Methods for detecting analytes with different abundances

Abstract

The present invention provides a method for detecting multiple analytes in a sample, wherein the analytes are found to be present at different levels in the sample. [Solution] The method comprises (i) preparing a plurality of aliquots from the sample, and (ii) detecting different subsets of the analyte in each aliquot by performing a separate multiplex assay on each aliquot, wherein the analyte of each subset is selected based on the predicted abundance in the sample.

Description

Technical Field

[0001] The present invention provides a method for detecting a plurality of analytes in a sample, wherein the levels of the amounts present in the sample are different. In this method, a plurality of aliquots of the sample are prepared, and in each aliquot, a subset of analytes selected based on the predicted amount present in the sample is detected. Further provided is a method for detecting analytes in a sample, wherein the analytes are detected by detecting reporter nucleic acid molecules specific to the analytes. In this method, a PCR reaction is performed to amplify the reporter nucleic acid molecules, and an internal control is used in this PCR. The method of the present invention is particularly useful in proximity extension assays (PEA).

[0002] Background In recent proteomic techniques, it is necessary to be able to detect many different proteins (or protein complexes) from a small amount of sample. For this, multiplex analysis needs to be performed. Common methods that can achieve multiplex detection of proteins in a sample include proximity extension assay (PEA) and proximity ligation assay (PLA). PEA and PLA are described in WO01 / 61037, and PEA is further described in WO03 / 044231, WO2004 / 094456, WO2005 / 123963, WO2006 / 137932, and WO2013 / 113699. However, as is often the case, when the target protein exists in a wide concentration range, the signal from the low-concentration protein is buried by the signal from the high-concentration protein, and as a result, the protein present at a low concentration cannot be detected, thus posing a problem.

[0003] The present invention provides a detection method that can reliably detect analytes (e.g., proteins) present in a sample in a wide concentration range and improve the accuracy of multiplex detection methods. The method of the present invention can be applied to the above PEA or PLA, but may also be applied to other techniques used in analyte multiplex detection.

[0004] PEA and PLA are proximity assays and rely on the principle of "proximity probing." In these methods, the analyte is detected by the binding of multiple probes (i.e., two or more, usually two or three). These probes generate a signal when they come into proximity with the analyte by binding (hence the name "proximity probes"). Typically, at least one of the proximity probes contains a nucleic acid domain (or nucleic acid moiety) linked to the analyte-binding domain (or analyte-binding portion) of the probe, and signal generation requires interaction between the nucleic acid moieties and / or between the nucleic acid moieties and further functional portions held by other probes. Thus, signal generation depends on the interaction between probes (more specifically, between the nucleic acid moieties / nucleic acid domains they hold or between other functional portions / functional domains), and therefore, a signal is generated only when the required probes have bound to the analyte. As a result, the specificity of the detection system is improved.

[0005] In PEA, the nucleic acid moieties linked to the analyte-binding domains of probe pairs hybridize with each other when the probes are in close proximity (i.e., when they bind to the target), and are then extended by nucleic acid polymerase. The extension product forms a reporter nucleic acid, and by detecting this, the presence of a specific analyte (the analyte to which the relevant probe pair is bound) in the sample of interest is demonstrated. In PLA, when the probes of a probe pair bind to the target, the nucleic acid moieties linked to the analyte-binding domains of the probe pairs come into close proximity, and ligation between the nucleic acid moieties may occur, or these nucleic acid moieties may function together as templates for the ligation of a separately added oligonucleotide that can hybridize to the nucleic acid domain when in close proximity. The ligation product is then amplified and acts as a reporter nucleic acid. Analyte multiplex detection using PEA or PLA may be achieved by including a unique barcode sequence in the nucleic acid moiety of each probe. The reporter nucleic acid molecule corresponding to a specific analyte may be identified by the barcode sequence it contains. The methods of the present invention are particularly useful in multiplex PEA and multiplex PLA methods.

[0006] The method of the present invention may be useful in at least one field where proteomics is utilized, and in particular, it may be useful in diagnosis from the viewpoint of biomarker identification and quantification. In modern personalized medicine, for example in the field of oncology, it is necessary to be able to evaluate large biomarker panels. As personalized medicine becomes more widespread, the importance of being able to accurately identify and quantify many biomarkers (across a wide concentration range) in a sample is increasing. The present invention addresses this need.

[0007] Summary of the Invention For this purpose, in a first embodiment, the present invention provides a method for detecting a plurality of analytes in a sample, wherein the analytes are present at different levels in the sample, and the method is (i) Prepare multiple aliquots from the sample, (ii) detecting different subsets of the analyte in each aliquot by performing a separate multiplex assay for each aliquot, wherein the analyte of each subset is selected based on the predicted abundance in the sample.

[0008] In a second embodiment, the present invention provides a method for detecting an analyte in a sample, wherein the analyte is detected by detecting a reporter nucleic acid molecule specific to the analyte, the method comprising: performing a PCR reaction to produce a PCR product of the reporter nucleic acid molecule; and detecting the PCR product. An internal control is prepared for the PCR reaction, and this internal control is (i) A control nucleic acid molecule present in a predetermined amount and amplified by the same primer as the reporter nucleic acid molecule, or a separate component that contains such a nucleic acid molecule or causes such a nucleic acid molecule to be generated, and / or (ii) A unique molecular identifier (UMI) sequence present in each reporter nucleic acid molecule and / or each control nucleic acid molecule.

[0009] In a third embodiment, the present invention provides a method for detecting an analyte in a sample, wherein the analyte is detected by detecting a reporter nucleic acid molecule for the analyte, the method comprising: performing a PCR reaction to produce a PCR product of the reporter nucleic acid molecule; and detecting the PCR product, wherein an internal control is included in the PCR reaction, the internal control is present in a predetermined amount and is a control nucleic acid molecule, contains a control nucleic acid molecule, or causes a control nucleic acid molecule to be produced, the control nucleic acid molecule contains a sequence that is the reverse sequence of the reporter nucleic acid molecule.

[0010] Detailed explanation As detailed above, a first aspect of the present invention provides a method for detecting multiple analytes in a sample, wherein the analytes are present at different levels in the sample. This method relies on performing separate assay sets, which are grouped according to the abundance of the analytes to be analyzed.

[0011] Therefore, from another perspective, the method disclosed herein is a method for detecting multiple analytes in a sample, wherein the analytes are present at different levels in the sample, and the method is A method may be defined as one which includes performing a separate block assay on each of several separate aliquots obtained from the sample in order to detect a subset of the analyte in each of the separate aliquots, wherein the analyte of each subset is selected based on the predicted abundance in the sample.

[0012] Therefore, each assay block performed on individual aliquots is a multiple assay. Thus, a multiple assay for detecting multiple analytes in an analyte subset (i.e., an analyte subset designated to be detected in any one particular aliquot) may be considered a “quantity block.” Thus, as used herein, the term “quantity block” refers to a set of assays (assay blocks) (or a set of assays) performed to detect a particular group or subset of analytes in a sample to be detected (i.e., analyzed), where the analytes are assigned to each assay block (or assay set) based on their abundance in the sample, i.e., expected or predicted abundance, or relative abundance. In other words, assays are grouped or “blocked” based on abundance. Thus, different aliquots or different quantity blocks may be designated for the detection of a particular analyte subset, for example, based on low-level abundances, high-level abundances, or intermediate-level abundances of varying degrees. This does not mean that the abundance of each analyte in an assay block or assay set is the same or nearly the same. Abundances may vary between different analytes / assays in a block or set, and / or between different samples.

[0013] As used herein (with respect to all aspects of the present invention), the term “analyte” means any substance (e.g., molecule) or entity that is desired to be detected by the method of the present invention. Thus, the analyte is the “target” of the assay method of the present invention, i.e., the substance to be detected or screened using the method of the present invention.

[0014] Therefore, the analyte may be a biomolecule or compound to be detected, for example, a peptide or protein, or a nucleic acid molecule or small molecule, and may include organic and inorganic molecules. The analyte may be a cell, or a microorganism including a virus, or a fragment or product thereof. Thus, it will be understood that the analyte can be any substance or object from which a specific binding partner (e.g., affinity binding partner) can be developed. The only requirement is that the analyte can simultaneously bind to at least two binding partners (more specifically, at least two analyte-binding domains of proximity probes).

[0015] Probe-based assays are particularly useful for detecting proteins or polypeptides. Therefore, specific target analytes include any proteinaceous molecules such as peptides, polypeptides, proteins, or prions, or any molecules or fragments thereof containing protein components or polypeptide components. In a particularly preferred embodiment of the present invention, the analyte is a completely proteinaceous molecule or a partially proteinaceous molecule, most preferably a protein. That is, the analyte is preferably a protein or contains a protein.

[0016] The analyte may be a single molecule or a complex containing two or more molecular subunits. The molecular subunits may or may not be covalently bonded to each other. The molecular subunits may be the same or different. Thus, such a complex analyte may be a protein complex, or a biomolecular complex containing a protein and one or more other biomolecules, in addition to cells or microorganisms. Thus, such a complex may be a homopolymer or a heteropolymer. Aggregates of molecules such as proteins, for example, aggregates of the same protein or aggregates of different proteins, can also be target analytes. The analyte may be a complex of a protein or peptide with a nucleic acid molecule such as DNA or RNA. A specific target may be the interaction between a protein and a nucleic acid, for example, the interaction between a regulatory factor such as a transcription factor and DNA or RNA. Thus, in a particular embodiment, the analyte is a protein-nucleic acid complex (e.g., a protein-DNA complex, or a protein-RNA complex). In another embodiment, the analyte is a non-nucleic acid analyte, which means an analyte that does not contain nucleic acid molecules. Non-nucleic acid analytes include proteins and protein complexes, small molecules, and lipids, as mentioned above.

[0017] The present invention relates to the detection of multiple analytes in a sample. The multiple analytes may be of the same type (for example, all analytes may be proteins or protein complexes) or of different types (for example, some analytes may be proteins and others may be protein complexes, lipids, protein-DNA complexes or protein-RNA complexes, etc., or any combination of such types of analytes).

[0018] As used in this disclosure, the term “multiple” means more than one (i.e., two or more) according to its standard definition. However, the method of the first aspect of the present invention requires separate multiple reactions for multiple (i.e., at least two) aliquots of a sample. As used herein, the term “multiplex” refers to an assay in which multiple (i.e., at least two) different analytes are analyzed simultaneously, more specifically in the same aliquot of a sample or in the same reaction mixture. Thus, it is clear that the minimum number of analytes to be detected according to the method of the first aspect of the present invention is four (two analytes to be detected in each of the two aliquots of the sample). However, it is preferable that considerably more than four analytes be detected according to the method. Preferably, at least 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 or more analytes are detected according to this method.

[0019] The terms “detecting” or “detected” are used herein to broadly include any means of determining the presence or absence of an analyte (i.e., determining whether or not a target analyte is present in the sample of interest). Therefore, even if the method of the present invention attempts to detect a specific analyte of interest in a sample, but the analyte is not detected because it is not present in the sample, the “detecting” step is still performed because the presence or absence of the analyte in the sample is being evaluated. The “detecting” step does not depend on the success of the detection, i.e., whether or not the analyte is actually detected.

[0020] The detection of an analyte may further, in any form, involve measuring the concentration or abundance of the analyte in the sample. The absolute concentration of the target analyte may be determined, or the relative concentration of the analyte may be determined for the purpose of comparing the concentration of the target analyte with the concentrations of other target analytes (or other multiple target analytes) in the sample or other samples.

[0021] Therefore, “detect” may include determining, measuring, evaluating, or analyzing the presence or amount of an analyte by any means. This includes quantitative and qualitative determinations, measurements, or evaluations, and semi-quantitative determinations. Such determinations, measurements, or evaluations may be relative or absolute, for example, if two or more different analytes are detected in the sample. Thus, when used in the context of quantifying a target analyte in a sample, the term “quantify” may refer to absolute quantification or relative quantification. Absolute quantification may be achieved by including one or more control analytes of known concentrations and / or by comparing the detection level of the target analyte with known control analytes (e.g., by creating a standard curve). Alternatively, relative quantification may be achieved by comparing the detected levels or amounts of two or more different target analytes, thereby providing relative quantification of each of the two or more different target analytes, i.e., quantification of each other. Methods by which quantification can be achieved in the methods of the present invention will be further described below.

[0022] The method of the present invention is for detecting multiple analytes in a sample. Any sample of interest can be analyzed according to the present invention. That is, any sample that contains or may contain the analyte of interest and which is desirable to be analyzed in order to determine whether or not it contains the analyte of interest and / or to determine the concentration of the analyte of interest in the sample.

[0023] Therefore, any biological sample or clinical sample may be analyzed according to the present invention. For example, not only any cell sample or tissue sample of an organism, or any cell sample or tissue sample derived from an organism, or any body fluid or preparation derived therefrom, but also samples such as cell cultures, cell specimens, cell lysates, etc. may be analyzed according to the present invention. Environmental samples such as soil samples and water samples, or food samples may also be analyzed according to the present invention. The sample may be newly prepared or may be pretreated in any convenient manner, for example, for storage.

[0024] Therefore, typical samples include any substance that may contain a biomolecule or other desired analyte or target analyte, for example, foods and their related products, clinical samples, and environmental samples. The sample may be a biological sample and may contain viral or cellular substances including prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts, and organelles. Thus, such biological substances may include all kinds of mammalian cells and / or non-mammalian animal cells, plant cells, algae including cyanobacteria, fungi, bacteria, protozoa, etc.

[0025] The sample is preferably a clinical sample, for example, whole blood, blood-derived products such as plasma, serum, buffy coat, and blood cells, urine, feces, cerebrospinal fluid or other body fluids (for example, respiratory secretions, saliva, milk, etc.), tissues, biopsy materials, etc. The sample is particularly preferably a plasma sample or a serum sample. Therefore, the method of the present invention may be used, for example, for the detection of biomarkers or for analyzing a sample for an analyte derived from a pathogen. The sample may particularly be of human origin, but the method of the present invention may be similarly applied to samples derived from non-human animals (i.e., veterinary samples). The sample may be pretreated and prepared by any convenient method or desired method, such as cell lysis or cell removal, for use in the method of the present invention.

[0026] A method according to a first aspect of the present invention is for detecting multiple analytes in a sample that are present at different levels in the sample. That is, the analytes are present in the sample at different concentrations or within a certain concentration range. It is not necessary for every analyte in the sample to be present at substantially different concentrations from all other analytes, and rather, not all analytes are present at substantially the same concentration. The analytes in the sample are present within a certain concentration range, but certain analytes may be present at very similar concentrations.

[0027] The analyte may be present in the sample across a concentration range spanning several orders of magnitude. For example, the highest concentration of an analyte present in the sample (or expected to be present) may be approximately 1000 times the concentration of the lowest concentration of an analyte present in the sample (or expected to be present). For example, the concentrations of analytes in a sample may differ from each other by approximately 10 times, 100 times, 1000 times, or more, and of course, the ratio may be any value in between. In clinical samples, the analyte may be present across a range of several orders of magnitude, for example, 3, 4, 5, or 6 or more orders of magnitude.

[0028] The levels or values of abundance used to block or group together different analytes, more specifically assays for different analytes, may not depend only on the absolute level or absolute concentration of the analyte(s) present (or expected to be present) in the sample. Other factors may be considered, including the nature of the assay method, differences in analytical performance for different analytes, etc. For example, in the case of a detection assay based on an antibody or other binding agent, it may depend on the affinity or avidity of the antibody for the analyte. Such variations between assays for different analytes may be considered. For example, the abundance may be that which reflects the amount of analyte detected in the assay, converted into the output value or measured value of the assay. Thus, the predicted abundance that serves as a criterion when selecting analytes in a subset may depend at least on the predicted level or predicted concentration of the analyte(s) in the sample, but further or alternatively may depend on the predicted level or predicted value of the abundance determined in a particular detection assay. In other words, the abundance of the analyte(s) in the sample may be an apparent abundance or a theoretical abundance that depends on the detection assay. The apparent abundance of the analyte may vary depending on the assay used, particularly depending on the sensitivity of that assay.

[0029] The method includes preparing a plurality (i.e., at least two) of aliquots of the sample. That is, a portion of the sample is prepared as separate multiple ones. The sample may be divided into a plurality of aliquots (such that the entire sample is aliquoted), or a portion of the sample may be prepared as an aliquot without using the entire sample. The aliquots may be of the same size or the same amount, or of different sizes or different amounts, or some aliquots may be of the same size and others of different sizes.

[0030] At least some of the aliquots may be diluted. For example, the sample may be diluted to a ratio of 1:2, 1:4, 1:5, 1:10, etc. In particular, the aliquots may be diluted in 10-fold increments. That is, one or more aliquots may be diluted 10-fold (1:10), one or more aliquots may be diluted 100-fold (1:100), and one or more aliquots may be diluted 1000-fold (1:1000). Further dilution (e.g., 1:10,000 or 1:100,000) may be performed if necessary, but generally, a maximum dilution of 1:1000 is expected to be sufficient. One or more aliquots may not be diluted (referred to as 1:1 in this specification).

[0031] In a particular embodiment, 10-fold dilutions may be performed sequentially to prepare aliquots diluted to 1:1, 1:10, 1:100, and 1:1000. In this embodiment, a 1:10 dilution is performed by diluting the undiluted sample 10-fold. Dilutions of 1:100 and 1:1000 may be performed by directly diluting the undiluted sample 100-fold and 1000-fold, respectively, or by sequentially diluting a 1:10 aliquot 10-fold (i.e., a 1:10 aliquot may be diluted 10-fold to obtain a 1:100 aliquot, and a 1:100 aliquot may be diluted 10-fold to obtain a 1:100 aliquot). The dilution of the sample (and, in fact, all dispensing steps throughout the method of the present invention) may be performed manually or using an automated dispensing robot (e.g., SPT Labotek Mosquito).

[0032] The sample may be diluted using any suitable diluent, which may depend on the type of sample being analyzed. For example, the diluent may be water, saline solution, or a buffer solution, particularly a buffer solution containing a biocompatible buffer compound (i.e., a buffer suitable for the detection assay used, e.g., a buffer suitable for PEA or PLA). Suitable buffer compounds include, for example, HEPES, Tris (i.e., tris(hydroxymethyl)aminomethane), and disodium phosphate. Suitable buffer solutions for use as diluents include PBS (phosphate-buffered saline), TBS (Tris-buffered saline), and HBS (HEPES-buffered saline). The buffer solution (or other diluent) used must be prepared with a purified solvent (e.g., water) so as not to contain contaminant analytes. Therefore, the diluent should be sterile, and if water is used as a diluent or as a base for a diluent, the water used is preferably ultrapure water (e.g., Milli-Q water).

[0033] Any appropriate number of aliquots may be provided from the sample. As stated above, at least two aliquots are provided, but in most embodiments, more than two will be provided. In a particular embodiment, as detailed above, four aliquots may be provided, these being an aliquot of the undiluted sample and aliquots of the sample diluted to 1:10, 1:100, and 1:1000. If it is desired to increase or decrease the dilution of the sample, more or fewer aliquots may be provided. Furthermore, depending on the requirements / demands of the particular assay being performed, one or more aliquots may be provided at each dilution ratio.

[0034] When multiple aliquots are provided from a sample, a separate multiplex assay is performed on each aliquot to detect a subset of the target analyte in each aliquot. The separate multiplex assay is performed on each aliquot so that each aliquot is analyzed separately (i.e., multiple aliquots do not mix during the multiplex reaction). When multiplex assays are performed on all prepared aliquots, all target analytes are detected. That is, assays are performed on all aliquots to determine whether or not each target analyte is present in the sample of interest. However, individual assays to detect a particular analyte may be performed on only one aliquot. Thus, different subsets of the analyte are detected in each aliquot. In other words, different analytes are detected in each aliquot. Preferably, the subsets detected in each aliquot are completely different. That is, each target analyte is detected in only one aliquot so that there is no overlap between analyte subsets. However, in some embodiments, a particular analyte may be detected in multiple aliquots if it is deemed appropriate. In this example, there is some overlap of analytes between subsets, with some analytes existing in multiple analyte subsets, while others exist in only one subset.

[0035] Each subset of analytes is selected based on its predicted abundance (i.e., concentration) in the sample. That is, analytes that are expected to be present in the sample at similar concentrations may be included in the same subset and analyzed in the same multiple reaction. Conversely, analytes that are expected to be present in the sample at different concentrations may be included in different subsets and analyzed in different multiple reactions. Each analyte is assigned to a subset of analytes that are expected to be present in the sample at similar concentrations (e.g., concentrations of a certain order of magnitude). Each analyte subset is then detected in sample aliquots diluted to an appropriate ratio, taking into account the expected concentrations of the analytes. Thus, the analyte expected to be present at the lowest concentration may be detected in an undiluted aliquot or in a low-dilution aliquot, the analyte expected to be present at the highest concentration may be detected in the most diluted aliquot, and analytes expected to be present at concentrations between these limits may be detected in aliquots of "intermediate" dilution ratios.

[0036] As described above, in some embodiments, a particular analyte may be included in multiple subsets. This may be, for example, when the expected concentration of the analyte is essentially between the expected concentrations of two subsets and does not clearly "belong" to either one. In this example, the analyte may be included in both subsets. If it is known that the analyte can be present in the sample over a remarkably wide concentration range, the analyte may be included in two (or more) subsets.

[0037] Assuming that the analytes in each subset are selected based on their predicted abundance in the sample, it will be understood that the number of analytes in each subset may differ. Alternatively, the number of analytes in each subset may, as appropriate, be the same.

[0038] The abundance / concentration of each analyte in a sample may be predicted based on known facts regarding the normal levels of each analyte in the sample species being analyzed. For example, if the sample is a plasma or serum sample (or other body fluid sample), the concentration of the analyte in it may be predicted based on the known concentrations of the species in these body fluids. Normal plasma concentrations of a wide range of possible analytes of interest are available at https: / / www.olink.com / resources-support / document-download-center / . However, as mentioned above, the abundance values ​​used to assign analytes to a particular subset (block) may be assay-dependent and may depend on the results obtained from that assay (e.g., measured values).

[0039] As detailed above, a multiple reaction is performed on each aliquot to detect all analytes in the subset of analytes to be analyzed within the aliquot. As stated above, the term "multiple" means an assay in which at least two different analytes are analyzed simultaneously. However, preferably, considerably more than two analytes are analyzed in each multiple reaction. For example, each multiple reaction may analyze at least 5, 10, 15, 20, 25, 30, 40, 50, 60, or more analytes. In a particular multiple reaction, more analytes may be analyzed, for example, at least 70, 80, 90, 100, 110, 120, 130, 140, 150, or more analytes.

[0040] In a particular embodiment of this aspect of the present invention, the analyte is detected by detecting a reporter nucleic acid molecule specific to each analyte in each aliquot. In this embodiment, the presence of a specific analyte in the sample causes a nucleic acid molecule having a specific nucleotide sequence known to correspond to that specific analyte to be produced during the detection assay. The detection of a specific nucleotide sequence indicates the presence of the analyte corresponding to that sequence in the sample. Therefore, a "reporter nucleic acid molecule" is a nucleic acid molecule whose synthesis during the detection assay indicates the presence of a specific analyte in the sample. The reporter nucleic acid molecule may be an RNA molecule or a DNA molecule. Preferably, it is a DNA molecule.

[0041] The reporter nucleic acid molecule may be generated by any means known in detection assays in the art. For example, it may be generated by ligation of two (or more) nucleic acids to form a specific nucleotide sequence that indicates the presence of the analyte in the sample. Alternatively, the reporter nucleic acid molecule may be generated by extension of a provided nucleic acid molecule along a template nucleic acid molecule. Extension and ligation may be used in combination.

[0042] Therefore, the reporter nucleic acid molecule is generated while a multiplex detection assay is performed on each aliquot. Any detection assay that works by generating such a nucleic acid molecule may be used to generate the reporter nucleic acid molecule. In certain embodiments, the reporter nucleic acid molecule is generated in a proximity extension assay (PEA). That is, multiplex PEA may be performed to detect the analyte in each aliquot and, consequently, in the sample. In other embodiments, the reporter nucleic acid molecule is generated in a proximity ligation assay (PLA). That is, multiplex PLA may be performed to detect the analyte in each aliquot. As described above, methods for performing PEA and PLA are known in the art. It is particularly preferable that the detection assay performed is a PEA.

[0043] After generating the reporter nucleic acid molecule, it is preferably amplified to facilitate detection. Amplification of the reporter nucleic acid molecule is preferably performed by PCR, but other nucleic acid amplification methods may be used, such as loop-mediated isothermal amplification (LAMP).

[0044] As described above, each reporter nucleic acid molecule is specific to a particular analyte. Therefore, the reporter nucleic acid molecule identifies the given analyte and, more specifically, may contain an identification (ID) sequence that can detect the analyte, or a sequence or domain that functions as a tag. The ID sequence may be detected, for example, by functioning as a binding site for a probe or primer, as will be further detailed below, or more directly, by sequencing. Thus, in other words, this specificity may be achieved by the presence of one or more barcode sequences in the reporter nucleic acid molecule. Generally, a barcode sequence can be defined as a nucleotide sequence within the reporter nucleic acid molecule that identifies the reporter and, consequently, the detected analyte. The entire reporter nucleic acid molecule generated in a detection assay may be unique, in which case the entire reporter nucleic acid molecule may be considered a barcode sequence. More generally, one or more smaller parts of the reporter nucleic acid molecule function as a barcode sequence.

[0045] Analytes in a sample are detected by detecting a specific barcode sequence within reporter nucleic acid molecules generated during a multiplex detection assay. This can be achieved in several ways. Firstly, the specific barcode sequence may be detected by sequencing all reporter nucleic acid molecules generated during the multiplex detection assay. Alternatively, all different reporter nucleic acid molecules generated may be identified by their barcode sequences by sequencing all of the generated reporter nucleic acid molecules, thereby allowing for the identification of all analytes present in the sample (this identification is based on whether or not a reporter nucleic acid molecule known to correspond to each target analyte is detected). Nucleic acid sequencing is a preferred method for the detection / analysis of reporter nucleic acids.

[0046] Other suitable methods for detecting reporter nucleic acid molecules include PCR-based methods. For example, quantitative PCR using TaqMan probes may be performed. In this example, the reporter nucleic acid molecule (or at least a portion of each reporter nucleic acid molecule containing the barcode sequence) is amplified, and a probe complementary to each barcode sequence is provided, but each different probe is bound to a different identifiable fluorescent substance. The presence or absence of each barcode (and thus the reporter nucleic acid molecule, and even the analyte) can then be determined based on whether a particular barcode was amplified. However, although the PCR-based methods described above are clearly only suitable for simultaneously analyzing a relatively small number of different sequences, combinatorial methods using probes to decode barcode sequences are known and may be used to extend the multiplexing capacity to some extent. Since there is virtually no limit to the number of sequences that can be identified in a single trial with nucleic acid sequencing methods, and because they allow for a higher level of multiplexing than detection using PCR, sequencing methods are preferred methods for detecting reporter nucleic acid molecules.

[0047] Preferably, a reporter nucleic acid molecule is detected using a form of high-throughput DNA sequencing. Synthetic sequencing is a preferred DNA sequencing method. Examples of synthetic sequencing methods include pyro-sequencing, reversible die-terminator sequencing, and ion-torrent sequencing, all of which can be used in this method. Preferably, the reporter nucleic acid is sequenced using a massively parallel DNA sequencing method. Massively parallel DNA sequencing can be applied in particular to synthetic sequencing methods (e.g., the reversible die-terminator sequencing, pyro-sequencing, or ion-torrent sequencing methods described above). Massively parallel DNA sequencing using a reversible die-terminator is a preferred sequencing method. Massively parallel DNA sequencing using a reversible die-terminator can be performed, for example, using the Illumina® NovaSeq® system.

[0048] As is well known in the art, ultra-parallel DNA sequencing is a technique for sequencing multiple (e.g., thousands, millions, or more) DNA strands in parallel, i.e., simultaneously. In ultra-parallel DNA sequencing, target DNA molecules need to be immobilized on a solid surface, such as the surface of a flow cell or on beads. Each immobilized DNA molecule is then sequenced individually. Typically, ultra-parallel DNA sequencing methods employing reversible dye-terminator sequencing utilize flow cells as the immobilization surface, while ultra-parallel DNA sequencing methods employing pyro-sequencing or ion-torrent sequencing utilize beads as the immobilization surface.

[0049] As is known to those skilled in the art, immobilization of DNA molecules on the surface in massively parallel sequencing is usually achieved by attaching one or more sequencing adapters to the ends of the molecule. Therefore, the method of the present invention may include attaching one or more sequencing adapters to a reporter nucleic acid molecule.

[0050] Generally, the sequencing adapter is a nucleic acid molecule (particularly a DNA molecule). In this example, a short oligonucleotide complementary to the adapter sequence is bound to the immobilization surface (e.g., the surface of a bead or flow cell) to enable annealing of the target DNA molecule to the surface via the adapter sequence. Alternatively, the target DNA molecule may be bound to the immobilization surface using other binding partner pairs, such as biotin and avidin / streptavidin. In this case, biotin may be used as the sequencing adapter, and a biotin sequencing adapter may be bound to the immobilization surface using avidin or streptavidin bound to the surface, or vice versa.

[0051] Therefore, the sequencing adapter may be a short oligonucleotide (preferably DNA), typically 10 to 30 nucleotides long (e.g., 15 to 25 nucleotides, or 20 to 25 nucleotides). As detailed above, the purpose of the sequencing adapter is to enable annealing of the target DNA molecule to the immobilized surface; therefore, the nucleotide sequence of the nucleic acid adapter is determined by the sequence of the binding partner bound to the immobilized surface. There are no other particular restrictions on the nucleotide sequence of the nucleic acid sequencing adapter.

[0052] The sequencing adapter may be attached to the reporter nucleic acid molecule of the present invention during PCR amplification. In the case of a nucleic acid sequencing adapter, this can be achieved by including the sequencing adapter nucleotide in one or both primers. Alternatively, if the sequencing adapter is a non-nucleic acid sequencing adapter (e.g., a protein / peptide or small molecule), the adapter may be bound to one or both PCR primers. Alternatively, the sequencing adapter may be attached to the reporter nucleic acid molecule by directly ligating or binding the sequencing adapter to the reporter nucleic acid molecule. Preferably, one or more sequencing adapters used in this method are nucleic acid sequencing adapters.

[0053] One or more nucleic acid sequencing adapters may be attached to the reporter molecule in one or more ligation and / or amplification steps. For example, if two sequencing adapters are attached to the reporter nucleic acid molecule (one at each end), they may be attached in one step (e.g., by PCR amplification using a pair of primers, both containing sequencing adapters) or in two steps. These two steps may be performed in the same way or in different ways. For example, the first sequencing adapter may be attached to the reporter nucleic acid molecule by ligation and the second sequencing adapter by PCR amplification, or vice versa. Alternatively, the first amplification reaction may be performed to attach the first sequencing adapter to the reporter nucleic acid molecule, and then the second amplification reaction may be performed to attach the second sequencing adapter to the reporter nucleic acid molecule.

[0054] As described above, one or more sequencing adapters may be attached to the reporter nucleic acid molecule. This means one or two sequencing adapters. Since sequencing adapters are attached to the ends of DNA molecules, the maximum number of sequencing adapters that can be attached to one DNA molecule (e.g., reporter nucleic acid) is two. Therefore, one sequencing adapter may be attached to one end of the reporter nucleic acid molecule, or two sequencing adapters may be attached, one to each end of the reporter nucleic acid molecule. In certain embodiments, Illumina P5 adapters and Illumina P7 adapters are used. That is, a P5 adapter is attached to one end of the reporter nucleic acid molecule, and a P7 adapter is attached to the other end. The sequence of the P5 adapter is shown in SEQ ID NO: 1 (AAT GAT ACG GCG ACC ACC GA), and the sequence of the P7 adapter is shown in SEQ ID NO: 2 (CAA GCA GAA GAC GGC ATA CGA GAT).

[0055] Therefore, in certain embodiments of the present invention, at least one first PCR amplification is performed on the reporter nucleic acid molecule in order to attach at least one first sequencing adapter to the reporter nucleic acid molecule. As described above, the reporter nucleic acid molecule is produced in the detection reaction in response to the presence of the corresponding target analyte (i.e., the analyte whose presence is indicated by the production of the reporter nucleic acid molecule). Furthermore, as described above, the reporter nucleic acid molecule is preferably amplified to enable or improve its detection.

[0056] Therefore, this amplification may be combined with the addition of one or more sequencing adapters to the reporter nucleic acid molecule. This may be achieved by amplifying the reporter nucleic acid molecule using a primer pair containing at least one sequencing adapter. In this example, at least one of the primers in the primer pair contains a sequencing adapter upstream of the sequence that binds to the reporter nucleic acid molecule. Thus, the sequencing adapter is typically located at the 5' end of one of the primers containing it.

[0057] In certain embodiments, the amplification step is performed using a primer pair in which one primer contains a sequencing adapter, such that one sequencing adapter is attached to one end of the reporter nucleic acid molecule.

[0058] In another embodiment, the amplification step is performed using a primer pair in which both primers contain sequencing adapters, such that the sequencing adapters are attached to each end of the reporter nucleic acid molecule in a single amplification step.

[0059] In another embodiment, sequencing adapters are attached to each end of a reporter nucleic acid molecule by performing two separate amplification reactions. In this case, each amplification step attaches a different sequencing adapter to a different end of the molecule.

[0060] In another embodiment, the initial amplification step is performed using primers that do not contain sequencing adapters. Subsequently, as described above, one or more further amplification reactions are performed on the amplified reporter nucleic acid molecule to add sequencing adapters to each end of the molecule.

[0061] As detailed above, each reporter nucleic acid molecule generated during the detection assay may contain a barcode sequence corresponding to a specific analyte. Therefore, reporter nucleic acid molecules with different sequences are generated in response to the presence of different analytes in the sample. Nevertheless, to facilitate multiplexing, it is preferable that all reporter nucleic acid molecules generated during the detection assay share a common primer binding site so that all different reporter nucleic acid molecules can be amplified using the same primer pair.

[0062] If a first PCR amplification is performed on a reporter nucleic acid molecule to attach only one sequencing adapter to the molecule, a second PCR amplification may be performed on the amplified reporter nucleic acid molecule (i.e., the product of the first PCR amplification) to attach a second sequencing adapter. Thus, in this embodiment, the first PCR amplification is performed using a primer pair in which one primer contains the sequencing adapter, thereby attaching the first sequencing adapter to one end of the reporter nucleic acid molecule. Subsequently, a second PCR amplification is performed using a different primer pair. In the second primer pair, one primer contains the second sequencing adapter. The second sequencing adapter is different from the first sequencing adapter, i.e., has a different sequence. The primer containing the second sequencing adapter binds to the reporter nucleic acid molecule at the end opposite to the end containing the first sequencing adapter, so that the second sequencing adapter is attached to the reporter nucleic acid molecule at the end opposite to the end containing the first sequencing adapter.

[0063] If necessary to amplify the product of the first PCR amplification, the second primer of the second primer pair may contain the sequence of the first sequencing adapter so that it can bind to the end of the reporter nucleic acid molecule to which the first sequencing adapter has been attached during the first PCR amplification. In certain embodiments, the primer containing the first sequencing adapter used in the first PCR amplification to attach the first sequencing adapter to the reporter nucleic acid molecule is also used in the second PCR amplification. That is, the same primer (containing the first sequencing adapter) may be used in both the first and second PCR amplifications.

[0064] In embodiments in which two PCR amplifications are performed consecutively to attach sequencing adapters to both ends of a reporter nucleic acid molecule, the product of the first PCR may be purified before performing the second PCR. Standard methods for purifying PCR products are known in the art.

[0065] As described above, the Illumina P5 sequencing adapter and the Illumina P7 sequencing adapter are preferred sequencing adapter pairs used in the present invention. In certain embodiments, the P5 sequencing adapter is attached to the reporter nucleic acid molecule in a first PCR amplification, and the P7 sequencing adapter is attached to the reporter nucleic acid molecule in a second PCR amplification. In other embodiments, the P7 sequencing adapter is attached to the reporter nucleic acid molecule in a first PCR amplification, and the P5 sequencing adapter is attached to the reporter nucleic acid molecule in a second PCR amplification.

[0066] It is preferable that at least one of the one or two PCR amplifications performed to attach a sequencing adapter to a reporter nucleic acid molecule is carried out to saturation. As is well known in the art, the amount of PCR amplification product with respect to the number of cycles follows an "S" curve. Initially, the amplicon concentration increases gradually, then an exponential amplification phase is reached, during which the amount of product (almost) doubles with each amplification cycle. After the exponential phase, a linear phase is reached, where the amount of product increases linearly rather than exponentially. Finally, a plateau is reached, where the amount of product reaches the maximum possible level, determined by the reaction setup and the concentrations of the components used.

[0067] In the present invention, saturated PCR may generally be considered as PCR beyond the exponential phase, i.e., PCR in the linear phase or having reached a plateau. In certain embodiments, as used herein, “saturated” means continuing the reaction until the maximum possible product is obtained (i.e., until the amount of product reaches a plateau), so that no further product can be produced even if the amplification cycle is repeated. Saturation can be reached when the reaction components are depleted, for example, when the primers or dNTPs are depleted. When the reaction components are depleted, the reaction rate decreases and then enters a plateau state. Less commonly, saturation can be reached when the polymerase is depleted (i.e., when the polymerase loses its activity). Saturation can also be reached when the amplicon concentration becomes so high that the DNA polymerase concentration is no longer sufficient to maintain exponential amplification, i.e., when there are more amplicon molecules than polymerase molecules. In this example, as long as there are enough primers and dNTPs remaining in the reaction mix, the amplification enters and maintains the linear phase.

[0068] In certain embodiments, two PCR amplifications are performed to attach the sequencing adapter to the reporter nucleic acid molecule, and both of these reactions are carried out to saturation. In other embodiments, only the first PCR amplification of the two is carried out to saturation. Alternatively, only the second PCR amplification of the two is carried out to saturation. It is particularly preferable that only the first PCR amplification of the two is carried out to saturation.

[0069] PCR amplification can be assumed to be saturated simply by performing many PCR amplification cycles until saturation is reached. For example, PCR amplification performed for at least 25, 30, 35, or more cycles can be assumed to have reached saturation by the end of the process, in that the exponential amplification phase ends by that stage. Alternatively, saturation can be measured by quantitative PCR (qPCR). For example, Tuckman PCR may be performed using a probe that binds to a sequence common to all reporter nucleic acid molecules, or qPCR may be performed using a dye that changes color when it binds to double-stranded DNA, such as SYBR green. By tracking the reaction in this way, the minimum number of amplification cycles required to reach saturation can be determined. In any case, if further processing of the amplified reporter nucleic acid molecule is required (before sequencing), it will be necessary to perform one of these experimental qPCRs to identify the saturation point in an aliquot separate from the one used in the experiment to generate the reporter nucleic acid molecule for sequencing. This is because Tuckman probes or intercalation dyes are prone to interfering with further steps in this method.

[0070] As detailed above, a separate multiple reaction is performed for each aliquot of the target sample. Each aliquot is used to detect analytes present at different levels in the sample. Initially, reporter nucleic acid molecules are produced in amounts corresponding to the amount of each analyte in the sample. Therefore, for analytes present at high concentrations, high concentrations of reporter nucleic acid molecules can be expected to be produced, and for analytes present at low concentrations, low concentrations of reporter nucleic acid molecules can be expected. The amount of reporter nucleic acid molecules produced can be expected to be proportional to the amount of the corresponding analyte present in the sample. For example, for a first analyte present in the sample at 10 times the concentration of a second analyte, it can be expected that 10 times the amount of reporter nucleic acid molecules for the first analyte will be produced compared to the amount for the second analyte. Therefore, a much larger amount of reporter nucleic acid molecules will be produced in an aliquot used to detect an analyte expected to be present at a high concentration in the sample than in an aliquot used to detect an analyte expected to be present at a low concentration in the sample.

[0071] If this difference in the amount of reporter nucleic acid carries over to the analysis step that identifies the reporter nucleic acid molecule (e.g., the sequencing step), the most abundant reporter nucleic acid molecule may "cancel out" the signal of the reporter nucleic acid molecule present in small amounts, resulting in insufficient detection of analytes present in small amounts in the sample.

[0072] In PCR performed to saturation, amplifying the reporter nucleic acid molecules obtained from each multiple reaction means eliminating the differences in reporter nucleic acid concentration between aliquots. Once saturation is reached, each aliquot will contain essentially the same amount of reporter nucleic acid molecules. This means that for each analyte present in the sample, a similar amount of reporter nucleic acid molecules is present, and consequently, when analyzing reporter nucleic acid molecules, all reporter nucleic acid molecules (and thus their corresponding analytes) should be detected.

[0073] As described above, the multiplex detection assay used in this method is performed separately on multiple aliquots of the target sample. Subsequently, the products of the multiplex detection assay are used to identify which of the target analytes are present in the sample. As detailed above, this may be achieved using reporter nucleic acid molecules that correspond to different analytes and are analyzed, for example, by sequencing, to determine which reporter nucleic acid molecules are present (and thus which analytes are present in the sample). It is possible to analyze each multiplex reaction performed on each sample aliquot separately. However, in a preferred embodiment of the present invention, the reaction products from each aliquot (i.e., the products of the multiplex detection assay) are pooled (i.e., mixed). In other words, in such a pooling step, separate "abundance-based blocks" can be considered to be pooled. This allows for more efficient analysis of the reaction products by enabling a single analytical reaction (e.g., a sequencing reaction) for all aliquots of the sample.

[0074] When the product of a multiplex detection assay is a reporter nucleic acid molecule, it is preferable to first amplify the reporter nucleic acid molecule (e.g., by PCR) and pool the amplified product. In some cases, further amplification steps may be performed in the pool. It is particularly preferable to perform a separate first PCR amplification on each reporter nucleic acid molecule generated by each separate multiplex detection assay, in which the first sequencing adapter is attached to the nucleic acid molecule, as described above, and then pool the products. In other words, in each separate aliquot, a detection assay is performed to generate a reporter nucleic acid molecule, and a first PCR reaction is performed on the reporter nucleic acid molecule to both amplify the reporter nucleic acid molecule and attach the first sequencing adapter to one end of it. The products of this first amplification reaction are pooled. If necessary, the products of each separately performed first PCR reaction may be purified before pooling. Alternatively, the products of the separate first PCR reactions may be pooled, and then all PCR products in the pool may be purified together. However, it is not necessary to purify the products of the first PCR amplification before proceeding to the second PCR amplification.

[0075] After pooling, a second PCR amplification is performed on the pooled product of the first PCR amplification. The second PCR is used to amplify the product of the first PCR and to add the second sequencing adapter to the reporter nucleic acid molecule, as detailed above. When pooling the product of the first PCR, it is important to perform the first PCR to saturate so that the amplified reporter nucleic acid molecule is present in approximately the same amount in each aliquot at the time of pooling. It is not important whether the second PCR amplification performed on the pooled product of the first PCR amplification is also performed to saturation, but it may be done if necessary. In a preferred embodiment, both the first and second PCR amplifications are performed to saturation.

[0076] In another embodiment, a separate multiplex detection assay is performed on each separate aliquot. Subsequently, a single PCR reaction is performed on each aliquot until saturation is reached, attaching a sequencing adapter to each end of the reporter nucleic acid molecule (one sequencing adapter is attached to each end of each reporter nucleic acid molecule). The products of this PCR reaction are then pooled and sequenced.

[0077] In yet another embodiment, a separate multiplex detection assay is performed on each separate aliquot. Subsequently, two PCR amplifications are performed on the reporter nucleic acid molecule generated in each aliquot. Both of these are performed separately for each aliquot. The first PCR is used to attach the first sequencing adapter to the reporter nucleic acid molecule, and the second PCR is used to attach the second sequencing adapter to the reporter nucleic acid molecule (the end of the reporter nucleic acid molecule opposite to the first sequencing adapter). The products of the second PCR are then pooled and sequenced. In this embodiment, it is important that at least one of the PCR amplifications is performed to saturation for each aliquot. Either the first PCR or the second PCR may be performed to saturation, or both PCRs may be performed to saturation, as long as the same reaction is performed to saturation in each aliquot.

[0078] When pooling amplified reporter nucleic acid molecules from separate multiple reactions, the amounts of amplification products from each separate multiple reaction added to the pool may be the same or different. Equal amounts of amplification products from each separate multiple reaction may be added to the pool. This may be achieved by adding the complete amplification reaction mixture from each multiple reaction to the pool, or by taking the same specified amount from each amplification reaction mixture and adding it to the pool. In this example, for instance, if three aliquots are prepared from the sample, and a separate multiple detection assay is performed on each, and the amplified reporter nucleic acid molecules from each aliquot are pooled, then one-third of the pool will come from each aliquot. Similarly, if four aliquots are prepared from the sample, then one-quarter of the pool will come from each aliquot.

[0079] Alternatively, amplification products obtained from each separate multiplex reaction may be added to the pool in different amounts. "Different amounts of amplification products" simply means that the amount of amplification product added to the pool is not the same across all aliquot / multiply detection assays. Thus, different amounts of amplification products obtained from each multiplex detection assay may be added to the pool, or equal amounts of amplification products may be added from some but not all aliquots, so that different amounts of amplification products are added from some aliquots. For example, if three aliquots are prepared from a sample, and a separate multiplex detection assay is performed on each, and the amplified reporter nucleic acid molecules from each aliquot are pooled, then different amounts of amplification products may be added to the pool from all three aliquots. Alternatively, equal amounts of amplification products may be added to the pool from two aliquots, and different amounts from the third aliquot. Similarly, if four aliquots are prepared from a sample, different amounts of amplification products may be added to the pool from all four aliquots. Alternatively, equal amounts of amplification product may be added to the pool from three aliquots, and a different amount of amplification product may be added from the fourth aliquot. If equal amounts of amplification product are added to the pool from two aliquots, different amounts of amplification product may be added from the other two aliquots, or the same amount (first amount) of amplification product may be added to the pool from two aliquots, and a different amount (second amount) of the same amplification product may be added from the other two aliquots.

[0080] When different amounts of amplification product are added to the pool from various aliquots, the amount added from each aliquot is preferably proportional to the number of analytes detected in each aliquot. For example, if twice as many analytes are detected in the first aliquot as in the second aliquot, then twice as much is added to the pool from the first aliquot. This can be considered as adding the same amount of amplification product to the pool for each analyte detected in the sample across all aliquots. For example, if 100 analytes are detected across three aliquots, with 50 in the first, 30 in the second, and 20 in the third, then the three aliquots are added to the pool in a ratio of 5:3:2, so that 50% of the pool comes from the first, 30% from the second, and 20% from the third.

[0081] The method of the first aspect of the present invention may be used to analyze multiple samples in parallel. When analyzing multiple samples in parallel, the samples may be of the same type or of different types. Preferably, all samples are of the same type, for example, all are plasma samples or all are saliva samples. The set of analytes detected in each sample may also be the same or different. Preferably, the same set of analytes is detected in each sample, and each of the specific analytes in all samples is identified using the same reporter nucleic acid molecule. Analyzing multiple samples in parallel means analyzing multiple samples simultaneously, while performing each step of the method essentially simultaneously for each sample.

[0082] When analyzing multiple samples in parallel, as detailed above, multiple aliquots are prepared from each sample, and a subset of the analyte is detected in each aliquot. Preferably, the same number of aliquots are prepared from each sample. For example, three aliquots may be prepared from each sample, or four aliquots may be prepared from each sample. However, this is not mandatory, and different numbers of aliquots may be prepared from different samples, for example, two aliquots from some samples, three from others, four from yet another, and five from yet another.

[0083] As described above, it is preferable that the same set of analytes is detected in each sample and that the same number of aliquots are prepared from each sample. It is even more preferable that the analytes in each sample are divided into aliquots in the same way so that the same subset of analytes is detected in each corresponding sample aliquot (i.e., aliquots from each sample with the same dilution ratio).

[0084] When analyzing multiple samples in parallel using the method of the first aspect of the present invention, the reporter nucleic acid molecule may be amplified as described above, and the amplified products from each specific sample may be pooled as described above to generate a first pool. Thus, the first pool may be generated separately for each sample, and each first pool contains the amplified products from all multiplex detection assays performed on that sample (i.e., the amplified products from all aliquots prepared for that sample).

[0085] In one embodiment, the separate first pools generated for each sample may be further pooled to facilitate subsequent analysis. In such an embodiment, after the first pooling step, a sample index is added to the amplified products of each first pool. The sample index is a nucleotide sequence that identifies the original sample from which the amplified product originates. Thus, different nucleotide sequences are used as sample index sequences for the amplified products derived from each sample. If sequencing of the amplified products is subsequently performed, the sample index will indicate which sample each individual reporter nucleic acid molecule comes from. Any nucleotide sequence may be used as the sample index. The sample index sequence may be of any length, but is preferably relatively short, for example, 3-12 nucleotides, 4-10 nucleotides, or 4-8 nucleotides.

[0086] Therefore, different sample index sequences are used to label the amplification product in each separate first pool. However, within each individual first pool, the sample index sequence is the same. The sample index sequence may be added to the amplification product by any appropriate method; for example, the sample index may be added during the amplification reaction (e.g., by PCR) or during the ligation reaction. In particular, if the amplified reporter nucleic acid molecule is analyzed by a massively parallel DNA sequencing method and requires sequencing adapters at both ends, the sample index sequence cannot be added in such a way that it ultimately lies at the ends of the reporter nucleic acid molecule.

[0087] As described above, it is preferable to perform a first PCR amplification on the reporter nucleic acid molecule, which includes attaching a first sequencing adapter to the reporter molecule, and then pool the reporter nucleic acid molecules to create a first pool. This is also the case when analyzing multiple samples in parallel. As described above, it is preferable to perform a first PCR amplification separately on each aliquot of each sample and attach the first sequencing adapter to one end of the reporter nucleic acid molecule. As described above, the aliquots of each sample are pooled separately to obtain a separate first pool for each sample.

[0088] Once a separate first pool is obtained, a sample index is added. As described above, this may be achieved by amplification or ligation. Regardless of how the sample index is added, it is added to the end of the reporter nucleic acid molecule opposite to the end having the first sequencing adapter. A ligation step may be performed to add the sample index to the end of each reporter nucleic acid molecule, but preferably, the addition of the sample index is achieved by amplification, which is usually performed by PCR. The sample index is added while amplification is performed using a primer pair in which one primer contains the sample index sequence, so that the sample index is incorporated into the amplified product.

[0089] The addition of the sample index may be performed in a dedicated amplification step carried out solely for the purpose of adding the sample index to the reporter nucleic acid molecule. Subsequently, a further amplification step may be performed as needed to add a second sequencing adapter to the reporter nucleic acid molecule. In this example, the second sequencing adapter is added to the reporter nucleic acid molecule at the same end where the sample index is located. Thus, typically, this results in the amplified and adapter-labeled reporter nucleic acid molecule being located internally to the second sequencing adapter, but immediately adjacent to it.

[0090] However, preferably, as detailed above, after pooling the products of the first PCR amplification to obtain a first pool, a second PCR amplification product is performed on the first pool (i.e., the products of the first PCR amplification) to add both the sample index and the second sequencing adapter to the reporter nucleic acid molecule. Thus, the second PCR amplification is performed separately for each of the first pools. In other words, the second PCR is performed separately for each sample that is analyzed.

[0091] In this embodiment, the second PCR amplification is performed using a primer pair in which one primer contains both the sample index sequence and the second sequencing adapter, such that both the sample index sequence and the second sequencing adapter are simultaneously attached to the reporter nucleic acid molecule. The primer containing the second sequencing adapter and the sample index sequence has the second sequencing adapter at its 5' end. The sample index sequence is located downstream of the second sequencing adapter, usually immediately downstream, but adjacent to the second sequencing adapter is not required. Thus, the product of the second PCR amplification contains two sequencing adapters (one at each end) and the sample index located inside the second sequencing adapter.

[0092] The second PCR may use a common first primer and unique second primers that differ across the multiple samples analyzed. In other words, one primer (the same primer) is used across all samples and binds to the end of the reporter nucleic acid molecule to which the first sequencing adapter was attached in the first PCR amplification. A different second primer is used for each sample, where the second primer contains a sample index sequence unique to each sample.

[0093] After the second PCR amplification, the indexed first pools generated for each sample are pooled together (i.e., added to or mixed with each other) to create a second pool. The second pool is used for DNA sequencing. Therefore, by performing a single DNA sequencing reaction, the reporter nucleic acid molecule generated for each sample can be identified. The sample index attached to the reporter nucleic acid molecule makes it possible to pinpoint the original sample from which each nucleic acid molecule originates, and as a result, it is possible to determine which analytes are present in each sample. Before DNA sequencing, it is preferable to purify the amplification product of the second PCR to remove excess primers and other residues remaining from the amplification reaction. This purification step may be performed regardless of whether one sample or multiple samples are analyzed in this method. When multiple samples are analyzed and the products of the second PCR amplification are pooled before sequencing, the products of the second PCR may be purified before or after pooling. That is, a second PCR may be performed on each of the first pools, the products may be pooled to create a second pool, and then the PCR products of the second pool may be purified together in a single purification reaction. Alternatively, a second PCR may be performed on each of the first pools, the products obtained from each second PCR may be purified separately, and then the purified second PCR amplification products may be pooled.

[0094] As described above, each reporter nucleic acid molecule contains at least one barcode sequence associated with a specific analyte. Therefore, each specific reporter nucleic acid molecule is usually detected by detecting its barcode sequence using sequencing. When analyzing a single sample using the method of the first embodiment of the present invention, all that is needed to detect all reporter nucleic acid molecules generated by the multiple detection assay is to detect their barcodes. The detection of each specific barcode indicates that the corresponding analyte is present in the sample. When analyzing multiple samples in parallel using this method, after amplification, each reporter nucleic acid molecule contains both a barcode sequence and a sample index. In this embodiment, the detection of each reporter nucleic acid molecule includes the detection of both the barcode sequence and the sample index. The detection of the sample index indicates which sample the reporter nucleic acid molecule originates from, and the detection of the barcode indicates the presence of a specific analyte in that sample. Therefore, by detecting the reporter nucleic acid molecules, it is possible to identify the analytes present in each analyzed sample.

[0095] As described above, sequencing for this method is typically performed by a massively parallel DNA sequencing method. For this purpose, the purified product (or an aliquot thereof) of the second PCR amplification is denatured, for example, with sodium hydroxide to obtain a single-stranded DNA molecule. The denatured (single-stranded) DNA may be diluted with a suitable buffer as needed. A suitable dilution buffer is generally provided with the DNA sequencing platform or by the manufacturer of the DNA sequencing platform. The denatured DNA is then placed on a solid support by hybridizing its sequencing adapter to a complementary sequence protruding from the solid support (e.g., beads or flow cell). Once the DNA is placed on the solid support, DNA sequencing is performed using a selected method.

[0096] The method described above makes it possible to detect each analyte in a sample. This method also makes it possible to compare the levels of analytes within each subset for each sample. In other words, it is possible to compare the levels of analytes in each specific sample aliquot that has been analyzed. In each individual aliquot, the level of each different reporter nucleic acid molecule generated is proportional to the level of each analyte (for example, if the first analyte is present at twice the level in a particular aliquot compared to the second aliquot, then twice the amount of reporter nucleic acid molecules corresponding to the first analyte will be generated compared to the reporter nucleic acid molecules corresponding to the second analyte). Such differences in reporter levels can be detected when performing reporter detection, such as sequencing, and this makes it possible to compare the relative amounts of analytes present in the sample, but this is only possible for analytes detected in the same aliquot.

[0097] It is advantageous to be able to compare the relative amounts of all analytes present in a sample (i.e., to compare analytes detected in different aliquots). It is even more advantageous to be able to compare the relative amounts of analytes present in different samples. This can be achieved by including an internal control in each aliquot. Each aliquot of each sample contains the same internal control. The internal control is included in each aliquot of the sample at different concentrations, depending on the dilution ratio of the aliquot. The concentration of the internal control is proportional to the dilution ratio of the aliquot. Therefore, for example, if an internal control is used at a specific given concentration in an aliquot of an undiluted sample, then in an aliquot of a 1:10 diluted sample, the internal control should be used at one-tenth the concentration used in the undiluted sample. This makes it possible to directly compare the relative concentrations of analytes between aliquots, while ensuring that the signal of the internal control does not drown out or be obscured by the signal of the analyte detected in the aliquot. This is because an internal control is present in each aliquot at a concentration appropriate for the analyte detected in each aliquot.

[0098] The internal control is either a control reporter nucleic acid molecule or something that results in the generation of a control reporter nucleic acid molecule. By comparing the amount of each reporter nucleic acid molecule with the control reporter, it is possible to compare the relative amounts of analytes analyzed with different aliquots, and / or the relative amounts of analytes from different samples. This is possible because the relative differences between each reporter nucleic acid molecule and the control reporter are comparable.

[0099] For example, if two different reporter nucleic acid molecules from different samples are present at the same relative level relative to the control reporter (e.g., half or one-third, or twice or three times), this indicates that the analytes represented by the two reporter nucleic acid molecules are present in the two samples at essentially the same concentration. Similarly, if the ratio of a particular reporter nucleic acid molecule to the control reporter is twice the ratio of the same reporter nucleic acid molecule from different samples to the control reporter (e.g., the reporter molecule is present at twice the level of the control reporter in the first sample, and at essentially the same level as the control reporter in the second sample), this indicates that the analyte represented by the particular reporter nucleic acid molecule is present in the first sample at approximately twice the level present in the second sample.

[0100] There are various options that can be used as internal controls. The appropriate control may depend on the detection technique used. In any detection assay, the internal control may be an added analyte, i.e., a control analyte added at a specified concentration to each aliquot being analyzed. The control analyte is added to the aliquots before the multiplex detection assay and is detected in each aliquot, just like other analytes in the sample. In particular, the detection of the control analyte may lead to the generation of a control reporter nucleic acid molecule specific to the control analyte, as described above. When a control analyte is used, it is an analyte that cannot be present in the sample of interest. For example, it may be an artificial analyte, or, if the sample is of animal (e.g., human) origin, the control analyte may be a biomolecule from a different species that is not present in the animal of interest. In particular, the control analyte may be a non-human protein. Examples of control analytes include fluorescent proteins such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), and cyan fluorescent protein (CFP).

[0101] Another example of an internal control is a double-stranded DNA molecule that has the same overall structure as the reporter nucleic acid molecule generated in the multiplex detection assay. That is, the DNA molecule includes a barcode sequence that identifies it as a control reporter nucleic acid molecule, and a common primer-binding site shared by all other reporter nucleic acids generated in response to the detection of the analyte, enabling the binding of primers used in the amplification reaction. In particular, the control DNA molecule does not include a sequencing adapter or a sample index. These sequencing adapters and sample indices are added to the control DNA molecule at the same time (e.g., in PCR amplification) they are added to the reporter nucleic acid molecule generated in response to the detection of the analyte, as described above.

[0102] The double-stranded DNA molecule used as a control in this manner is referred to as the detection control in this specification. This is because it is useful not only for evaluating the analyte concentration (by comparing it with the control concentration, as described above), but also for confirming that the reporter nucleic acid molecule generated during analyte detection is amplified, labeled, and detected (e.g., by sequencing), as described above. If the detection control is not detected when analyzing the reporter nucleic acid molecule (e.g., by sequencing), this indicates that the detection method has failed. For example, the amplification step may have failed, or the sequencing reaction may have failed. The detection control is preferably added to each aliquot before performing the multiplex detection assay.

[0103] In a particular embodiment of this method, both the control analyte and the detection control are added to each aliquot. In this example, the barcode sequence for the control analyte is distinctly different from the barcode sequence for the detection control, and as a result, the two internal controls can be identified individually.

[0104] As described above, the multiplex detection assay is preferably a multiple proximity extension assay or a multiple proximity ligation assay, and most preferably a multiple proximity extension assay. These are briefly explained above. As described above, both of these techniques rely on the use of paired proximity probes.

[0105] In this specification, a proximity probe is defined as an entity comprising an analyte-specific analyte-binding domain and a nucleic acid domain. "Analyte-specific" means that the analyte-binding domain specifically recognizes and binds to a particular target analyte, i.e., it binds to the target analyte with higher affinity than it would to other analytes or other parts. The analyte-binding domain is preferably an antibody, particularly a monoclonal antibody. Antibody fragments or derivatives of antibodies containing an antigen-binding domain are also suitable for use as analyte-binding domains. Examples of such antibody fragments or derivatives include molecules such as Fab, Fab', F(ab')2, and scFv.

[0106] A Fab fragment consists of the antigen-binding domain of an antibody. An individual antibody may be considered to contain two Fab fragments, each of which consists of a light chain and the N-terminal portion of the heavy chain to which it is bound. Therefore, a Fab fragment consists of a complete light chain and the N-terminal portion of the heavy chain to which it is bound. H Domain and C H It contains one domain. The Fab fragment may be obtained by digesting the antibody with papain.

[0107] The F(ab')2 fragment consists of two Fab fragments of the antibody and a hinge region of the heavy domain, containing a disulfide bond that links the two heavy chains. In other words, the F(ab')2 fragment can be considered as two Fab fragments linked by a covalent bond. The F(ab')2 fragment may also be obtained by digesting the antibody with pepsin. Reducing the F(ab')2 fragment yields two Fab' fragments, which can be considered Fab fragments containing additional sulfhydryl groups that may be useful for binding the fragments to other molecules. The ScFv molecule is a synthetic construct produced by fusing the variable domains of the light and heavy chains of an antibody. Typically, this fusion is achieved recombinantly by designing the antibody gene to produce a fusion protein containing both the heavy and light chain variable domains.

[0108] The nucleic acid domain of the proximity probe may be a DNA domain or an RNA domain. Preferably, it is a DNA domain. The nucleic acid domains of the proximity probes in each proximity probe pair are typically designed to hybridize with each other or to hybridize with one or more common oligonucleotide molecules (both nucleic acid domains of a pair of proximity probes may hybridize). Therefore, the nucleic acid domains must be at least partially single-stranded. In one embodiment, the nucleic acid domain of the proximity probe is entirely single-stranded. In other embodiments, the nucleic acid domain of the proximity probe is partially single-stranded and includes both single-stranded and double-stranded portions.

[0109] The proximity probes are typically provided as proximity probe pairs, each specific to the target analyte. As described above, the target analyte may be a single entity, and in particular, a single protein. In this embodiment, both probes of the proximity probe pair bind to the target analyte (e.g., a protein), but they have different epitopes. The epitopes do not overlap, and as a result, binding of one probe of the proximity probe pair to an epitope does not interfere with or inhibit the binding of the other probe of the proximity probe pair to the epitope. Alternatively, as described above, the target analyte may be a complex, such as a protein complex, in which case one probe of the proximity probe pair binds to one element of the complex, and the other probe of the proximity probe pair binds to the other element of the complex. The probes bind to proteins in the complex at a site different from the protein interaction site (i.e., the site of the proteins interacting with each other).

[0110] As described above, the proximity probes are provided as proximity probe pairs, each specific to the target analyte. This means that in each proximity probe pair, both probes contain analyte-binding domains specific to the same analyte. Since the detection assays used are multiplex assays, multiple different probe pairs are used in each detection assay, and each probe pair is specific to a different analyte. That is, the analyte-binding domains of each different probe pair are specific to different target analytes.

[0111] The nucleic acid domains of each proximity probe are designed according to the method in which the probes are used. A typical example of a proximity extension assay is schematically shown in Figure 1, and these embodiments are described in detail below. Typically, in a proximity extension assay, when a pair of proximity probes bind to the target analyte, the nucleic acid domains of the two probes come into close proximity and interact with each other (i.e., hybridize directly or indirectly). The interaction between the two nucleic acid domains results in a nucleic acid duplex containing at least one free 3' end (i.e., at least one of the nucleic acid domains in the duplex has an extendable 3' end). Addition of a nucleic acid polymerase enzyme to the assay mix, or activation of a nucleic acid polymerase enzyme in the assay mix, causes the at least one free 3' end to extend. Thus, at least one of the nucleic acid domains in the duplex extends using its paired nucleic acid domain as a template. The resulting extension product is the reporter nucleic acid molecule used herein and contains a barcode sequence indicating the presence of the analyte to which the proximity probe pair that produced the extension product is bound.

[0112] Version 1 of Figure 1 shows a "conventional" proximity extension assay, where the nucleic acid domain of each proximity probe (indicated by arrows) is attached at its 5' end to an analyte-binding domain (indicated by an inverted "Y"), leaving two free 3' ends. When the proximity probes bind to their respective analytes (analytes not shown in the figure), the nucleic acid domains of the probes, which are complementary at their 3' ends, can hybridize and interact, i.e., form a double helix. By adding a nucleic acid polymerase enzyme to the assay mixture, or by activating a nucleic acid polymerase enzyme within the assay mixture, each nucleic acid domain can be extended using the nucleic acid domain of the other proximity probe as a template. As detailed above, the resulting extension product is a reporter nucleic acid molecule that, when detected, detects the analyte to which the probe pair is bound.

[0113] Version 2 of Figure 1 shows a different proximity extension assay, where the nucleic acid domain of the first proximity probe is attached to the analyte-binding domain at its 5' end, and the nucleic acid domain of the second proximity probe is attached to the analyte-binding domain at its 3' end. This results in the nucleic acid domain of the second proximity probe having a free 5' end (indicated by a blunt arrow), which cannot be extended using a typical nucleic acid polymerase enzyme (which only extends the 3' end). The 3' end of the second proximity probe is effectively "blocked"; that is, this 3' end is bound to the analyte-binding domain and is thus blocked, and therefore cannot be "free" and extended. In this embodiment, when the proximity probes bind to their respective analyte-binding targets on the analyte, the nucleic acid domains of the probes, which share complementary regions at their 3' ends, can hybridize and interact, i.e., form a double helix. However, in contrast to version 1, only the nucleic acid domain of the first neighboring probe (with a free 3' end) can be extended using the nucleic acid domain of the second neighboring probe as a template to obtain the extension product (i.e., the reporter nucleic acid molecule).

[0114] In version 3 of Figure 1, as in version 2, the nucleic acid domain of the first proximity probe is attached to the analyte-binding domain at its 5' end, and the nucleic acid domain of the second proximity probe is attached to the analyte-binding domain at its 3' end. As a result, the nucleic acid domain of the second proximity probe has a free 5' end (indicated by a blunt arrow), which cannot be extended. However, in this embodiment, the nucleic acid domains attached to the analyte-binding domain of each proximity probe do not have complementary regions and therefore cannot directly form a double helix. Instead, a third nucleic acid molecule is provided that has a region homologous to the nucleic acid domain of each proximity probe. This third nucleic acid molecule acts as a "molecular bridge" or "splint" between the nucleic acid domains. This "splint" oligonucleotide bridges the gaps between nucleic acid domains, causing them to interact indirectly. That is, each nucleic acid domain forms a double helix with the splint oligonucleotide.

[0115] Therefore, when the proximity probe binds to each analyte-binding target on the analyte, each nucleic acid domain of the probe interacts with the sprint oligonucleotide by hybridizing, i.e., forming a double helix with the sprint oligonucleotide. Thus, the third nucleic acid molecule or sprint can be considered as a second strand of a partially double-stranded nucleic acid domain provided on one side of the proximity probe. For example, one side of the proximity probe may have a partially double-stranded nucleic acid domain, which is attached to the analyte-binding domain via the 3' end of one strand, and the other (unattached) strand has a free 3' end. Thus, such a nucleic acid domain has a terminal single-stranded region including a free 3' end. In this embodiment, the nucleic acid domain of the first proximity probe (having a free 3' end) may be extended using the "sprint oligonucleotide" (or the single-stranded 3' terminal region of the other nucleic acid domain) as a template. Alternatively, the free 3' end of a sprint oligonucleotide (i.e., an unattached chain or a 3' single-stranded region) may be extended using the nucleic acid domain of the first nearby probe as a template.

[0116] As is clear from the above description, in one embodiment, the sprint oligonucleotide may be provided as a separate element of the assay. In other words, the sprint oligonucleotide may be added separately to the reaction mix (i.e., added separately to the sample containing the analyte, apart from the proximity probe). Even in this case, it can still be considered a chain of nucleic acid domains that is partially double-stranded, since it hybridizes to a nucleic acid molecule that is part of the proximity probe, and hybridizes upon contact with such a nucleic acid molecule. Alternatively, the sprint may be pre-hybridized to one of the nucleic acid domains of the proximity probe; that is, it may be hybridized before the proximity probe is brought into contact with the sample. In this embodiment, the sprint oligonucleotide can be directly considered as part of the nucleic acid domain of the proximity probe. In other words, the nucleic acid domain is a partially double-stranded nucleic acid molecule. For example, a nearby probe may be created by linking a double-stranded nucleic acid molecule to an analyte-binding domain (preferably, the nucleic acid domain is single-stranded and bound to the analyte-binding domain), and then modifying the nucleic acid molecule to generate a partially double-stranded nucleic acid domain (having a single-stranded protrusion that can hybridize to the nucleic acid domain of another nearby probe).

[0117] Therefore, the extension of the nucleic acid domain of the proximity probe as defined herein also includes the extension of the "sprint" oligonucleotide. Conveniently, when the extension product is produced by the extension of the sprint oligonucleotide, the resulting extended nucleic acid chain is linked to the proximity probe pair only by interaction between the two strands of the nucleic acid molecule (by hybridization of the two nucleic acid chains). Therefore, in these embodiments, the extension product can be dissociated from the proximity probe pair using denaturing conditions such as, for example, an increase in temperature or a decrease in salt concentration.

[0118] The sprint oligonucleotide depicted in version 3 of Figure 1 is shown to be complementary to the entire length of the nucleic acid domain of the second neighboring probe, but this is merely illustrative. The sprint only needs to be able to form a double helix with the terminal (or near-terminal) portion of the nucleic acid domain of the neighboring probe, that is, it only needs to form a bridge between the nucleic acid domains of the two probes.

[0119] In another embodiment, the sprint oligonucleotide may be provided as the nucleic acid domain of a third proximity probe, as described in WO2007 / 107743, incorporated herein by reference, which has been demonstrated to further improve the sensitivity and specificity of the proximity probe assay.

[0120] Version 4 of Figure 1 is a variation of version 1, in which the nucleic acid domain of the first neighboring probe contains a sequence at its 3' end that is not entirely complementary to the nucleic acid domain of the second neighboring probe. Therefore, when the neighboring probes bind to their respective analytes, the nucleic acid domains of the probes can hybridize and interact, i.e., form a double helix. However, the outermost 3' end of the nucleic acid domain of the first neighboring probe (the part of the nucleic acid molecule containing the free 3' hydroxyl group) cannot hybridize to the nucleic acid domain of the second neighboring probe and therefore exists as a single-stranded, unhybridized "flap." Addition or activation of nucleic acid polymerase enzymes can cause only the nucleic acid domain of the second neighboring probe to be extended using the nucleic acid domain of the first neighboring probe as a template.

[0121] Version 5 of Figure 1 may be considered a variation of version 3. However, in contrast to version 3, the nucleic acid domains of both proximity probes are attached to their respective analyte-binding domains at their 5' ends. In this embodiment, the 3' ends of the nucleic acid domains are not complementary, and therefore the nucleic acid domains of the proximity probes cannot interact or directly form double helixes. Instead, a third nucleic acid molecule is provided having a region homologous to the nucleic acid domain of each proximity probe. This third nucleic acid molecule acts as a "molecular bridge" or "sprint" between the nucleic acid domains. This "sprint" oligonucleotide bridges the gap between the nucleic acid domains, causing them to interact indirectly. That is, each nucleic acid domain forms a double helix with the sprint oligonucleotide. Thus, when the proximity probes bind to each analyte, each of the nucleic acid domains of the probes hybridizes and interacts with the sprint oligonucleotide, i.e., forms a double helix with the sprint oligonucleotide.

[0122] According to version 3, the third nucleic acid molecule or sprint can be considered as a second strand of a partially double-stranded nucleic acid domain provided on one side of the proximity probe. In a preferred example, one side of the proximity probe may have a partially double-stranded nucleic acid domain, which is attached to an analyte-binding domain via the 5' end of one strand, and the other (unattached) strand has a free 3' end. Thus, such a nucleic acid domain has a terminal single-stranded region containing at least one free 3' end. In this embodiment, the nucleic acid domain of the second proximity probe (having a free 3' end) may be extended using the “sprint oligonucleotide” as a template. Alternatively, the free 3' end of the sprint oligonucleotide (i.e., the unattached strand, or the 3' single-stranded region of the first proximity probe) may be extended using the nucleic acid domain of the second proximity probe as a template.

[0123] As described above in relation to version 3, the sprint oligonucleotide may be provided as a separate element of the assay. Alternatively, since it hybridizes to a nucleic acid molecule that is part of the proximity probe, and hybridizes upon contact with such a nucleic acid molecule, it can still be considered a chain of a partially double-stranded nucleic acid domain even if added separately. Or, the sprint may be pre-hybridized to one of the nucleic acid domains of the proximity probe; that is, it may be hybridized before the proximity probe is brought into contact with the sample. In this embodiment, the sprint oligonucleotide can be directly considered as part of the nucleic acid domain of the proximity probe. That is, the nucleic acid domain is a partially double-stranded nucleic acid molecule, and for example, the proximity probe may be created by linking a double-stranded nucleic acid molecule to an analyte-binding domain (preferably the nucleic acid domain is single-stranded and bound to the analyte-binding domain), and modifying the nucleic acid molecule to produce a partially double-stranded nucleic acid domain (having a single-stranded protrusion that can hybridize to the nucleic acid domain of another proximity probe).

[0124] Therefore, the extension of the nucleic acid domain of the proximity probe as defined herein also includes the extension of the "sprint" oligonucleotide. Conveniently, when the extension product is produced by the extension of the sprint oligonucleotide, the resulting extended nucleic acid chain is linked to the proximity probe pair only by interaction between the two strands of the nucleic acid molecule (by hybridization of the two nucleic acid chains). Therefore, in these embodiments, the extension product can be dissociated from the proximity probe pair using denaturing conditions such as, for example, an increase in temperature or a decrease in salt concentration.

[0125] The sprint oligonucleotide depicted in version 5 of Figure 1 is shown to be complementary to the entire length of the nucleic acid domain of the first neighboring probe, but this is merely illustrative. The sprint only needs to be able to form a double helix with the terminal (or near-terminal) portion of the nucleic acid domain of the neighboring probe, that is, it only needs to form a crosslink between the nucleic acid domains of the neighboring probe.

[0126] In another embodiment, the sprint oligonucleotide may be provided as the nucleic acid domain of a third proximity probe, as described in WO2007 / 107743, incorporated herein by reference, which has been demonstrated to further improve the sensitivity and specificity of the proximity probe assay.

[0127] Version 6 of Figure 1 represents the most preferred embodiment of the present invention. As shown, both probes of the probe pair are bound to a partially single-stranded nucleic acid molecule. The short nucleic acid strands are bound to the analyte-binding domain via their 5' ends. The short nucleic acid strands bound to the analyte-binding domain are not hybridized to one another. Rather, each of the short nucleic acid chains hybridizes to a longer nucleic acid chain, which has a single-stranded protrusion at its 3' end (i.e., the 3' end of the long nucleic acid chain extends beyond the 5' end of the short chain bound to the analyte-binding domain). The protrusions of the two long nucleic acid chains hybridize to each other to form a double helix. If the 3' ends of the two long nucleic acid molecules are completely hybridized to each other, the double helix contains two free 3' ends, as shown in the figure. However, the 3' ends of the long nucleic acid molecules may be designed as in version 4, such that the outermost end of one 3' end of the long nucleic acid molecule is not complementary to the other, forming a flap, i.e., the double helix contains only one free 3' end. Two interacting long nucleic acid molecules can be considered sprint oligonucleotides in that they together form a bridge between two short oligonucleotides directly bound to the analyte-binding domain.

[0128] The addition or activation of nucleic acid polymerase elongates the free 3' ends of one or both sprint oligonucleotides. In particular, one sprint oligonucleotide elongates using the other sprint oligonucleotide as a template. Therefore, when one sprint oligonucleotide is elongated, the other "template" sprint oligonucleotide moves away from the short chain bound to the analyte-binding domain.

[0129] In a preferred embodiment, the short nucleic acid chain directly bound to the analyte-binding domain is a “common chain.” That is, the same chain is directly bound to all proximity probes used in a multiplex detection assay. Therefore, each sprint oligonucleotide includes a “common site” consisting of a sequence that hybridizes to the common chain, and a “proprietary site” containing a barcode sequence specific to the probe. Such proximity probes and methods for creating them are described in WO2017 / 068116.

[0130] In all proximity detection assay techniques, it is preferable that each nucleic acid domain of an individual proximity probe contains a unique barcode sequence that identifies a specific probe (as described above for PEA version 6). In this case, the reporter nucleic acid molecule (or extension product in a proximity extension assay) contains the unique barcode sequence of each proximity probe. Thus, these two unique barcode sequences together form the barcode sequence of the reporter nucleic acid molecule. In other words, the barcode sequence of the reporter nucleic acid molecule is a combination of or contains the two probe barcode sequences, and the reporter nucleic acid molecule is generated by combining the barcode sequences of the proximity probes. Thus, a specific reporter nucleic acid molecule is detected by detecting a specific combination of the two probe barcode sequences.

[0131] When using a multiple proximity extension assay to detect an analyte, it is preferable to use an extension control, which is an additional internal control. The extension control is a single probe containing an analyte-binding domain bound to a nucleic acid domain that includes a double helix having an extendable free 3' end. Preferably, the extension control has a structure that is essentially equivalent to the double helix formed between two experimental probes when bound to the target analyte, except that it contains only one analyte-binding domain. The analyte-binding domain used in the extension control does not recognize any analytes that may be present in the sample of interest. Suitable analyte-binding domains are commercially available polyclonal isotype control antibodies, such as goat IgG, mouse IgG, or rabbit IgG.

[0132] Figure 2 shows examples of extension controls that can be used in the present invention. Parts A through F correspond to extension controls that can be used in versions 1 through 6 of the PEA assay in Figure 1, respectively. The extension controls are used to ensure that the extension step is performed as intended. By extending the extension control, a reporter nucleic acid molecule is obtained that contains a unique barcode so that it can be identified as the extension control reporter nucleic acid molecule. When multiple PEA is used in the method of the first embodiment of the present invention, it is preferable that all of the control analyte, extension control, and detection control are used in the assay (i.e., added to each aliquot). In other embodiments, only two internal controls are used, for example, the control analyte and the extension control, the control analyte and the detection control, or the extension control and the detection control.

[0133] As detailed above, in a proximity extension assay, a reporter nucleic acid molecule is generated by the extension of the nucleic acid domains of one or both proximity probes using the nucleic acid domain of the other proximity probe as a template. In a preferred embodiment, the extension reaction is carried out in PCR amplification. In other words, a single reaction, including PCR amplification, performs both the extension of the nucleic acid domains of the proximity probes that generate the reporter nucleic acid molecule and the amplification of the reporter molecule, including the addition of a first sequence adapter to the generated reporter nucleic acid molecule. In this embodiment, the reaction is started from the extension step in which the reporter nucleic acid molecule is generated, rather than starting from the denaturation step (which is common in PCR). Subsequently, the reporter nucleic acid molecule is amplified by performing a standard PCR that starts with the denaturation of the reporter molecule. As detailed above, the PCR is performed using common primers that bind to a common sequence at the ends of the reporter nucleic acid molecule, one of which contains a sequencing adapter. Alternatively, the PCR may be performed using primers, each containing a sequencing adapter, in order to add a sequencing adapter to each end of the reporter nucleic acid molecule in a single run, as detailed above.

[0134] In some cases, it is desirable to detect more analytes in a sample than are available in the different reporter nucleic acid barcode sequences. In this case, multiple panels of proximity probes (i.e., at least two panels) may be used. Each panel contains a different set of proximity probe pairs; that is, the proximity probe pairs in each panel bind to a different set of analytes. Typically, the proximity probe pairs in each panel bind to completely different sets of analytes; that is, the analytes to which the proximity probe pairs of different panels bind do not overlap. Thus, each panel of proximity probes is intended for detecting different groups of analytes.

[0135] As described above, each panel of proximity probes contains a different set of proximity probe pairs. In each individual panel, each probe contains a different nucleic acid domain (i.e., each probe contains a nucleic acid domain with a different sequence). Therefore, each probe pair contains a different nucleic acid domain pair, and thus, a unique reporter nucleic acid molecule is generated for each probe pair in the panel. However, in each different panel, the same nucleic acid domain (and usually the same nucleic acid domain pairing) is used for the probe pair. That is, in different panels, the probe pair contains the same nucleic acid domain pair. This means that the same reporter nucleic acid molecule is generated in each panel. However, since the reporter nucleic acid molecule is generated by each panel using different probe pairs, the same reporter nucleic acid molecule indicates that different analytes are present in each panel of probes. Since each panel of the probe generates the same reporter nucleic acid molecule, a multiplex detection assay using each panel of the probe requires the preparation of separate sample aliquots. That is, a multiplex detection assay is performed using each panel of the probe, while a multiplex detection assay using different probe panels is performed using different aliquots of the sample. As detailed above for a single probe panel, for each panel of the probe, multiple sample aliquots are prepared at different dilutions, and different subsets of the analytes from each panel are detected in each aliquot. As detailed above, the subset of the analytes detected in each aliquot is determined based on the predicted concentration in the sample.

[0136] Reporter nucleic acid molecules generated using each separate probe panel are processed (i.e., amplified and possibly labeled with a sequencing adapter) and detected as detailed above. In certain embodiments, as detailed above, the reporter nucleic acid molecules are amplified by PCR, sequencing adapters are attached to both ends of the reporter nucleic acid molecules, and a sample index is attached to each reporter nucleic acid molecule. In this embodiment, it is preferable to perform a first PCR separately on each aliquot to amplified the reporter nucleic acid molecule and to attach a first sequencing adapter to one end of the reporter nucleic acid molecule, as described above. Subsequently, as described above, the amplified reporter nucleic acid molecules from each sample generated using the specific probe panel are pooled to generate a plurality of separate first pools. Each of the separate first pools contains the product of the first PCR amplification performed on all aliquots of the specific sample analyzed using the specific probe panel.

[0137] Subsequently, a second PCR amplification is performed in each of the separate first pools, and a second sequencing adapter and sample index are added to each reporter nucleic acid molecule. After the second PCR, the PCR products themselves, generated from different samples using the same probe panel, are pooled into a second pool known as the panel pool. The entirety of each first pool may be combined to form the panel pool, or only a portion of each first pool may be combined. Thus, each panel pool contains reporter nucleic acid molecules generated from all the analyzed samples using a specific probe panel.

[0138] Subsequently, the amplified reporter nucleic acid molecule, including the sequencing adapter and sample index, is sequenced as described above. Each panel pool is sequenced separately. This is because, as described above, although the same reporter nucleic acid molecule is generated in each probe panel, these reporter nucleic acid molecules represent different analytes in each probe panel. It is impossible to distinguish at the sequence level the same reporter nucleic acid molecule generated using different probe panels and thus representing different analytes. Therefore, in this embodiment, it is essential to sequence each panel pool separately.

[0139] In another embodiment of this method, a panel index sequence is added to the reporter nucleic acid molecule during one PCR amplification. The same panel index sequence is used to identify all reporter nucleic acid molecules (across all samples) generated using a specific proximity probe panel. Combining the panel index and sample index makes it possible to accurately identify which analytes are present in each sample across all probe panels used in the detection assay. Therefore, when both the panel index and sample index are added to each reporter nucleic acid molecule, all PCR products generated in the detection assay across all samples and probe panels can be pooled and sequenced together.

[0140] Alternatively, the reporter nucleic acid molecules generated using each probe panel may be labeled with different sample indices. Different sample index sequences are selected and used for each different sample so that each sample index used is unique to a particular sample. However, in any given sample, the reporter nucleic acid molecules generated using each different probe panel are labeled with different sample indices. Therefore, in this embodiment, the sample index serves two functions for each reporter nucleic acid molecule: to identify both the sample and the probe panel. Thus, a specific sample index present in a reporter nucleic acid molecule associates that reporter with a specific probe panel, and the combination of the sample index and barcode sequence of the reporter nucleic acid molecule functions to identify the analyte that led to the generation of the reporter nucleic acid molecule.

[0141] In a further embodiment of this method, as detailed above, the same nucleic acid domain is used as the probe in each probe panel. However, each panel contains different pairs of nucleic acid domains so that each panel generates a different reporter nucleic acid molecule. As described above, each probe's nucleic acid domain contains a unique barcode sequence. By pairing the nucleic acid domains differently in each panel, different combinations of barcode sequences are paired in the reporter nucleic acid molecules generated in the detection assay. This means that a different reporter nucleic acid molecule is generated for each panel. This method has the advantage that, since each probe panel generates a different reporter nucleic acid molecule, they can be distinguished at the sequence level without the need for a panel index sequence at all. In this embodiment, as detailed above, all PCR products from each sample are pooled and sample indices are added, and then all indexed PCR products from all samples and probe panels are combined into a single pool and sequenced.

[0142] As described above, the advantage of this embodiment is that all reporter nucleic acid molecules from all samples and panels can be pooled and sequenced together without requiring a panel index to identify which reporter nucleic acid molecule originates from each panel. However, the advantage of using probe pairs with the same nucleic acid domain pair for each panel so that each panel generates the same reporter nucleic acid molecule is that any nucleic acid molecule resulting from the hybridization of two non-paired nucleic acid molecules can be identified as nonspecific background. If each probe panel generates a different reporter nucleic acid molecule, it is no longer possible to accurately determine which of the generated nucleic acid molecules is background.

[0143] As described above, in a second embodiment, the present invention provides a method for detecting an analyte in a sample, wherein the analyte is detected by detecting a reporter nucleic acid molecule specific to the analyte, the method comprising: performing a PCR reaction to produce a PCR product of the reporter nucleic acid molecule; and detecting the PCR product. An internal control is prepared for the PCR reaction, and this internal control is (i) A control nucleic acid molecule present in a predetermined amount and amplified by the same primer as the reporter nucleic acid molecule, or a separate component that contains such a nucleic acid molecule or causes such a nucleic acid molecule to be generated, and / or (ii) A unique molecular identifier (UMI) sequence that is present in each reporter nucleic acid molecule.

[0144] All details of this second aspect of the present invention may be the same as those of the first aspect (e.g., analytes, samples, reporter nucleic acid molecules and the techniques used to produce them, detection of reporter nucleic acid molecules, etc.).

[0145] In this second embodiment, the internal control is a component or sequence present in the PCR performed to generate the PCR product of the reporter nucleic acid molecule. As described above, the internal control may be a control nucleic acid molecule present in a predetermined amount and amplified by the same primer as the reporter nucleic acid molecule, or it may be a separate component that contains such a nucleic acid molecule or causes such a nucleic acid molecule to be generated.

[0146] If the internal control is a separate component present in a predetermined amount during the reaction, the internal control may, as described above, be a control analyte, an extension control, or a detection control. As detailed above, the control analyte is an analyte that is added to the sample and detected by detecting a control reporter nucleic acid molecule specific to the control analyte.

[0147] In the method of the second aspect of the present invention, the analyte is preferably detected using a proximity probe, for example, PEA or PLA as detailed above, most preferably PEA. Therefore, when a control analyte is used as an internal control, a proximity probe for detecting the control analyte must be included. A control reporter nucleic acid molecule is generated when a control-specific proximity probe binds to the control analyte.

[0148] As described above, an extension control may be used. As detailed above, the extension control is a single control probe that generates a control reporter nucleic acid molecule during the extension phase of PEA.

[0149] Generally speaking, an internal control can be a single molecule or multiple molecules, and it is added to the sample to generate a control reporter nucleic acid molecule that is later amplified in a PCR reaction.

[0150] Furthermore, as described above, a detection control may be used. As detailed above, the detection control is a control reporter nucleic acid molecule that is added to the sample and amplified by the PCR reaction. The detection control is a double-stranded DNA molecule having the same overall structure as the reporter nucleic acid molecule that is produced in response to the presence of the analyte. As with the first aspect of the present invention, it is preferable that the control analyte, extension control, and detection control are all used in this method. In certain embodiments, two types of internal controls may be used, and the options for such controls are described above.

[0151] As detailed above, the control analyte, extension control, and detection control all generate or are the control reporter nucleic acid molecule. In certain embodiments of the present invention, the control reporter nucleic acid molecule has a sequence that is the reverse sequence of the reporter nucleic acid molecule generated in response to the detection of the analyte. In particular, the control reporter nucleic acid molecule has the reverse sequence of the reporter nucleic acid molecule generated in response to the detection of the analyte, but does not have a reverse complementary sequence. Since the control reporter nucleic acid molecule simply has the reverse sequence of the reporter nucleic acid molecule generated in response to the detection of the analyte, the control reporter nucleic acid molecule cannot hybridize to the reporter nucleic acid molecule in question. This makes it possible to maintain the maximum level of similarity between the control reporter nucleic acid molecule and the reverse-sequenced reporter nucleic acid molecule generated in response to the detection of the analyte, while preventing unwanted hybridization interactions between the control reporter nucleic acid molecule and the reporter nucleic acid molecule generated in response to the detection of the analyte, which is an advantage in PCR amplification. A control reporter nucleic acid molecule having a sequence that is the reverse sequence of the reporter nucleic acid molecule generated in response to the detection of the analyte is preferably also used in the method of the first embodiment of the present invention.

[0152] As described above, the method of this aspect of the present invention preferably uses a control analyte, an extension control, and a detection control as internal controls. It is clear that for these three controls to function together, the control reporter nucleic acid molecules generated / provided by the controls must be distinguishable from each other, i.e., they must all have different sequences. It is preferable that the control reporter nucleic acid molecules used in the method of the present invention each have the reverse sequence of a reporter nucleic acid molecule generated in response to the detection of the analyte. In this case, obviously, each control reporter nucleic acid molecule has the reverse sequence of a different reporter nucleic acid molecule generated in response to the detection of the analyte.

[0153] Alternatively, the internal control may not be a separate component of the amplification reaction, but rather a unique molecular identifier (UMI) sequence present in each reporter nucleic acid molecule, which is unique to each molecule. This means that each individual reporter nucleic acid molecule produced during analyte detection contains a UMI sequence. More specifically, it will be understood that each individual reporter nucleic acid molecule has a different UMI. The UMI is appended to any sequence present in the reporter nucleic acid molecule, such as a barcode, as a means of detecting or identifying the analyte. As detailed above, the analyte is preferably detected by a proximity extension assay according to the method of the second aspect of the present invention. The PEA, including the probes that can be used therefor, is described above. As detailed above, the analyte is detected using a proximity probe pair, each of which binds to the analyte. Both probes in the probe pair contain nucleic acid domains containing a barcode sequence specific to the analyte recognized by the probe.

[0154] Typically, when performing PEA, multiple identical probe pairs are applied to the sample for each analyte to be detected. "Identical" probe pairs mean that all of the probe pairs contain the same analyte-binding molecule pair and the same nucleic acid domain pair, so that each identical probe pair that binds to the target analyte generates the same reporter nucleic acid molecule that indicates the presence of that analyte in the sample.

[0155] When using the UMI sequence as an internal control, the probes used to detect each specific analyte are not identical. While specific analyte-binding molecule pairs are used, each individual probe—that is, each probe containing at least one specific analyte-binding molecule from the pair—contains a different, unique nucleic acid domain. Each nucleic acid domain is unique because it contains the UMI sequence. This means that each specific probe pair that binds to a particular analyte molecule generates a unique reporter nucleic acid molecule. A unique reporter nucleic acid molecule is generated for each individual analyte molecule to which a neighboring probe pair binds. This allows for the absolute quantification of the amount of analyte present in the sample because the exact number of detected analyte molecules can be counted based on the number of unique reporter nucleic acid molecules generated for a particular analyte.

[0156] UMI can be advantageous not only because it enables quantification, but also because it improves the resolution of measurements by allowing the number of reporter nucleic acid molecules generated in a detection assay to be calculated backward. UMI makes it possible to determine how many times a reporter nucleic acid molecule (e.g., the extension product of PEA) has been amplified. Therefore, it is possible to detect differences in UMI levels for reporter molecules for the same analyte. For example, each individual reporter nucleic acid molecule for the same analyte may have the same barcode sequence but different UMIs. By detecting differences in different UMI levels, any bias that may occur in a PCR reaction can be detected and explained.

[0157] Improved resolution may also be useful or beneficial in control nucleic acid molecules. Therefore, UMI may be included in control nucleic acid molecules as an alternative or addition. Thus, UMI may be included in each of the individual control reporter nucleic acid molecules, such as the detection control molecules described above (it will be understood that each individual control nucleic acid molecule will have a different UMI). Alternatively, UMI may be appropriately included for each different IC control mode, such as extension control or control analyte, so that UMI is included in the resulting control reporter nucleic acid molecule. For example, UMI may be included in the nucleic acid sequence of the nucleic acid domain of the extension control that acts as a template for the extension reaction, or in the sequence of a part of the domain that acts as a primer for the extension reaction. Similarly, in the case of a control analyte, UMI may be included in one or both of the nucleic acid domains of the proximity probe used to detect the control analyte so that it is incorporated into the control reporter nucleic acid.

[0158] If UMIs are included in the control nucleic acids, they can be used to improve the resolution of normalization. For example, UMIs can account for all PCR bias, as mentioned above. This can sometimes allow for the use of very precise values ​​in normalization. Therefore, UMIs may be used as a tool to improve or guarantee data quality.

[0159] In one exemplary embodiment, the control reporter nucleic acid molecule includes a sequence which is the reverse sequence of the reporter nucleic acid molecule generated in response to the detection of the analyte, and a UMI.

[0160] The UMI array may be used in a proximity probe used in the method of the first aspect of the present invention.

[0161] The method of a second aspect of the present invention may be applied to the detection of multiple analytes in the same sample (and is actually preferred). As detailed above, multiple analytes may be detected in multiple detection assays. Each of the different analytes is detected based on the detection of a reporter nucleic acid molecule specific to the analyte. As detailed above, each reporter nucleic acid for each different analyte has a unique barcode sequence that confers specificity to the analyte, but it is preferable that all reporter nucleic acids contain a common primer binding site so that all reporter nucleic acid molecules can be amplified in a single PCR using the same primer. PCR amplification of the reporter nucleic acid molecule may include adding at least one (i.e., one or two) sequencing adapters to the end of the reporter nucleic acid molecule, as detailed above.

[0162] As detailed above, when detecting multiple analytes in the same sample, different subsets of the analytes may be detected in different aliquots of the sample, as detailed above, based on the predicted abundance of the analytes in the sample. In this embodiment, PCR is performed separately on each aliquot. As detailed above, the PCR products may then be pooled.

[0163] The method of the second aspect of the present invention may be used to detect one analyte in a plurality of samples, or to detect multiple analytes. In this embodiment, PCR is performed separately to amplify the reporter nucleic acid molecule generated from each sample. When detecting different analyte subsets in separate aliquots of each sample, PCR is performed separately for each separate aliquot of each sample. Using the same primers, the reporter nucleic acid molecules generated for all analytes in all samples are amplified.

[0164] When multiple PCR amplifications are performed separately on multiple different samples and / or multiple different sample aliquots, if the internal control is a separate component present in the PCR mix, then that component is present in each aliquot at a concentration proportional to the dilution ratio of the aliquot, as described above. The concentration of the internal control differs between aliquots with different dilution ratios, while the concentration of the internal control is the same for aliquots from different samples with the same dilution ratio (as is the case in the first embodiment of the present invention). This makes it possible to compare the relative amounts of each analyte present in each sample / aliquot, as detailed above.

[0165] In the method of the second aspect of the present invention, it is preferable that the PCR reaction be carried out to saturation. The saturation state of the PCR reaction is described above. This is particularly advantageous when using this method to detect multiple analytes with different levels of abundance in one or more samples, while performing detection assays on multiple aliquots of each sample and detecting a subset of the analyte in each aliquot. Combining carrying out PCR to saturation with using a separate component of the PCR mix as an internal control is a particularly preferred embodiment of the present invention. As detailed above, carrying out PCR to saturation eliminates differences in the concentration of reporter nucleic acid molecules between different sample aliquots. Once saturation is reached, the total concentration of reporter nucleic acid molecules present in each reaction becomes essentially the same. Including an internal control during the reaction ensures that the relative levels of analytes detected in different aliquots or different samples can be reliably compared.

[0166] As described above, in the second embodiment of the present invention, it is particularly preferable to detect one or more analytes using analyte-specific probes. When such probes are used to detect analytes, the internal control (if it is a separate component of the PCR mixture) is usually added to the sample before or at the same time as the probe is added to the sample. Alternatively, as described above, the internal control may constitute a UMI sequence present in each probe. Preferably, one or more analytes are detected by proximity assay (e.g., PEA or PLA, particularly PEA) that generates a reporter nucleic acid molecule specific to each analyte. In this embodiment, it is preferable that at least an extension control is included. As described above, it is most preferable that all of the control analytes, extension controls, and detection controls are included.

[0167] In a preferred embodiment of a second aspect of the present invention, the method is for detecting multiple analytes in a sample that are present at different levels in the sample, the method is (i) Prepare multiple aliquots from the sample, (ii) A separate multiplex assay is performed on each aliquot to detect a subset of the analyte in each aliquot, wherein the analyte of each subset is selected based on the predicted abundance in the sample. Each aliquot includes at least one internal control.

[0168] All parts of this embodiment may be as defined above with respect to a first aspect of the present invention. The internal control may be any internal control as defined above.

[0169] As described above, when detecting an analyte subset in multiple aliquots with different dilution ratios relative to the original sample, different amounts of internal control are added. The amount of internal control added to each aliquot is determined by the predicted abundance of the analyte subset to be detected in that aliquot. As detailed above, this means that, in practice, the amount of internal control used in each aliquot is proportional to the dilution ratio of the aliquot.

[0170] The reporter nucleic acid molecule produced by the method of the second aspect of the present invention (i.e., more precisely, the PCR product obtained as a result of the amplification of the reporter nucleic acid molecule) is preferably detected by DNA sequencing. Most preferably, as described above, a massively parallel DNA sequencing method is used.

[0171] A third aspect of the present invention provides a method for detecting an analyte in a sample, wherein the analyte is detected by detecting a reporter nucleic acid molecule for the analyte, the method comprising: performing a PCR reaction to produce a PCR product of a reporter nucleic acid molecule; and detecting the PCR product, wherein an internal control is included in the PCR reaction, is present in a predetermined amount, and is a control nucleic acid molecule, or contains a control nucleic acid molecule, or causes a control nucleic acid molecule to be produced, and the control nucleic acid molecule contains a sequence that is the reverse sequence of the reporter nucleic acid molecule.

[0172] All features of the third aspect of the present invention may be as described in relation to the first and / or second aspects of the present invention.

[0173] The present invention may be further understood by referring to the following non-limiting embodiments and drawings. [Brief explanation of the drawing]

[0174] [Figure 1]Figure 1 shows conceptual diagrams of six different versions of the proximity extension assay described in detail above. The inverted "Y" shape represents the antibody as the analyte-binding domain of the exemplary proximity probe. [Figure 2] Figure 2 shows a conceptual diagram of examples of elongation controls that may be used in a proximity elongation assay. Parts A through F show appropriate elongation controls used in versions 1 through 6 of Figure 1, respectively. Parts B through E show alternative options (i) and (ii) for different possible elongation controls used in versions 2 through 5 of Figure 1, respectively. The captions for Figure 1 also apply to Figure 2. [Figure 3] Figure 3 shows the counts (accurately paired barcodes) obtained from 367 assays performed on a single plasma sample, displayed on a Log10 scale. Comparisons were made between contacting the sample with a probe pool containing all 367 assays and contacting the sample with the same probe set divided into four abundance-based blocks. Assays in Block A and Block B showed a significant increase in counts compared to assays with lower counts without the abundance-based blocks, enabling higher detection in the corresponding assays. Similarly, counts in Block D decreased compared to assays with higher counts without the abundance-based blocks, mitigating the reduction in flow cell real estate. [Figure 4]Figure 4 shows the counts (accurately paired barcodes) obtained from 367 assays performed on a single plasma sample, displayed on an equally spaced scale. A comparison was made between contacting the sample with a probe pool containing all 367 assays and contacting the sample with the same probe set divided into four abundance-based blocks. Assays in Block A and Block B showed a significant increase in counts compared to assays with lower counts without the abundance-based blocks, enabling higher detection in the corresponding assays. Similarly, counts in Block D decreased compared to assays with higher counts without the abundance-based blocks, mitigating the reduction in space on the flow cell. [Figure 5] Figure 5 shows a box plot of results from 54 plasma samples exposed to a probe pool of 372 assays, divided into four abundance-based blocks and classified by the median count within each block. The abundance-based blocks allow for detection of a wide range of protein abundances across samples without sacrificing detection or risking that low-end assays with high inter-sample variability will fall below robust count detection. The dashed line represents 100 counts as a threshold for sufficient count detection. [Examples]

[0175] Example 1 - Exemplary Experimental Protocol Step 1 - Sample preparation and incubation Sixteen aliquots from each of 48 to 96 plasma samples are incubated in a 96-well or 384-well incubation plate with up to 16 proximity probe pools (four abundance-based blocks for each of the four 384 probe-versus-panels). For probe pools that include assays requiring prior dilution, samples may be pre-diluted to 1:10, 1:100, 1:1000, and 1:2000. • Dilution and dispensing of plasma samples into incubation solution can be done manually or by a dispensing robot, such as Labotec's Mosquito® HTS. The incubation solution is dispensed into the wells of the plate. • Add 1 μl of sample to 3 μl of incubation mix at the bottom of each well, seal the plate with adhesive film, rotate at 400 × g for 1 minute at room temperature, and incubate overnight at 4°C. • When using the above dispensing robot, the volume of the sample may be reduced to 0.2 μl and the volume of the incubation mix to 0.6 μl (one-fifth).

[0176] The table below shows an example reagent formulation. The probe solution may contain other components, such as other blocking agents. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0177] Step 2 - Proximity Extension and PCR1 Amplification PwoDNA polymerase is used for extension and amplification. PCR 1 is performed using common primers for amplification of all extension products. Bring the incubation plate (from Step 1) to room temperature and centrifuge at 400 × g for 1 minute. Add the extension mix (containing ultrapure water, DMSO, PwoDNA polymerase, and PCR1 solution) to the plate, then seal the plate, gently vortex it, and centrifuge at 400 × g for 1 minute. Then, place it on a thermal cycler for the PEA reaction and pre-amplification (20 minutes at 50°C, 5 minutes at 95°C, (30 seconds at 95°C, 1 minute at 54°C, 1 minute at 60°C) × 25 cycles, hold at 10°C). Preferably, the extension mix may be dispensed into the plate using a dispensing robot, such as a Thermo Scientific® MultiDrop® Combi-Regent Dispenser. The forward common primer contains the Illumina P5 sequencing adapter sequence (SEQ ID NO: 1). [Table 6] [Table 7]

[0178] Step 3 - Pooling blocks by quantity The PCR1 products obtained from each of the four abundance-based blocks derived from the 384 probe-pair panel are pooled together. This allows for a maximum of four PCR1 pools per sample for each of the 384 probe-pair panels. Different amounts can be taken from each block to equalize the relative levels of assays between blocks. PCR1 products can be pooled manually or by a dispensing robot.

[0179] Step 4 - PCR2 Indexing Prepare a primer plate containing 48 to 96 reverse primers (typically one primer per well in a 96-well plate). Each reverse primer contains the "Illumina P7" sequencing adapter sequence (SEQ ID NO: 2) and a sample index barcode. Use a unique barcode sequence for each PCR1 product from different samples. Preferably, each of up to four PCR1 pools containing the same plasma sample (384 probe-pairs per panel) is assigned the same sample index to facilitate identification and data processing. Add a forward common primer containing the "Illumina P5" sequencing adapter sequence (the same forward primer used in PCR1) to the PCR2 solution. Each PCR1 pool is brought into contact with a PCR2 solution containing a forward common primer, a single reverse (sample index) primer from the primer plate, and DNA polymerase (TaqDNA polymerase or PwoDNA polymerase). Amplification is performed by PCR until the primers are depleted (3 minutes at 95°C, 10 cycles of (30 seconds at 95°C, 1 minute at 68°C), and held at 10°C). The theoretical final concentration of the pooled PCR1 product is 1 μM (using all primers). For PCR2, the PCR1 amplicon is diluted 1:20, and the starting concentration in each PCR2 reaction is 50 nM. The concentration of each PCR2 primer is 500 nM. Therefore, the PCR2 primers should be depleted after 3.3 cycles (10x amplification). [Table 8] [Table 9] [Table 10]

[0180] Step 5 - Final Pool All 48 to 96 indexed sample pools belonging to the same 384 probe-pair panel are pooled together, with equal amounts added from each sample. This results in a maximum of four pools (i.e., libraries) for each 384 probe-pair panel.

[0181] Step 6 - Purification and Quantification (Optional) The library is purified separately using magnetic beads, and the total DNA concentration of the purified library is determined by qPCR using a DNA calibration curve. AMPureXP beads (Beckman Coulter, USA), which preferentially bind to longer DNA fragments, may be used according to the manufacturer's protocol. AMPureXP beads bind to long PCR products but not to short primers. Therefore, it becomes possible to purify the PCR product from the remaining primers. The depletion of PCR2 primers means that this purification step may not be necessary in some cases.

[0182] Step 7 - Quality Control (Optional) Small aliquots from each (purified) library are analyzed using an Agilent Bioanalyzer (Agilent, USA) according to the manufacturer's instructions to confirm the success of DNA amplification.

[0183] Step 8 - Sequence Determination The libraries are sequenced using an Illumina platform (e.g., the NoveSeq platform). Up to four libraries (obtained from each of the 384 probe-versus-panel) are routed in separate "lanes" of the flow cell. Depending on the size and model of the flow cell and sequencer used, the up to four libraries may be sequenced in parallel or sequentially (one at a time) in different flow cells.

[0184] Step 9 - Data Output The sequences of barcodes (derived from each reporter nucleic acid molecule) and sample indices (derived from sample index primers) are identified, counted, and summed in the data, and then aligned / labeled according to a known barcode-assay-sample key. • "Matching barcodes" represent interactions between two paired PEA probes. The count correlates with the number of interactions in the PEA. • Counts for each assay and sample must be normalized using an internal reference control to allow for comparison between samples. Each of the four existence-based blocks has its own unique internal reference control. Each of the 384 probe-versus-panel pairs is separated based on the lane being read. Each panel includes the same 96 sample indices, the same 384 barcode combinations, and an internal reference control.

[0185] Example 2 - PEA with and without the use of quantity-specific blocks Multiple PEA (using probes containing antibodies bound to nucleic acid domains having the structure described in version 6 above) was performed to detect 367 proteins in plasma samples. Each probe contained a unique barcode sequence. A proximity probe pool containing all 367 assays was incubated with the samples, and for comparison, four aliquots from each plasma sample were incubated in 96-well or 384-well incubation plates with each of the four proximity probe pools (four abundance-based blocks containing all 367 assays). PEA was performed as described above, except that step 3 was omitted in the case of a proximity probe pool without abundance-based blocks. During amplification of the extension products, P5 and P7 sequencing adapters were attached to each end of the product, along with unique sample indices for reporter nucleic acid molecules from each different sample. All extension products were sequenced using a massively parallel DNA sequencing method employing reversible dye-terminator sequencing technology with the Illumina Novasek platform. Extension products from a probe pool containing 367 assays and extension products from a total of 367 assays obtained from pooled abundance-based blocks were sequenced in separate flow cells at separate times.

[0186] The results for one of the plasma samples can be seen in Figures 3 and 4. The table below shows the ratio between the highest assay (count) and the lowest assay (count) for the same plasma sample, with and without the use of abundance-based blocks. The ratio with abundance-based blocks is significantly lower than the ratio for all 367 assay pools, which means that the measurements for these assays are obtained using space on the flow cell in a more suitable manner (higher counts for low-abundance assays and lower counts for high-abundance assays). [Table 11]

[0187] Example 3 - PEA assay of samples using blocks with different abundances Multiplex PEA (using probes containing antibodies bound to nucleic acid domains with the structure described in version 6 above) was performed to detect 372 proteins in 54 plasma samples. Each probe contained a unique barcode sequence. Four aliquots from each plasma sample were incubated in 96-well or 384-well incubation plates with each of the four proximity probe pools (four abundance-based blocks containing 372 assays). As described above, PEA was performed. During the amplification of the extension products, P5 and P7 sequencing adapters were attached to each end of the product, along with unique sample indices for reporter nucleic acid molecules from each different sample. All extension products were then sequenced using a massively parallel DNA sequencing method employing reversible dye-terminator sequencing technology on the Illumina Novasek platform. The results in Figure 5 demonstrate that assays with low levels of proteins showing large variability between samples, or assays with relatively low abundances across all 54 samples, can be used to detect protein targets with a wide range of abundances in the samples without sacrificing signal reduction (robust levels, e.g., counts below 100 counts).

Claims

1. A method for detecting multiple analytes in a sample, wherein the analytes exist at different levels in the sample, and the method is (i) Prepare multiple aliquots from the sample, and (ii) A method comprising detecting different subsets of the analyte by performing a separate multiplex assay on each aliquot, wherein the analyte of each subset is selected based on the predicted abundance in the sample.

2. The method according to claim 1, wherein the analyte is a non-nucleic acid analyte.

3. The method according to claim 1 or 2, wherein the analyte is a protein or contains a protein.

4. The method according to any one of claims 1 to 3, wherein the analyte is detected by detecting a reporter nucleic acid molecule specific to each analyte in each aliquot.

5. The method according to claim 4, wherein the reporter nucleic acid molecule is generated by the multiple detection assay performed on each aliquot.

6. The method according to claim 4 or 5, wherein the reporter nucleic acid molecule is amplified by PCR and preferably detected by nucleic acid sequencing.

7. The method according to claim 6, wherein one or more sequencing adapters are attached to the reporter nucleic acid molecule in one or more amplification and / or ligation steps.

8. The method according to claim 6 or 7, wherein at least a first PCR reaction is performed on the reporter nucleic acid molecule to attach at least a first nucleic acid sequencing adapter.

9. The method according to claim 8, wherein a second PCR reaction is performed on the PCR product of the first PCR reaction to add a second nucleic acid sequencing adapter.

10. The method according to any one of claims 6 to 9, wherein at least one PCR reaction is carried out until saturation.

11. The method according to any one of claims 1 to 10, wherein the reaction products of the separate multiple assays, or, if the reaction product is a nucleic acid molecule, the amplified product thereof, are pooled to create a first pool, and the reaction product or amplified product is amplified in the first pool.

12. The reaction product of the above multiplex assay is a reporter nucleic acid molecule, and the above method is The method according to claim 11, comprising: amplifying the reporter nucleic acid molecule in a first PCR reaction performed separately for each individual aliquot to produce a first PCR product; pooling the first PCR products from the individual aliquots to create a first pool; and performing a second PCR reaction on the first pool.

13. The method according to claim 11 or 12, wherein the reaction product or its amplification product is added to the first pool in different amounts.

14. The method according to any one of claims 11 to 13, wherein the method is performed separately and in parallel on a plurality of different samples to produce a reaction product or an amplified product for each sample, a separate first pool is created for each sample, and a sample index is added to the products of the first pool by an amplification reaction and / or a ligation reaction.

15. The method according to claim 14, wherein the separate first pool prepared for each sample contains a first PCR product, and in the second PCR reaction performed on the first pool for each sample, a sample index is added to the first PCR product.

16. The method according to claim 14 or 15, wherein the first indexed pools generated for each of the aforementioned samples are pooled together to create a second pool for nucleic acid sequencing.

17. The method according to any one of claims 6 to 16, wherein the PCR reaction includes an internal control in each aliquot.

18. The method according to any one of claims 4 to 17, wherein the reporter nucleic acid molecule is generated in a proximity probe detection assay, particularly a proximity extension assay (PEA).

19. The reporter nucleic acid molecule comprises at least one barcode sequence, and the detection of the reporter nucleic acid molecule comprises detecting the at least one barcode sequence, and optionally detecting it together with a sample index. Preferably, the reporter nucleic acid molecule comprises a combination of barcode sequences derived from the nucleic acid domains of a pair of proximity probes, and the detection of the reporter nucleic acid molecule comprises the detection of the combination of barcode sequences, according to any one of claims 4 to 16.

20. The method according to any one of claims 1 to 19, wherein the sample is a plasma sample or a serum sample.

21. The analyte is detected using a pair of proximity probes, and each proximity probe is (i) an analyte-specific analyte-binding domain, (ii) Nucleic acid domains and, Both probes in each probe pair contain analyte-binding domains specific to the same analyte, and each probe pair is specific to a different analyte. Each probe pair is designed such that when the pair of proximity probes bind to each analyte, the nucleic acid domains of the proximity probes interact to generate a reporter nucleic acid molecule. At least two proximity probe-to-panel pairs are used, each panel for detecting different groups of the analyte, and separate aliquots of the sample are prepared for each panel to detect different subsets of the analyte within the group. The method according to any one of claims 18 to 20, wherein (a) in each panel, each probe pair comprises a different nucleic acid domain pair, and (b) in different panels, the probe pair comprises the same nucleic acid domain pair.

22. A method for detecting analytes from different samples, wherein a sample index is added to the PCR product generated by amplifying the reporter nucleic acid molecule generated for each sample. The PCR products generated from each of the different samples using the same proximity probe pair panel are pooled in a nucleic acid sequencing panel pool, and the PCR products generated using each panel are pooled in separate panel pools. The method according to claim 21, wherein each panel pool is arranged separately.

23. The method according to any one of claims 7 to 22, wherein the nucleic acid sequencing method is a massively parallel DNA sequencing method.

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