to a specific target protein when the biological sample containing the target protein is exposed to the aptamer, thereby forming an aptamer-containing eluent; a plurality of capture probes, each capture probe configured to hybridize with a specific aptamer, wherein the plurality of capture probes includes a first capture probe having a portion having hybridization with a first portion of the specific aptamer, and a second capture probe having a portion having hybridization with a second portion of the specific aptamer, a DNA primer region, and an aptamer ID sequence corresponding to the aptamer; a means of exposing the aptamer in the eluent to the capture probes to form a plurality of three-molecule complexes; and a means of sequencing the aptamer ID sequence to determine the abundance of the target protein in the biological sample.

[0177] B. A quantitative system for the abundance of two or more target proteins in a biological sample, comprising a plurality of aptamers, each aptamer configured to capture a specific protein in the sample, thereby forming an elution buffer containing the aptamer after isolating and capturing an aptamer of a target protein in the biological sample; a plurality of first probes, each first probe hybridizing with a corresponding first portion of a specific aptamer; a plurality of second probes, each second probe hybridizing with a corresponding second portion of a specific aptamer, each second probe including at least one DNA primer region and an aptamer ID sequence corresponding to the specific aptamer; means for sequencing the aptamer ID sequence; and means for quantifying the abundance of the target protein based on the sequenced aptamer ID sequence.

[0178] C. A detection system for a target protein in a biological sample, comprising a plurality of aptamers, each aptamer configured to capture a specific protein; a plurality of first probes, each first probe including a portion that hybridizes to a corresponding first portion of one of the aptamers that has captured the target protein; a plurality of second probes, each second probe including a portion that hybridizes to a corresponding second portion of one of the aptamers that has captured the target protein, at least one DNA primer region, and an aptamer ID sequence corresponding to the aptamer that has captured the target protein; means for amplifying the aptamer ID sequence; and means for sequencing the aptamer ID sequence to identify the aptamer that has captured the target protein and the target protein.

[0179] The systems described in any of the above paragraphs also include methods for normalizing the compensated read counts across samples.

[0180] The system described in any of the preceding paragraphs also includes means for dynamically compressing aptamer abundance and / or aptamer ID sequences prior to sequencing and counting.

[0181] Advantages, features and benefits The various embodiments and examples of methods and systems described herein for detecting and quantifying the presence of target molecules in biological samples offer numerous advantages over previously known approaches. For instance, the illustrative embodiments and examples described herein enable the quantification of aptamer-based protein detection using next-generation sequencing by simplifying the sequencing target from the aptamer to the aptamer recognition sequence.

[0182] In addition, among other benefits, the exemplary embodiments and examples described herein enable accurate detection of the abundance of target molecules across multiple orders of magnitude by dividing the assay eluent into multiple dilution groups at one or more stages of assaying and then recombining them before next-generation sequencing.

[0183] In addition, among other benefits, the exemplary embodiments and examples described herein enable the correction of errors caused by limited sequencing reads by normalizing compensated readings across samples by adding a quantitative peak reporter factor to the assay eluent.

[0184] Existing systems or devices cannot achieve these functions. However, not all embodiments and examples described herein have the same advantages or the same degree of advantage.

[0185] in conclusion The above disclosure may include multiple different embodiments, each with its own practical applicability. While each has been disclosed in its preferred form, the specific embodiments disclosed and described herein should not be considered in a limiting sense, as many variations are possible. The headings of the various sections used in this disclosure are for organizational purposes only. The subject matter of this disclosure includes all novel and non-obvious combinations and sub-combinations of the various elements, features, functions, and / or properties disclosed herein. The following claims specifically point to certain combinations and sub-combinations considered novel and non-obvious. Other combinations and sub-combinations of features, functions, elements, and / or properties may be claimed from priority applications or related applications. These claims, whether broader or narrower in scope than the original claims, identical or different, are also considered to be included within the scope of this disclosure.

Claims

1. A system for quantifying the abundance of a target protein in a biological sample, comprising: Multiple aptamers, each configured to bind to a specific target protein when a biological sample containing the target protein is exposed to the aptamer; An aptamer-containing eluent, comprising an aptamer that binds to the target protein, wherein the aptamer-containing eluent is divided into multiple dilution groups, each dilution group containing a specific concentration of aptamer based on the expected relative abundance of aptamer in each dilution group; Multiple capture probes, each configured to hybridize with a specific aptamer among the multiple aptamers, wherein each capture probe includes a DNA primer region and an aptamer ID sequence corresponding to the specific aptamer; Wherein, the aptamer ID sequence is used for sequencing to determine the abundance of the target protein in the biological sample; and The plurality of capture probes include a plurality of different capture probe sets, each set corresponding to an aptamer in one of the plurality of dilution sets.

2. The system according to claim 1, wherein, The plurality of dilution groups specifically includes two dilution groups.

3. The system according to claim 2, wherein, Each of the two dilution groups is diluted in a ratio of 1:2, 1:4, or 1:

16.

4. The system according to claim 1, wherein, The multiple dilution groups exactly include three dilution groups.

5. The system according to claim 4, wherein, Each of the three dilution groups is diluted at a ratio of 1:2, 1:4, or 1:

16.

6. The system according to claim 1, wherein, The plurality of dilution groups includes at least four dilution groups.

7. The system according to claim 6, wherein, Each of the plurality of dilution groups is diluted in a ratio of 1:2, 1:4, or 1:

16.

8. A system for detecting a target protein in a biological sample, comprising: Multiple aptamers, each configured to capture a specific protein; Biological sample, wherein the plurality of aptamers can be exposed to the biological sample to capture one or more target proteins in the biological sample using the respective aptamers; An aptamer-containing eluent, comprising an aptamer that binds to the target protein, wherein the aptamer-containing eluent is divided into multiple dilution groups, each dilution group containing a specific concentration of aptamer based on the expected relative abundance of aptamer in each dilution group; and Multiple probes, each probe including at least one DNA primer region and an aptamer ID sequence, the aptamer ID sequence corresponding to one of the plurality of aptamers to which the probe is configured to hybridize; The aptamer ID sequence is used to amplify and sequence to identify the aptamer ID sequence, capture the aptamer of the target protein, and the target protein; and The plurality of probes includes a plurality of different probe sets, each set corresponding to an aptamer contained in one of the plurality of dilution sets.

9. The system according to claim 8, wherein, The plurality of dilution groups specifically includes two dilution groups.

10. The system according to claim 9, wherein, Each of the two dilution groups is diluted in a ratio of 1:2, 1:4, or 1:

16.

11. The system according to claim 8, wherein, The multiple dilution groups exactly include three dilution groups.

12. The system according to claim 11, wherein, Each of the three dilution groups is diluted at a ratio of 1:2, 1:4, or 1:

16.

13. The system according to claim 8, wherein, The plurality of dilution groups includes at least four dilution groups.

14. The system according to claim 13, wherein, Each of the plurality of dilution groups is diluted in a ratio of 1:2, 1:4, or 1:

16.

15. A system for quantifying the abundance of a target protein in a biological sample, comprising: Multiple aptamer-containing samples, each sample containing an aptamer that has previously bound to a target protein upon exposure to multiple target proteins in a biological sample, wherein each of the multiple samples contains a specific concentration of the aptamer based on the expected relative abundance of the aptamer in each sample; and Multiple probes, each probe including at least one DNA primer region and an aptamer ID sequence, the aptamer ID sequence corresponding to one of a plurality of aptamers with which the probe is used to hybridize, wherein the aptamer ID sequence is used to be amplified and sequenced to identify the aptamer ID sequence, the aptamer previously bound to the target protein, and the target protein; and The plurality of probes includes multiple different probe sets, each set corresponding to an aptamer contained in one of the multiple samples.

16. The system according to claim 15, wherein, The plurality of samples specifically includes two samples, wherein each of the two samples is diluted in a ratio of 1:2, 1:4, or 1:

16.

17. The system according to claim 15, wherein, The plurality of samples specifically includes three samples, wherein each of the three samples is diluted in a ratio of 1:2, 1:4, or 1:

16.

18. The system according to claim 15, wherein, The plurality of samples includes at least four samples, and each of the plurality of samples is diluted in a ratio of 1:2, 1:4 or 1:

16.

19. The system according to claim 15, wherein, At least one of the plurality of samples was diluted, and at least one of the plurality of samples was not diluted.

20. The system according to claim 19, wherein, Each of the diluted samples was diluted at a ratio of 1:2, 1:4, or 1:16.

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