Enhancing NGS detection of low-abundance targets

Abstract

This disclosure pertains to multiplexed nucleic acid-based-detection assays where a signal specific for each target is generated by enzymatically connecting two or more oligonucleotide tags to produce a specific unified nucleic acid marker. Varied concentrations of adapters are used to increase the efficiency of next-generation sequencing detection of lower-abundance targets.

Description

ENHANCING NGS DETECTION OF LOW- ABUNDANCE TARGETSFIELD OF THE INVENTION

[0001] The invention relates to assays for detecting protein and / or DNA.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] The present application claims the benefit of U. S. Provisional Patent Application Serial No. 63 / 737, 263 filed December 20, 2024 and U. S. Provisional Patent Application Serial No. 63 / 913,626 filed November 7, 2025, the contents of which are hereby incorporated by reference in its entirely.BACKGROUND

[0003] Detecting low-abundance targets (LATs) amidst high- abundance targets (HATs) is challenging due to the wide dynamic range of plasma and other biofluid proteomes and other biomarkers, both non-nucleic acid and nucleic acid. Next-generation sequencing (NGS) technologies coupled with affinity-based probes, used in multiplexed biomarker detection assays, tend to disproportionately allocate sequencing reads to high-abundance targets, limiting the detection of low-abundance targets. Ensuring sufficient reads for reliable measurement of each biomarker being detected is crucial and thus low-abundance biomarkers are vulnerable to incorrect quantification or non-detection when they are not “read” at a sufficient level. However, higher read count options, if they are available, are costly and often do not solve this issue. Accordingly, methods of enhancing efficiency of NGS detection of LATs are needed.SUMMARY

[0004] In one aspect, the disclosure provides a method for increasing efficiency of nextgeneration sequencing (NGS) detection of one or more lower-abundance targets within a sample comprising a plurality of targets, the plurality of targets comprising lower-abundance targets and higher-abundance targets, the method comprising the steps of a) providing a plurality of sets of probes, each set comprising at least two probes for a target, wherein each probe binds specifically to the target and comprises an oligonucleotide tag comprising a unique identifier sequence associated with the target, and a plurality of adapters, each adapter being specific for an individual target or group of targets, wherein each adapter can specifically interact with a complex comprising one set of probes bound to their target; b) contacting the sample with the plurality of sets of probes, thereby allowing the plurality of targets and the plurality of sets of probes to interact to form a plurality of target-probe complexes, and the plurality of adapters,thereby allowing the plurality of adapters and the plurality of target-probe complexes to interact to form a plurality of target-probe-adapter complexes, wherein the plurality of adapters comprises a relatively low concentration of adapters specific for at least one higher-abundance target compared to the concentration o f adapters specific for lower-abundance targets, c) performing an enzymatic reaction that connects probes of a set of probes directly or indirectly together, thereby producing a unified nucleic acid marker (UNAM) from each of the plurality' of target-probe-adapter complexes, thereby producing a plurality of UNAMs, and d) detecting the plurality of UNAMs by parallel sequencing.[00051 In some embodiments, a) a first oligonucleotide tag and a second oligonucleotide tag of a set of probes that binds specifically to a target each comprise a double-stranded nucleic acid with an overhang, b) an adapter specific for the individual target or group of targets is an insert comprising a double-stranded nucleic acid w'ith a first overhang on a first end and a second overhang on a second end, c) the first overhang of the adapter comprises a nucleotide sequence complementary to a nucleotide sequence of the overhang of the first oligonucleotide tag, d) the second overhang of the adapter comprises a nucleotide sequence complementary to a nucleotide sequence of the overhang of the second oligonucleotide tag, and e) the enzymatic reaction is mediated by a ligase.

[0006] In some embodiments, a) a first oligonucleotide tag and a second oligonucleotide tag of a set of probes that binds specifically to a target each comprise a nucleic acid with a singlestranded end, and b) an adapter specific for the individual target or group of targets comprises a linker comprising a nucleotide sequence complementary' to the end of the first oligonucleotide tag and a nucleotide sequence complementary to the end of the second oligonucleoti de tag, and c) the enzymatic reaction is mediated by a polymerase.

[0007] In some embodiments, a) a first oligonucleotide tag and a second oligonucleotide tag of a set of probes that binds specifically to a target each comprise a nucleic acid with a singlestranded end, b) an adapter specific for the individual target or group of targets is a splint comprising a sequence complementary to a sequence of the first oligonucleotide tag and a sequence complementary to a sequence of the second oligonucleotide tag, and c) the enzymatic reaction is mediated by a ligase.

[0008] In some embodiments, a) a first oligonucleotide tag and a second oligonucleotide tag of a set of probes that binds specifically to a target each comprise a nucleic acid with a singlestranded end, b) an adapter specific for the indi vidual target or group of targets comprises a linker hybridized to a splint, c) a first end of the splint comprises a nucleotide sequence complementary to a nucleotide sequence of the overhang of the first oligonucleotide tag, d) asecond end of the splint comprises a nucleotide sequence complementary to a nucleotide sequence of the overhang of the second oligonucleotide tag, and e) the enzymatic reaction is mediated by a ligase.

[0009] In some embodiments, a) a first oligonucleotide tag and a second oligonucleotide tag of a set of probes that binds specifically to a target each comprise a nucleic acid with a singlestranded end, b) the target comprises nucleic acid comprising a binding region, c) the single stranded end of the first oligonucleotide tag comprises a sequence complementary to a first portion of the binding region of the target nucleic acid, d) the single stranded end of the second oligonucleotide tag comprises a sequence complementary to a second portion of the binding region of the target nucleic acid, e) the first and second portions of the binding region are separated by a gap, f) an adapter specific for the individual target or group of targets is a linker comprising a nucleic acid sequence complementary to a nucleotide sequence of the gap, and g) the enzymatic reaction is mediated by a ligase. In some embodiments, a single nucleic acid comprises the first probe and the second probe, and the UNAM produced from a target-probe-adapter complex comprising the two probes is circular.

[0010] In some embodiments, a target of the plurality of targets comprises a nucleic acid, a first probe for tire target comprises a nucleic acid sequence complementary to a nucleotide sequence of a first portion of the target, and a second probe for the target comprises a nucleic acid sequence complementary to a nucleotide sequence of a second portion of the target.

[0011] In some embodiments, a target of the plurality of targets comprises a protein and a probe that binds specifically to the target comprises an antibody, an antigen-binding fragment of an antibody, an aptamer, or a peptide.

[0012] In some embodiments, each adapter of the plurality of adapters is specific for an individual target. In some embodiments, an oligonucleotide tag of at least one probe of the plurality of sets of probes comprises a locked nucleic acid base. In some embodiments, the method further comprises a step of amplifying the plurality of UNAMs before detecting the plurality of UN AMs by parallel sequencing. In some embodiments, a higher-abundance target is 5-fold to 500-fold, 500-fold - 50,000-fold, 50,000-fold - 1,000,000-fold higher in concentration than a lower-abundance target. In some embodiments, the method further comprises a step of enriching a lower-abundance target before or after the contacting.

[0013] In some embodiments, the targets are classified as the at least one higher-abundance target or lower-abundance targets and the concentration of adapters for the at least one higher-abundance target is reduced by at least 50%, 90%, 95% or 98% compared to the concentration of adapters for the lower-abundance targets; or the targets are classified as the at least onehigher-abundance target, medium-abundance targets, or lower-abundance targets, the concentration of adapters for the at least one higher-abundance target is reduced by at least 95%, 98% or 99% compared to the concentration of adapters for the lower-abundance targets, and the concentration of adapters for the medium abundance targets is reduced by at least 50% or 90% compared to the concentration of adapters for the lower-abundance targets. In some embodiments, the abundances of the targets are classified according to the percentages of UNAMs per target from a method identical to the method of claim 1 except wherein the plurality of adapters comprises identical or more similar concentrations of adapters for each of the plurality of targets.

[0014] In another aspect, the method comprises the steps of a) providing a plurality of sets of probes, each set comprising at least two probes for a target, wherein each probe comprises a target-binding moiety connected to an oligonucleotide tag, the oligonucleotide tag comprising a unique identifier sequence associated with the target, and a plurality of adapters, each adapter being specific for a target, wherein each adapter can specifically interact with one set of probes, a target of the one set of probes, another adapter for the one set of probes, or a combination thereof, b) contacting the sample with the plurality of sets of probes, thereby allowing the plurality of targets and the plurality of sets of probes to interact to form a plurality of targetprobe complexes, and the plurality o f adapters, thereby allowing the plurality of sets of probes and the plurality' of adapters to interact to form a plurality of target-probe-adapter complexes, wherein the sample is contacted with a lower concentration of adapters specific for higher-abundance targets, and the sample is contacted wi th a higher concentration of adapters specific for lower-abundance targets, c) performing an enzymatic reaction that connects probes of a set of probes directly or indirectly together, thereby producing a unified nucleic acid marker (UNAM) from each of the plurality of target -probe-adapter complexes, thereby producing a plurality of UNAMs, and d) detecting the plurality of UNAMs by parallel sequencing.

[0015] In some embodiments, the target comprises a protein or a nucleic acid. In some embodiments, the target comprises a protein. In some embodiments, the target comprises an antigen. In some embodiments, the target comprises DNA or RNA. In some embodiments, the target-binding moiety comprises a nucleic acid, an antibody or a fragment thereof, an aptamer, a peptide, or a combination thereof. In some embodiments, the target- binding moieties of two probes of a set of probes comprise nucleic acid, the two probes are covalently connected to each other, and the UNAM produced from a target-probe-adapter complex comprising the two probes is circular. In some embodiments, each oligonucleotide tag comprises a nucleic acid selectedfrom double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), locked nucleic acid (LNA), and a combination thereof.

[0016] In some embodiments, each adapter is selected from: an insert comprising dsDNA and overhangs on both sides of the dsDNA (e.g., wherein each strand comprises an overhang or one strand comprises both overhangs), each overhang being complementary to one probe of one set of probes; a linker comprising ssDNA, wherein the linker is complementary to one target, one splint, one target and one splint, or two probes; and a splint comprising ssDNA, wherein the splint is complementary to at least one probe, at least one linker, or a combination thereof.

[0017] In some embodiments, a first probe and a second probe of a set of probes each comprises dsDNA and an overhang, the adapter is an insert complementary to the overhangs of the first and second probes, and the insert hybridizes to the first and second probes, thereby forming a target-probe-adapter complex. In some embodiments, a first probe and a second probe of a set of probes each comprise ssDNA complementary to non-adjacent portions of a target or a splint, the adapter is a linker complementary to a portion of the target or the splint that connects the non-ad jacent portions of the target or the splint, and the first and second probes and the linker hybridizes adjacently’ to the target or the splint, thereby forming a target-probe-adapter complex. In some embodiments, two probes of a set of probes each comprise ssDNA, tire adapter is a splint complementary to the two probes, and the splint hybridizes to the two probes, thereby forming a target-probe-adapter complex. In some embodiments, the splint is further complementary to a linker and further hybridizes to the linker. In some embodiments, non-adjacent portions of the splint are complementary to the two probes. In some embodiments, performing the enzymatic reaction that connects probes of a set of probes directly or indirectly together comprises enzymatic ligation, enzymatic extension, or both. In some embodiments, the method further comprises a step of amplifying the plurality of UNAMs before detecting the plurality of UNAMs by parallel sequencing.

[0018] In some embodiments, a higher-abundance target is 5-fold to 1,000,000- fold higher in concentration than a lower-abundance target. In some embodiments, the method further comprises an additional step before or following step b), comprising enriching a lower-abundance target. In some embodiments, the parallel sequencing comprises short read sequencing or long read sequencing.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0020] FIG. 1 depicts a DNA padlock probe. Regions of complementarity of the probe may hybridize adjacently to a target nucleic acid and may subsequently be ligated to each other to generate a circular unified nucleic acid marker (UNAM).

[0021] FIG. 2 depicts use of DNA padlock probes and a linker in an approach for increasing the efficiency of NGS detection of lower-abundance targets. Ligation of two ends of a padlock via a linker forms a UNAM. One copy of the linker is added to each assay. Although five copies of the padlock probe are formed for the high-abundance target, the amount of linker is limiting, so only one copy of UNAM is formed for types of targets.

[0022] FIG. 3 depicts use of a pair of (i.e. separate) nucleic acid-based probes and a linker that are ligated to produce a linear UNAM.

[0023] FIG. 4 depicts use of a pair of nucleic acid-based probes and a splint. Ligation of two probes mediated by the splint forming the UNAM. In a similar embodiment, DNA padlock probes can be used instead of the pair of probes shown.

[0024] FIG. 5 depicts use of a pair of nucleic acid-based probes and an insert. Ligation of two probes via insert forming the UNAM. In a similar embodiment, DNA padlock probes can be used instead of the pair of probes shown.

[0025] FIG. 6 depicts use of a pair of nucleic acid-based probes, a linker, and a splint. Ligation of two probes mediated by Splint and via Linker forming the UNAM. In a similar embodiment, DNA padlock probes can be used instead of the pair of probes shown.

[0026] FIG. 7 depicts use of multiplex paired-antibody amplified detection (MP AD), including a pair of antibody-based probes and an insert. Liga tion of two probes via Insert forming the unified nucleic acid marker (UNAM).

[0027] FIG. 8 depicts use of a proximity ligation assay (PLA), including a pair of antibodybased probes and a splint.

[0028] FIG. 9 depicts use of a PLA, including a pair of antibody-based probes, a splint, and a linker.

[0029] FIG. 10 depicts use of a proximity extension assay (PEA), including a pair of antibody-based probes and a linker. Extension of the linker form the UNAM.

[0030] FIG. 11 presents qPCR titration curves for target CA19-9 corresponding to the experiment shown in Table 3 below. The data demonstrate that decreasing the concentration of the adapter for CAI 9-9 proportionally reduces the qPCR signal for target CAI 9-9, while maintaining overall assay performance without detectable deterioration in signal integrity.DETAILED DESCRIPTIONAbbreviations

[0031] cDNA — complementary DNA

[0032] dNTP — deoxyribonucleoside triphosphate

[0033] dsDNA — double-stranded DNA

[0034] HAT — high- or higher- abundance target

[0035] LAT — low- or lower- abundance target

[0036] LNA — locked nucleic acid

[0037] MDISA — multiplex DNA immune-sandwich assay; synonymous to MP AD

[0038] MP AD — multiplex paired-antibody amplified detection; synonymous to MDISA

[0039] NGS — next-generation sequencing

[0040] nt — nucleotide

[0041] PBST — phosphate-buffered saline with Tween

[0042] qPCR — quantitative polymerase chain reaction

[0043] RCA — rolling circle amplification

[0044] ssDNA — single-stranded DNA

[0045] UN AM — unified nucleic acid markerDefinitions

[0046] Several aspects of the disclosure are described below, with reference to examples for illustrative purposes only. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the disclosure. One having ordinary skill in the relevant art, however, will readily recognize that the disclosure can be practiced without one or more of the specific details or practiced with other methods, protocols, reagents, instrumentation, samples, and subjects. The present disclosure is not limited by the illustrated ordering of acts or events, as acts may occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts, steps, or events are required to implement a methodology in accordance with the present disclosure. Many of the techniques and procedures described, or referenced herein, are well understood and commonly employed using conventional methodology by those skilled in the art.

[0047] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In various cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusionof such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and / or as otherwise defined herein.

[0048] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0049] As used herein, the indefinite articles “a,” “an,” and “the” should be understood to include plural reference unless the context clearly indicates otherwise.

[0050] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of, e.g., a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. When used herein, the term “comprising” can be substituted with the term “containing” or “including.”

[0051] As used herein, “consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and no vel characteristics of the claim. Any of the terms “comprising,” “containing,” “including,” and “having,” whenever used herein in the context of an aspect or embodiment of the disclosure, can in some embodiments, be replaced with the term “consisting of,” or “consisting essentially of’ to vary' the scope of the disclosure.

[0052] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability" of the second element without the first. A third option refers to the applicability" of the first and second elements together. Any one of these options is understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall w ithin the meaning, and, therefore, satisfy the requirement of the term “and / or.”

[0053] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every' combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.”

[0054] As used herein, “protein” refers to any of numerous naturally occurring extremely complex substances (as an enzyme or antibody) that consist of amino acid residues joined by peptide bonds, contain the elements carbon, hydrogen, nitrogen, oxygen, usually sulfur. In general, a protein comprises 50 - 1000 or more amino acids.

[0055] As used herein, “locked nucleic acid” (LNA), also known as bridged nucleic acid (BNA), refers to a synthetic nucleic acid analog wherein the ribose ring is conformationally locked by a methylene bridge connecting the 2’-0 to the 4’C.

[0056] As used herein, “nuclei c aci d,” refers to a polymer comprising of nucleotides. A nucleic acid can be an oligonucleotide or polynucleotide; can be single-stranded, doublestranded or a combination of single-stranded and double-stranded; and can comprise DNA, RNA, LNA, or combinations thereof.

[0057] The terms “oligonucleotide” and “oligo” are used interchangeably herein and refer to short single-stranded or double-stranded nucleic acids.

[0058] The term “portion,” when used in reference to a nucleic acid, refers to fragments that may range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue.

[0059] As used herein, “antibody” refers to an immunoglobulin that binds to an immunogen (antigen). It is desired that the antibody demonstrates specificity to epitopes contained in the immunogen. In some embodiments, the antibody is a fragment of a complete antibody that retains the antigen binding domain. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, single chain antibody molecules (e.g., scFv), disulfide-linked scFv (dsscFv), diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD), single variable domain antibodies (e.g., camelid antibodies, alpaca antibodies), and single variable domain of heavy chain antibodies (VHH).

[0060] As used herein, “sample” is used in its broadest sense and includes any complex biological sample comprising two or more targets at different concentrations. Examples of samples include environmental samples (, e.g., soil and water), samples isolated from an animal, plant, or microorganism, bodily fluids (nasopharyngeal discharge, saliva, milk, urine, cerebrospinal fluid, blood, plasma and serum), and extracts from organelles, virions, cells, tissues, organs, foods or stool.

[0061] As used herein, methods related to increasing efficiency of NGS detection of lower-abundance targets are considered a means of “read balancing,” e.g., balancing the allocation of sequencing reads within the read number constraints of next-generation sequencing (NGS). The disclosed methods promote detection of a higher proportion of copies of UNAMs of low-abundance targets (LATs) and reduce the formation and detection of UNAMs (or correct UNAMs) of high-abundance targets (HATs).

[0062] As used herein, “target” refers to an analyte of interest that is measured or detected according to the methods disclosed herein. The target can be any molecule or complex to which two probes can bind specifically and simultaneously. The target can be a nucleic acid, a protein, a polypeptide, a protein complex, a virus, an organelle, a cell, or a chemical compound. A target may refer to an individual target (e.g., a single protein) or to a group of targets (e.g, to a family of similar proteins). In some embodiments, assays disclosed herein are multiplexed, i.e., are configured to detect two or more targets. In some embodiments, assays disclosed herein are configured to detect tens, hundreds, or thousands of targets.

[0063] As used herein, “abundance” refers to an apparent concentration determined by an assay described herein. The apparent concentration of a target depends upon its actual concentration and additional factors including properties of the probes, adapters, enzymes, and UNAMs used for detecting the target (e.g. binding affinity, binding kinetics, and non-specific binding). An apparent concentration may be a relative or absolute concentration.

[0064] As used herein, “probe” and “binding probe” are used interchangeably to refer to an element that binds specifically to a target and comprises an oligonucleotide tag comprising a unique identifier sequence associated with the target. A probe is capable of binding to (e.g., hybridizing with) a target. In some embodiments, target-binding is mediated by an antibody or antibody fragment and the oligonucleotide tag is conjugated to the antibody (see, e.g., FIGs, 7, 8, 9, 10). In some embodiments, target-binding is mediated by a nucleic acid and the unique identifier sequence of the oligonucleotide tag comprises all or part of the same sequence that hybridizes to the target (see, e.g., FIGs. 2, 3, 4, 5, 6). Unique identifier sequences are used during analysis of NGS data to assign each read from a UNAM to a target. In some embodiments, the two probes that bind the same target are separate (see, e.g., FIGs. 3-7). In some embodiments, two probes that bind the same target are joined (see, e.g., FIG. 2).

[0065] As used herein, “linker” refers to a single-stranded oligonucleotide that is complementary to and hybridizes with a target nucleic acid (see, e.g., FIGs. 2, 3) or a splint (see, e.g., FIGs. 6, 9). When oligonucleotide tags of one or more binding probes adjacently hybridize to a target nucleic acid or splint, the linker may be ligated to the adjacent oligonucleotide tags.

[0066] As used herein, “splint” refers to a single-stranded oligonucleotide that is complementary to and hybridizes with a portion of at least two nucleic acids, e.g., oligonucleotide tags of binding probes, linkers, and / or target nucleic acids. A splint facilitates the ligation of, but is not itself ligated to, the combination of oligonucleotide tags and linkers towhich it is hybridized. For example, a splint can facilitate ligation of two oligonucleotide tags to each other (see, e.g., FIGs. 4, 8) or ligation of two oligonucleotide tags to either end of a linker (see, e.g., FIGs. 6, 9).

[0067] As used herein, “insert” refers to a dsDNA with an overhang on each side of the dsDNA (e.g., wherein each strand comprises an overhang or one strand comprises both overhangs). One or both overhangs are complementary to and hybridize with oligonucleotide tag components of target-binding probes. An insert may be ligated to two complementary' oligonucleotide tags of binding probes (see, e.g., FIGs. 5, 7).

[0068] The term “adapter” is used herein to refer to one or more functional elements used in methods disclosed herein. For example, “adapter” may refer to any one or any combination of linkers, splints, and inserts.Example Embodiments

[0069] The method s disclosed herein relate to balancing reads of biomarkers in proximitybased assays and other specific DNA / nucleic acid detection assays. Balancing the distribution of reads across all targets in an NGS run can enhance detection efficiency and scientific insight.

[0070] Current methods to address read balance in detecting low-abundance biomarkers include: 1) stratifying sample dilutions into sub-panels based on analyte concentration, 2) modifying detection probe concentrations for specific targets, and 3) using "cold" competing antibodies to reduce signal for high-abundance targets. These strategies have pros and cons. Sub-panel stratification is effective but cumbersome and expensive. Modifying antibody probe concentrations can lower dynamic range, limiting assay usability. Adding cold antibodies effectively reduces HAT signal but, depending on the magnitude of required read balancing, the “cold antibody7” quenching can impact signal integrity and significantly increase costs.

[0071] The “variable adapter concentration method” provided herein for balancing reads involves adjusting the concentration of specific adapters, (inserts, linkers, and / or splints) that facilitate the ligation of oligonucleotide tags in multiplexed protein and other biomarker assays. For high-abundance targets, reducing adapter concentration selectively lowers DN A product formation from one or more oligonucleotide tags and, in some embodiments, one or more adapters. For low-abundance targets, using a saturated adapter concentration ensures maximized ligation of the oligo tags. Alternatively, adapter blocking elements can be added to inhibit the incorporation of specific adapters into DNA products. Read balancing is achieved by partially inhibiting the fomiation of UN AMs specific for high-abundance targets. This approach balances DNA product levels across targets of different abundances in a sample, facilitating more- u -balanced read allocation during NGS readout and improving the detection of low-abundance biomarkers.

[0072] This method is especially suited for multiplexed protein assays such as the MPAD procedure (multiplex paired-antibody amplified detection; also known as multiplex DNA immuno-sandwich assay or MDISA) that utilizes DNA tags as a readout element (see U. S. Patent 11,459,598 to MacKenzie et al.). This method is similarly suited for a variety of other assays (see, e.g., U. S. Patent 10,669,569 to Gullberg et al. (see also WO 2012 / 049316), U. S. Patents 7,914,987 and 8,580,504 to Fredriksson and Davis (see also WO 2005 / 123963), Nilsson et al. (Padlock Probes: Circularizing Oligonucleotides for Localized DNA Detection. Science.1994; 265(5181):2085-2088), and Szemes et al. (Diagnostic application of padlock probes — multiplex detection of plant pathogens using universal microarrays. Nucleic Acids Research. 2005; 33(8):e70), the contents of which are incorporated herein by reference in their entirety), for example, splint-based proximity ligation assays and variations of proximity extension assays. This method is suited for any assay where there are 1) one or more sets of at least two binding probes — some embodiments of which will not be able to connect together without an insert or a linker (see, e.g., FIGs. 2, 3, 5, 6, 7, 9, 10), and some embodiments of which can connect to each other directly with the assistance of a splint (see, e.g., FIGs. 4, 8, and 2), for each set of binding probes, at least one additional element (including an adapter, e.g., an insert, a linker, or a splint) that is specific to a target (or group of targets) and can be varied (e.g., the concentration of which can be varied within a sample) by target.

[0073] To illustrate the principle of the methods, each of these figures show's one copy of a low-abundance target and five copies of a high-abundance target. The adapters have one copy for the low-abundance target and one copy for the high abundance target. In practice, there will be multiple copies of the low'-abundance target and thousands or even millions of copies of the high-abundance target. Furthermore, there ’will be multiple copies of the adapter for the low' abundance target and few'er copies of the adapter for the high-abundance target such that UNAM production for the high-abundance target is limited by the lower number of adapters.

[0074] Additionally, the methods can be adapted for detecting low abundance genes / nucleic acids using Padlock probes by addressing gene imbalances and improving NGS read allocation. Tire target can be either single-stranded or double stranded. If the target is double-stranded DNA, it must be transformed (e.g. melted) into single-stranded DNA before detection with Padlock probes. By introducing a gap between Padlock probes that requires an adapter for ligation, the method modulates DNA product formation. Adjusting adapter concentrations or inhibiting adapter function limits HAT template formation while ensuring LAT templates areadequately formed. This results in balanced template formation and subsequent read allocation during NGS readout. The approach enhances detection resolution, coverage, and depth, and is applicable to diagnostics, spatial transcriptomics, and single-cell RNA sequencing, improving NGS efficiency for targets with differential expression (FIG. 11).

[0075] In one aspect, the disclosure provides a method (e.g., a variable adapter concentration method) for increasing efficiency of next-generation sequencing (NGS) detection of one or more lower-abundance targets within a sample comprising a plurality of targets e.g., a multiplexed assay). In some embodiments, the disclosure provides a variable adapter concentration method for use in a proximity ligation assay (PLA). In some embodiments, the disclosure provides a variable adapter concentration method for use in a proximity extension assay (PEA).

[0076] The plurality of targets comprises lower-abundance targets (LATs) and higher-abundance targets (HATs). In some embodiments, a higher-abundance target is 5-fold to 1,000,000-fold higher in concentration than a lower-abundance target. In plasma, for example, insulin-like growth factor 1 (IGF-1 ), angiopoietin-2, and soluble transferrin receptor (sTfR) are relatively high-abundance proteins (approximately 1 ng / mL to 1 pg / mL), and vascular endothelial growth factor (VEGF), and interleukins are relatively low-abundance proteins (approximately 1 pg / mL to 1 ng / mL). In some embodiments, a higher-abundance target is a high-abundance target present at a higher level (e.g., concentration) than the mean abundance of targets in the sample. In some embodiments, a lower-abundance target is a low'-abundance target present at a lower level (e.g., concentration) than the mean abundance of targets in the sample.

[0077] The difference between adapter concentrations could be several orders of magnitude. For example, in a cell extract a particular target (high-expressing housekeeping, e.g., p-tubulin), a HAT, had an expected concentration range 1000x-10,000x higher than the expected concentration range for a different target (p53 transcription factor, a LAT); therefore, in order to balance the NGS reads, the adapter for p53 can be added at the standard concentration of 5 nM and the adapter for p-tubulin can be added at a concentration 100 times lower than the standard concentration. Adapter concentrations may further be optimized experimentally. The example above is a good place to start the optimization process.

[0078] Other examples of proteins whose detection in multiplex could benefit from this technology may include;• Ribosomal proteins are produced in very high abundance relative to certain regulatory proteins in cells. Ribosomal proteins can number in the millions per cell, while regulatory proteins like transcription factors often exist in hundreds to thousands of copies per cell.• Actin, a major cytoskeletal protein, is expressed in extremely high quantities relative to many signaling proteins. Ratios can exceed 1: 1000 depending on the cell type and functional context.

[0079] The method comprises steps of providing and contacting the sample with a plurality of sets of probes and a plurality of adapters, performing an enzymatic reaction that connects probes, and detecting unified nucleic acid markers (UNAMS) by parallel sequencing. In some embodiments, the method comprises one or more additional steps that may occur before, after, and / or between the steps of providing, contacting, performing an enzymatic reaction, and detecting.

[0080] In some embodiments, the method further comprises an additional step of enriching one or more lower-abundance targets. In some embodiments, the enrichment step takes place prior to contacting the sample with probes and adapters. In embodiments where enrichment occurs prior to contacting the sample with probes and adapters, the sample may undergo a selective concentration or isolation procedure designed to increase the relative abundance of low-abundance targets. Examples include immunoaffinity capture using antibodies or aptamers specific for the target class, depletion of interfering species, size-based or charge-based fractionation, or chemical precipitation methods. Performing enrichment at this stage increases the effective concentration of the targets before probe engagement, thereby improving downstream detection sensitivity.Probes and Adapters

[0081] The method comprises a step of providing a plurality of sets of probes and a plurality of adapters.

[0082] Each probe set is specific for a target and comprises at least two probes for that target. In some embodiments, a probe set comprises three probes as described in W02005123963 A2. Each set (e.g., pair, triad, etc.) of probes may form a probe-target complex with its specific target.

[0083] Each probe comprises a target-binding moiety connected to an oligonucleotide tag (z.., oligo tag). The oligonucleotide tag comprises a unique identifier sequence associated with the target (or a group of targets). In some embodiments, the target-binding moiety comprises a nucleic acid, an antibody or a target binding fragment thereof, an aptamer, a peptide, or a combination thereof. In some embodiments (e.g. a modified Padlock assay), the target-binding moieties of two probes of a set of probes comprise nucleic acid, the two probes are co valently connected to each other, and the UNAM produced from a target-probe-adapter complexcomprising the two probes is circular. In some embodiments, each oligonucleotide tag comprises a nucleic acid selected from double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), locked nucleic acid (LNA), and a combination thereof.

[0084] In some embodiments, each adapter is specific for an individual target, / .e.. can specifically interact with one set of probes, a target of the one set of probes, another adapter for the one set of probes, or a combination thereof. In some embodiments, an adapter comprises a unique identifier sequence specific to the target. In some embodiments, each adapter is specific for a group of targets, z.e., can specifically interact with a group of sets of probes and comprises a unique identifier sequence specific to the group of targets. Targets can be grouped by concentration (e.g. high, medium, and low), biological pathway (e.g. inflammatory) or biological function (e.g. kinases, cytokines).

[0085] In some embodiments, the adapter is an insert comprising dsDNA with an overhang on each side of the dsDNA e.g., wherein each strand comprises an overhang or one strand comprises both overhangs), each overhang being complementary to one probe of one set of probes (i.e., the overhang on one end of the insert hybridizes to the overhang of the first probe and the overhang on the other end of the insert hybridizes to the overhang of the second probe).

[0086] In some embodiments, the adapter is a linker comprising ssDNA, wherein the linker is complementary to one target, one splint, one target and one splint, or two probes.

[0087] In some embodiments, the adapter is a splint comprising ssDNA, wherein the splint is complementary to at least one probe, at least one linker, or a combination thereof.Proximity-Based Interactions

[0088] The method comprises a step of contacting the sample with the plurality of sets of probes and the plurality of nucleic acid adapters. Contacting the sample with the plurality of sets of probes allows the plurality of targets and the plurality of sets of probes to interact to form a plurality of target-probe complexes. Contacting the sample with the plurality of adapters allows the plurality of sets of probes and the plurality of adapters to interact to form a plurality of target-probe-adapter complexes i.e., the adapters specific to a target interact with that target and / or probes for that target, as designed). Furthermore, each adapter links the oligonucleotide tags of the two probes of a set of probes, to form a tag-adapter-tag complex. Addition of an enzyme (e.g. a ligase or polymerase) converts the tag-adapter-tag complex into a unified nucleic acid marker (UNAM).

[0089] The sample is contacted with a lower concentration of adapters that are specific for higher-abundance targets. In some embodiments, the probes bound to higher-abundance targetsare not saturated by the lower concentration of adapters, i.e., not all copies of higher-abundance target-bound probes are bound by adapters and form the unified nucleic acid markers (UNAMs). The sample is contacted with a higher concentration of adapters that are specific for lower-abundance targets, thus maximizing lower-abundance target UNAM formation. In some embodiments, the probes bound to lower-abundance targets are saturated by the higher concentration of adapters, i.e., a higher number of copies of lower-abundance target-bound probes are bound by adapters. In some embodiments, w ithin the context of limited reads in NGS, this approach leads to reduced read allocation to UNAMs associated with higher-abundance targets and increased read allocation to UNAMs associated wdth lower-abundance targets.

[0090] In some embodiments, the difference between adapter concentrations for higher-abundance targets and lower-abundance targets are up to several orders of magnitude. In some embodiments, the concentration of each adapter can be increased or decreased (i.e., can be made higher or lower) independent from the concentration of any other adapters. In some embodiments, the difference between adapter concentrations for higher-abundance targets and lower-abundance targets is up to several orders of magnitude. For example, where a low'-abundance target is assigned a nominal adapter concentration of about 1.5 nM, a mediumabundance target may be assigned an adapter concentration in the range of about 0.015-0.15 nM (approximately 10-100-fold lower), and a high-abundance target may be assigned an adapter concentration in the range of about 0.0015-0.015 nM (approximately 100-1000-fold lower).

[0091] In some embodiments, a target-probe-adapter complex includes a target-bound probe (probe A) comprising dsDNA with an overhang (on one ofthe strands at the 5’-end or 3’ end), a second target-bound probe (probe B) comprising dsDNA with an overhang (on one of the strands at the 5 ’-end or 3’ end), and an adapter that is an insert complementary to those probes (i.e., complementary to the overhangs of probes A and B). The insert comprises dsDNA with an overhang on each side (e.g., wherein each strand comprises an overhang or one strand comprises both overhangs), wherein the overhangs are complementary to (i.e., compatible with or can be hybridized to) the overhangs of probes A and B. The dsDNA insert hybridizes with the probes to form a target-probe-adapter complex. Such embodiments of a target-probe-adapter complex enables ligation of the probes to the insert (i.e., a UNAM would comprise a sequence of: probe A oligo tag — insert — probe B oligo tag).

[0092] In some embodiments, a target-probe-adapter complex includes two target-bound probes comprising ssDNA complementary to non-adjacent portions of the target or a splint, and an adapter that is a linker complementary to a portion of the target or the splint that connects thenon-adjacent portions of the target or splint. The first and second probes hybridize non-adjacently to the target or splint. The linker hybridizes to the target or splint between the hy bridized probes, thereby forming a target-probe-adapter complex. Such embodiments of a target-probe-adapter complex enables ligation of the probes to the link er (z.e., a UNAM would comprise a sequence of: probe A oligo tag — linker — probe B oligo tag).

[0093] In some embodiments, a target-probe-adapter complex includes a splint and two target-bound probes comprising ssDNA complementary to the splint. The target-bound probes hybridize to the splint, thereby forming a target-probe-adapter complex. Such embodiments of a target-probe-adapter complex enables ligation of the probes directly or indirectly to each other i.e., a UNAM would comprise a sequence of: probe A oligo tag — probe B oligo tag or probe A oligo tag — linker — probe B oligo tag, respectively).

[0094] In some embodiments, a target-probe-adapter complex includes a linker and two target-bound probes comprising ssDNA complementary to the linker. The probes hybridize non-adjacently to the linker, enabling formation of a UNAM via an extension step in which the linker is used as a template for extension of an oligo tag of a probe, and the oligo tag of the probe is similarly used as a template for extension of the linker. In some embodiments, a target-probe-adapter complex enables a proximity-extension assay (PEA).

[0095] In some embodiments, the splint is further complementary to a linker and further hybridizes to the linker. In some embodiments, non-adjacent portions of the splint are complementary to the two probes. In some embodiments, the probes hybridize to non-adjacent portions of the splint and a linker hybridizes to the splint between the hybridized probes, thereby enabling ligation of the probes indirectly to each other (i.e., a UNAM would comprise a sequence of: probe A oligo tag — linker — probe B oligo tag).

[0096] In some embodiments, a target-probe-adapter complex includes more than two target-bound probes (i.e., there are more than two probes in the set of probes for the target) and more than one adapter (e.g., a splint and a linker or two inserts). For example, probe B may comprise a dsDNA with an overhang at its 5’-end, a target-binding moiety7, and a dsDNA with an overhang at its 3 ’-end. Probe B could be used in conjunction with probe A comprising dsDNA with an overhang at its 5’-end, probe C comprising dsDNA with an overhang at its 3’-end, insert A-B comprising 5’ -end overhangs complementary to probes A and B, and insert B-C comprising 3’-end overhangs complementary to probes B and C. In such embodiments, the probes hybridize to the inserts, thereby enabling ligation of the probes indirectly to each other (i.e., a UN AM would comprise a sequence of: probe A oligo tag — insert A-B — probe B oligo tag — insert B-C — probe C oligo tag).Adapter Blocking Elements

[0097] In some embodiments, read balancing is achieved by introducing adapter blockers that selectively impede proper UN AM formation for HATs. The adapter blockers are added at concentrations selected to partially inhibit UNAM formation for the HATs, thereby reducing but not eliminating the number of UNAMs generated from those targets. Adapter blockers may comprise oligonucleotides, modified nucleic acids, or other binding elements that compete with, occlude, or otherwise interfere with adapter hybridization or ligation for a given target. By¬ introducing adapter blockers specific to a high-abundance or high-signal target, DNA template formation for that target is reduced, thereby lowering the number of sequencing reads or signal units allocated to that target. Adapter blockers may be introduced in controlled molar ratios relative to their corresponding adapters. In this manner, adapter blockers function as a tunable mechanism for suppressing over-represented targets without altering the concentrations of adapters for other targets, enabling redistribution of reads or signal toward lower-abundance targets in a multiplexed assay. By selectively applying blocker elements to high-abundance or high-signal targets, the effective concentration of productive nucleic acid templates is reduced, thereby decreasing sequencing read allocation or signal output for those targets and redistributing analytical capacity toward low-er-abundance targets.

[0098] For inserts, adapter blockers may hybridize to ligation junctions or other parts of the insert, impeding insert hybridization, amplification, circularization, and formation of the UNAM. Blockers may also sterically hinder the access of polymerase or ligase or competitively sequester reaction components required for productive template generation.

[0099] For linkers, adapter blockers may hybridize to all or a portion of the linker sequence, thereby preventing or reducing linker hybridization to the target nucleic acid or oligonucleotide tags of the probes, and / or inhibiting ligation of the l inker to adjacent oligonucleotide tags. By reducing productive linker incorporation, the forma tion of downstream amplifiable templates associated with a given target is decreased.

[0100] For splints, an adapter blocker may comprise a single-stranded oligonucleotide configured to interfere with splint-mediated ligation of oligonucleotide tags. In some embodiments, a splint-specific blocker hybridizes to all or a portion of the splint, thereby- preventing proper alignment of the oligonucleotide tags required for ligation. In other embodiments, the blocker hybridizes to one or more of the oligonucleotide tags of the probes, thereby preventing productive interaction with the splint. In either case, inhibition of splint-mediated alignment reduces ligation efficiency- and lowers the effective yield of complete nucleic acid constructs for selected targets.

[0101] In proximity extension assays (PEA), a linker-specific blocker may comprise a single-stranded oligonucleotide that hybridizes to the linker, to one or more linker-binding regions, or to one or more oligonucleotide tags of the probes, thereby preventing or reducing proper alignment, extension, or ligation of the oligonucleotide tags via the linker. Such inhibition reduces linker-mediated construct formation efficiency and lowers the effective yield of complete nucleic acid constructs, and thus UNAMs, for selected targets without fully preventing signal generation.Unified Nucleic Acid Markers (UNAMs)

[0102] The method comprises a step of performing an enzymatic reaction that connects the oligonucleotide tags of the probes of a set of probes directly or indirectly together, thereby producing a unified nucleic acid marker (UNAM) from each of the plurality of target-probe-adapter complexes, thereby producing a plurality of UNAMs. In some embodiments, performing an enzymatic reaction that connects probes of a set of probes directly or indirectly together comprises enzymatic ligation, enzymatic extension, or both, hi some embodiments, performing an enzymatic reaction that connects probes of a set of probes directly or indirectly together comprises enzymatic ligation. In some embodiments, performing an enzymatic reaction that connects probes of a set of probes directly or indirectly together comprises enzymatic extension. In some embodiments, performing an enzymatic reaction that connects probes of a set of probes directly or indirectly together comprises enzymatic ligation and enzymatic extension. Suitable enzymes for performing enzymatic ligation (e.g., T4 DNA ligase) or enzymatic extension (e.g., Phi29 DNA polymerase) are known and readily available to those of skill in the art.

[0103] In some embodiments, the method comprises a step of producing a unified nucleic acid marker (UN AM) from each of the plurality of target-probe-adapter complexes, thereby producing a plurality of UNAMs. A UNAM is an example of a DNA detection template. In some embodiments, a UNAM can be amplified (e.g., by PCR). A UNAM can be detected by various methods, including NGS, Sanger sequencing, qPCR, or other nucleic acid detection methods.

[0104] In some embodiments, producing the UNAM from each of the plurality of targetprobe-adapter complexes comprises a ligation step.

[0105] In some embodiments, producing the UNAM from each of the plurality of target¬ probe-adapter complexes comprises a cleavage step (e.g., wherein non-UN AM complex components are removed from the UNAM). In such embodiments, a cleavable linkage is provided betw een the UNAM and one or more portions of the complex or probes. Upon application of a suitable cleavage condition (for example, enzymatic, chemical, thermal, orphotolytic cleavage), the UNAM is released from the target-probe-adapter complex, thereby generating a liberated nucleic acid molecule that is no longer covalently attached to the complex. The cleaved UNAM can then be collected and subjected to further processing, such as amplification, barcoding, sequencing, or quantitative detection, without interference from the original probe or target components.

[0106] In some embodiments, producing the UNAM from each of the plurality of targetprobe-adapter complexes comprises an amplification step. In such embodiments, the nucleic acid portion of the complex (e.g., the adapter, probe, or a joined sequence derived from both) serves as a template for enzymatic amplification, such as PCR, isothermal amplification, rollingcircle amplification, or another nucleic acid amplification method. During amplification, the UNAM sequence is replicated to generate multiple copies of the molecule, thereby increasing its abundance relative to other components of the complex. The amplification products may include the UNAM itself, extended forms of the UNAM, or amplicons incorporating the UNAM sequence. Once amplified, these products can be isolated and subjected to downstream processing, such as quantification, sequencing, barcoding, or read allocation analysis.Amplification- based production of the UN AM provides a means of enhancing detection sensitivity and enabling robust downstream workflows even when tire initial abundance of the target-probe-adapter complex is low.Detection by Next-Generation Sequencing (NGS)

[0107] The method provides a step of detecting the plurality of UNAMs by next-generation sequencing (NGS). As used herein, NGS refers to any method or technology that comprises simultaneous (z.e., parallel) sequencing of DNA fragments, e.g., massively-parallel sequencing. In some embodiments, NGS comprises short-read sequencing. In some embodiments, NGS comprises long-read sequencing (e.g., PacBio Single-molecule real-time (SMRT®) sequencing by synthesis, Oxford Nanopore DNA sequencing by detection through electrical impedance).

[0108] Detecting the plurality of UNAMs by NGS comprises sequencing the plurality of UNAMs by any NGS technology or method. Examples of different NGS technologies and methods are described, for example, in Satam et al. (Next-Generation Sequencing Technology: Current Trends and Advancements. Biology (Basel). 2023 Jul 13;12(7):997). Examples of shortread sequencing NGS technologies and methods include, for example, sequencing by synthesis (e.g., ION TORRENT® semiconductor chip-based sequencing, ILLUMINA® flow cell-based sequencing, Roche 454® pyrosequencing, Helicos™ single-molecule sequencing), sequencing by ligation (e.g., ABI / Life Technologies Sequencing by Oligonucleotide Ligation and Detection(SOLiD™), BGI / Complete Genomics DNA nanoball sequencing), and sequencing by binding (e.g., PacBio ONSO®). In some embodiments, detection by NGS comprises targeted sequencing, wherein specific sequences of interest (e.g., UNAMs) are selectively amplified prior to sequencing.

[0109] In some embodiments, the method further comprises a step of amplifying the plurality of UNAMs prior to a detection step or a l ibrary preparation step. In some embodiments, the UNAM from each of the plurality of target-probe-adapter complexes (i.e., the plurality of UNAMs) undergoes an NGS library preparation step prior to sequencing. In some embodiments, an additional amplification step is performed prior to or at the NGS library preparation step.

[0110] In some embodiments, an additional step of analysis allows interpretation of the sequencing data, generated by NGS. In some embodiments, the sequencing data reads are demultiplexed and mapped to reads that correspond to sequences of specific UNAMs. The read counts for these UN AMs represent the expression level of the target. In some embodiments, the abundance of UNAMs detected in a sample is ascertained by comparison to the abundance of UNAMs in a control sample.Application to Groups of Targets

[0111] In certain embodiments, read-balancing is implemented at the level of groups of targets that share common properties. For such embodiments, a single adapter (or adapter family) may be assigned to and adjusted for an entire group of targets, rather than for each individual target. Examples of grouping strategies include, without limitation: (i) grouping by¬ target concentration or abundance, (ii) grouping by antibody or conjugate non-specific binding -“stickiness,” and (iii) grouping by functional or biological relatedness.

[0112] Grouping of targets can be implemented in two di stinct ways. In one approach, a single adapter concentration is assigned to an entire group of targets, while each target within the group may still have its own unique adapter sequence. In this case, the grouping determines only the amount of adapter used (e.g., reduced, standard, or elevated concentration), allowing multiple targets to share the same concentration profile for read-balancing purposes.

[0113] In another approach, the group of targets may be associated with a single, universal adapter sequence, such that all targets in the group utilize the same adapter species. This allows a single adapter to act as a proxy for the entire group, enabling collective modulation of signal or read allocation by7adjusting the concentration of that universal adapter.

[0114] Thus, grouping can be achieved either through shared concentration (with distinct sequences) or through shared sequence (with a single concentration), depending on the desired level of control and multiplexing efficiency.

[0115] (i) Grouping Targets by Abundance or Concentration: Targets may be sorted into discrete abundance classes, where each class is associated with its own adapter concentration. This enables coarse-grained read balancing while reducing the number of distinct adapter concentrations required and thus simplifying optimization experiments. For example, in one embodiment, targets are assigned to three abundance groups:• High-abundance group: targets whose measured or estimated abundance exceeds a defined upper threshold T. In certain examples, the high-abundance group may include targets with expression or molecular counts more than 10x above the median of all targets in a typical sample.• Medium-abundance group: targets with abundance between thresholds Ti and T2. In some embodiments, this may include targets within 0.1 x to 10* the median target abundance. • Low-abundance group: targets whose abundance is below threshold Ti, for example less than 0.1 * the median abundance. The specific numeric values of Ti and T2 may be determined empirically for a given panel, platform, or sample type. These embodiments use different adapter concentrations - for example, a reduced adapter concentration for high- abundance targets, an intermediate concentration for medium -abundance targets, and an elevated concentration for low-abundance targets — thereby enabling controlled redistribution of sequencing reads across the target space.

[0116] (ii) Grouping by Functional or Biological Relatedness: In certain embodiments, targets may be grouped according to shared biological function, path way, cell-type association, or biomarker class. This grouping can be advantageous when a set of related targets is expected to behave similarly across sample types or when balancing of entire biological categories is desired. In such embodiments, the adapter concentration assigned to a functional target group can be tuned based on expected abundance patterns of that group, clinical relevance or required sensitivity for that class, panel-specific optimization goals.EXEMPLIFICATION EXAMPLE 1Increasing Efficiency of LAT Detection with MPAD

[0117] A scheme comparing detection of a low-abundance and high-abundance targets in an MPAD assay with unique adapters for each probe pair is shown in Fig. 7. Plasma samples weremixed with a protease / phosphatase inhibitor. Pairs of antibodies against 16 proteins of interest were chosen. Each of the antibodies was conjugated to an oligonucleotide tag with a sequence that contained a unique identifier sequence (antibody signature sequence) adjacent to the overhang at the distal end. Antibody conjugates for a multiplex paired-antibody amplified detection (MP AD) system were diluted to appropriate concentrations for the assay.

[0118] The initial incubation involved mixing samples with antibody conjugate solutions and incubating overnight at 4°C. allowing the conjugated antibodies to specifically bind to their target protein(s). Streptavidin magnetic beads were used to capture biotinylated antibody conjugate-protein complexes and washed with phosphate-buffered saline / Tween (PBST) buffer.

[0119] Beads with captured antibody conjugate-protein complexes were incubated with a DNA adapter mix and ligation reagents. The adapter mix was made up such that the adapters for high-abundance targets (HATs) had concentrations lower than a standard concentration, and which were set for each specific HAT depending on the expression level. Adapters for low- abundance targets (LATs) had standard concentrations. Ligation was done for 15 minutes at 22°C, forming ligation products comprising two antibody-oligonucleotide tag conjugates and an adapter, resulting in a unified nucleic acid marker (UNAM).

[0120] Samples underwent pre-amplification PCR and were further prepared for nextgeneration (NGS) sequencing using Illumina NGS barcodes using the NEB Ultra II DNA library preparation kit. Libraries were pooled and NGS sequencing was performed. The abundance of targets within a sample was ascertained by comparison to a control sample.

[0121] Results for a high abundance target (HAT) from three iterative experiments (Expl, Exp2, Exp3) are shown in Table 1. Upon addition of lower concentrations of adapter specific for “Target A” the NGS read counts allocated to “Target A” took up a lower percentage of total reads in the NGS run, allowing lower-abundance targets (LATs) to be sequenced with increasingly sufficient read allocation (Table 1). Because approximately 10,000 total reads per sample were obtained in the NGS run, the read allocation for LAT targets in Expl was on the order of about 5--3O reads per target, which is below the read level needed for reliable quantification. In Exp3, increased read allocation resulting from the read-balancing adjustments led to an increase in reads for these targets to greater than 50 reads per target, which is sufficient for reliable quantification.

[0122] As a result of decreasing the adapter concentration for Target A, Targets B-R, LATs that were detected at very low levels under the conditions of Experiment 1 (Expl), were sequenced with increasingly sufficient read allocation in Experiment 3 (Exp3; see Table 2).Table 1. Increased Read Allocation for LATsExp 1 Exp2 Exp3Target Reads Adapter Reads Adapter Reads Adapter(%) (nM) (%) (nM) (%) (nM)Target A (HAT) 94 1.5 47 0.3 32 0.15All LATs 6 1.5 53 1.5 68 1.5Reads (%R percentage of total read a locationTable 2. Increased Detection of Lower- Abundance TargetsExpl Exp2 Exp31.5 nM adapter 0.3 nM adapter 0.15nM adapter Abundance Target for HAT for HAT for HAT Reads Adapter Reads Adapter Reads Adapter (%) (nM) (%) (nM) (%) (nM) HAT A 94 1.5 47 0.3 32 0.15B 3.25 1.5 28.73 1.5 36.83 1.5 C 0.53 1.5 4.65 1.5 5.96 1.5 D 0.16 1.5 1.38 1.5 1.77 1.5 E 0.05 1.5 0.42 1.5 0.53 1.5 F 0.08 1.5 0.69 1.5 0.89 1.5 G 0.12 1.5 1.07 1.5 1.37 1.5 H 0.09 1.5 0.83 1.5 1.06 1.5 I 0.15 1.5 1.37 1.5 1.76 1.5 LATJ 0.09 1.5 0.78 1.5 1.00 1.5 K 0.27 1.5 2.36 1.5 3.03 1.5 L 0.08 1.5 0.71 1.5 0.91 1.5 M 0.21 1.5 1.86 1.5 2.39 1.5 N 0.39 1.5 3.45 1.5 4.43 1.5 O 0.45 1.5 3.95 1.5 5.06 1.5 P 0.09 1.5 0.78 1.5 1.00 1.5R 0.18 1.5 1.63 1.5 2.10 1.5 Reads (%), percentage of total read allocation

[0123] Table 3 shows results from a series of experiments performed using the readbalancing approach of FIG. 7 with three targets. The intended use of this read-balancing method described herein is for next-generation sequencing (NGS); however, quantitative PCR (qPCR) may be used as a modeling, optimization, and quality-control tool due to its wide dynamic range capability. In an initial experiment, adapters for all targets were loaded at equimolar concentrations, and the assay was read out by qPCR, resulting in a wide range of signal outputs. Under these conditions, target CAI 9-9 exhibited a high signal, target 322 exhibited a medium signal, and target 305 exhibited a low signal. Based on the qPCR readout, adapter concentrations were selectively adjusted — specifically reduced for the high-signal target — followed by rerunning the assay with qPCR for verification. Table 3 and FIG. 11 illustrate three experimentalconditions in which the adapter for CAI 9-9 was loaded at different concentrations, with the percentage of total signal shown for each target. When the CAI 9-9 adapter was present at a standard concentration of 1.5 nM, equal to the adapter concentrations for targets 322 and 305, the CAI 9-9 signal dominated the total signal output. Reducing the CAI 9-9 adapter concentration to 0.15 nM resulted in a corresponding decrease in the CA19-9 qPCR signal and a relative increase in the percentage of total signal attributable to targets 322 and 305. A further reduction of the CA19-9 adapter concentration to 0.015 nM produced an even greater decrease in CAI 9-9 signal and a further increase in the proportion of signal attributable to targets 322 and 305, demonstrating the use of qPCR-guided adapter tuning to balance target representation prior to NGS readout.Table 3. Increased percentage signal from LATs (305 and 322) upon lowering the concentration of the adapter for HAT (CAI 9-9)Target Abundance CAI 9-9 Adapter concentration1.5 nM 0.15 nM 0.015 nMCAI 9-9 High 98.4% 92.7% 47.6%322 Medium 1.5% 7% 49.4%305 Low' 0.09% 0.3% 3%EXAMPLE 2 (Prophetic)Increasing Efficiency of LAT Detection with Splint Proximity Ligation Assay (PL A)

[0124] Schemes comparing detection of a low-abundance and high-abundance targets in PLA assays with unique splints for each probe pair are shown in Fig. 8 and Fig. 9. The assay is performed using the procedures described for proximity ligation assay using a splint as described by Fredriksson and Davis (WO 2005 / 123963), the contents of which are incorporated herein by reference in their entirety.

[0125] Proximity probes are incubated with target analytes at 37°C for one hour to reach binding equilibrium in a 5 pL volume (20 mM Tris-HCl pH 7.3, 150 mM NaCl, 0.1 % bovine serum albumin (BSA) or 1 % BSA). 15 pL is then added of a mix of splints of varying concentrations and design containing 0.1 Units of AMPLIGASE® thermostable DNA ligase, 0.3 mM nicotinamide adenine dinucleotide (NAD), 2 mM MgCh, in a IX PCR buffer II and.Ligation proceeds for 30 minutes at 37°C.

[0126] Splints are designed to be specific for each target. The splint mix is made up such that the splints for HATs have concentrations lower than standard concentrations, and which are set for each specific HAT, or group of HATs, depending on the expression level; splints for LATs have standard concentrations. The difference between splint concentrations for HATs and LATs could be more than 2 orders of magnitude.

[0127] Ligation is performed for 15 minutes at 22°C, forming ligation products comprising two antibody-oligonucleotide tag conjugates and an adapter, resulting in a unified nucleic acid marker (UNAM).

[0128] Samples undergo pre-amplification PCR. Quality control is performed via quantitative PCR (qPCR).

[0129] Samples are further prepared for next-generation sequencing (NGS) readout using barcoded adapters using a DNA library preparation kit compatible with the NGS sequencer. Libraries are pooled and NGS sequencing is performed. The abundance of targets within a sample is ascertained by comparison to a control sample.EXAMPLE 3 (Prophetic)Increasing Efficiency ofLAT Detection with Proximity Extension Assay (PEA) + Linker

[0130] A scheme comparing detection of a low-abundance and high-abundance targets in a PEA assay with unique linkers for each probe pair is shown in Fig. 10. The assay is performed using the procedures described for proximity extension assay with an addition of a splint as described by Fredriksson and Lundberg (U. S. Patent 10,781,473), the contents of which are incorporated herein by reference in their entirety.

[0131] Probes in this Example are designed such that their oligonucleotide tags cannot anneal to each other. In order to anneal, they need a linker. Linkers are designed to be specific for each target. The linker is designed such that when annealed to one of the oligos, it serves as a primer, that is, the linker and the oligo act as templates for extension.

[0132] The linker mix is made up such that the linkers for HATs have concentrations lower than standard concentrations, and which are set for each specific HAT, or group of HATs, depending on the expression level; and linkers for LATs have standard concentrations. The difference between linker concentrations could be up to several orders of magnitude.

[0133] Proximity extension is performed according to protocol described in Assarsson et al. (Homogenous 96-plex PEA immunoassay exhibiting high sensitivity’, specificity, and excellent scalability. PLoS One. 2014 Apr 22:9(4):e95192), the contents of which are incorporated herein by reference in their entirety.

[0134] Samples undergo pre-amplification PCR. Quality7control is performed via quantitative PCR (qPCR).

[0135] Samples are further prepared for next-generation sequencing (NGS) readout using barcoded sequencing adapters using a DNA library preparation kit compatible with the NGS sequencer. Libraries are pulled and NGS sequencing is performed. The abundance of targets within a sample is ascertained by comparison to a control sample.EXAMPLE 4 (Prophetic)Increasing Efficiency of LAT Detection with Modified Padlock Probes

[0136] DNA or RNA is extracted from samples. In the case of RNA, RNA is reverse transcribed to generate complementary7DNA (cDNA).

[0137] Padlock probes (i.e., two connected probes) or a pair of probes (z.e., two separate probes), are custom designed for each target (synthesized by vendor). The design is such that there is a gap of about 20 nucleotides (nt) between the target sequences of the padlock probes (FIG. 2) or a pair of probes (FIG. 3). These 20 nt are filled by a linker oligonucleotide. These linker oligos are also custom designed (synthesized by vendor). They are specific for each target.

[0138] The linkers are mixed such that for HATs, the amount of linker added is lower than standard concentration depending on the expression level and linkers for LATs had standard concentrations. The difference between linker concentrations could be several orders of magnitude.

[0139] Each target DNA / RNA sample (approximately 10-50 ng) is incubated with padlock probes or a pair of probes and linkers at 42-65°C for 30 minutes, facilitating hybridization.

[0140] Following hybridization, DNA ligase (e.g. T4 DNA ligase) is added to the reaction mix in ligation buffer in order to ligate the padl ock probes or a pair of probes and linkers around the target sequence.

[0141] Reactions are incubated at 37°C for 1 hour, followed by enzyme inactivation at 65CC for 10 minutes.

[0142] Amplification is performed using rolling circle amplification (RCA) or PCR.

[0143] If RCA is used, DNA polymerase (e.g. Phi29) is added to the ligated product in amplification buffer containing dNTPs. The reaction is maintained at 37°C for 1-2 hours, allowing RCA of the padlock probe.

[0144] Quality control of amplified products is performed by qPCR. NGS libraries are prepared using a DNA library preparation kit compatible with the NGS sequencer. Libraries are pulled and NGS sequencing is performed.EXAMPLE 5Next-generation sequencing (NGS) methods

[0145] Illumina Library Preparation. Manufacturer’s instructions for NEBNEXT® Ultra II DNA Library Prep Kit for ILLUMINA ® were followed (New England Biolabs, Inc., Ipswich, MA; Catalog Nos. E7645S, E7103S).

[0146] An end prep reaction mixture wras prepared by mixing 500 pg-1 pg DNA (z'.e., UNAMs), NEBNEXT® Ultra II End Prep Enzyme Mix, and NEBNEXT® Ultra II End Prep Reaction Buffer, then incubating at 20°C for 30 minutes followed by 65°C for 30 minutes. A ligation mixture was prepared by mixing NEBNEXT® Adaptor for ILLUMINA®, NEBNEXT® Ultra 11 Ligation Master Mix, and NEBNEXT® Ligation Enhancer with the End Prep Reaction Mixture. The ligation mixture wras incubated at 20°C for 15 minutes. Samples with >50 ng of fragmented DNA were subjected to size selection using SPRIselect size selection beads (Beckman Coulter, Inc., Indianapolis, IN) or NEBNEXT® Sample Purification Beads. Samples were cleaned up with SPRIselect or NEBNEXT® Sample Purification Beads.

[0147] Adapter-ligated DNA was enriched by PCR using NEBNEXT® Ultra II Q5 Master Mix and either Index Primer Mix or Intex Primer / i7 Primer and Universal PCR Primer / i5 Primer. Initial denaturation was performed at 98°C for 30 seconds, followed by 3-15 cycles of denaturation at 98°C for 10 seconds and annealing / extension at 65CC for 75 seconds. A final extension step was performed at 65°C for 5 minutes. PCR product was cleaned up with SPRIselect or NEBNEXT® Sample Purification Beads.

[0148] Illumina Next-Generation Sequencing (NGS). NGS is described, for example, in Vecera et al. (Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long noncoding RNAs. PLoS One. 2019 Feb I l;14(2):e0211978).

[0149] The concentration of prepared DNA libraries was measured using Qubit 2.0 Fluorometer and Qubit dsDNA HS Assay Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA). Libraries were analyzed on Agilent 2200 TapeStation in with Agilent High Sensitivity D1000 ScreenTape System (Agilent Technologies, Santa Clara, CA) according to the manufacturer's protocol. The molarity of individual libraries was calculated using determined concentrations and modal lengths using weight-to-moles conversion.

[0150] Paired-end sequencing of library with a read length of 200 was performed with NEXTSEQ® 2000 Sequencing System using NEXTSEQ 2000 P3® XLEAP-SBS Reagent kit (200 cycles) (Illumina, San Diego, CA). PhiX Control v3 (Illumina) wras added at 1% to the library as an internal control before the sequencing.

[0151] Sequencing data was processed with tools in R-based Bioconductor software. FastQC (see www.bioinformatics.babraham.ac.uk / projects / fastqc) was used to perform pre-alignment quality control (QC) of sequencing data contained in FASTQ files. NGS library adaptors were trimmed from the sequences with the command-line tool cutadapt (see Release 1.17; media.readthedocs.org / pdftcutadapt / stable / cutadapt.pdf). Quality control and quantification was performed with Picard (see broadinstitute.github.io / picard). Numerical and graphical output was gathered and exported via MultiQC (Ewels et al., MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016 Oct I;32(19):3047-8). The tool dupRadar was used for assessment of the distribution of RPK values per target, relation of duplication rates to target abundance (RPK), and generating histograms and 2D density scatter plots (Sayols et al., dupRadar: a Bioconductor package for the assessment of PCR artifacts in RNA-Seq data. BMC Bioinformatics. 2016 Oct 21; 17(1):428).

[0152] Nanopore Sequencing. Oxford Nanopore sequencing (OXFORD NANOPORE TECHNOLOGIES®, Oxford, United Kingdom) is described, for example, in Lu et al. (Oxford Nanopore MinlON Sequencing and Genome Assembly. Genomics Proteomics Bioinformatics.2016 Oct; 14(5):265-279).EXAMPLE 6 (Prophetic Example)

[0153] Adapter concentrations for optimal read balancing can be determined empirically. Probe sets and adapters are designed for 100 targets. In a preliminary assay, the same concentration of adapter is added for each target. The resulting UNAMs are quantified by NGS. More than 98% of the reads are generated from Targets 1 and 2. The other 2% of the reads are for Targets 3-50, and Targets 51-100 are not detected. In a second assay, the concentrations of adapters for Targets 1 and 2 are reduced 100-fold wdthout changing the concentrations of other adapters. The number of reads for Targets I and 2 is reduced to 20%, number of reads for Targets 3-50 is increased 10-fold, Targets 51-74 are detected, and Targets 75-100 are still not detected. In the second assay, 50% of the reads are from Targets 3-10. In a third assay, the concentrations of adapters for Targets 1 and 2 are reduced an additional 10-fold, the concentrations of adapters for Targets 3-10 are reduced 10-fold, and the concentrations of adapters for Targets 11-100 remain the same. In the third assay, the number of reads for Targets1-10 is reduced to 20%, the number of reads for Targets 11-74 is increased an additional 10-fold, and Targets 75-100 are detected with a quantifiable percentage of reads.

[0154] In a second experiment, Probe sets and adapters are designed for 100 targets. In a preliminary assay, the same concentration of adapter is added for each target. The resulting UNAMs are quantified by qPCR. A UNAM is detected for every target, but the percentage of UNzXMs per target varies over 1,000,000-fold. Targets are assigned to three UNAM signal abundance groups: high (1-10% of targets), middle (4-20% of targets), and low (70-95% of targets). In the second assay, the concentration of adapters is reduced 100-fold for high UN AM targets and 10-fold for middle abundance targets, with no change to the concentration of adapters for low abundance targets. In the second assay, the percentage of UNAMs per target varies by less than 1000-fold as determined by qPCR or NGS, and the percentage of UNAMs for every target is quantifiable by NGS.(00155] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety'.

[0156] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method for increasing efficiency of next-generation sequencing (NGS) detection of one or more lower-abundance targets within a sample comprising a plurality of targets, the plurality of targets comprising lower-abundance targets and higher-abundance targets, the method comprising the steps of:a) providing:i) a plurality of sets of probes, each set comprising at least two probes for a target, wherein each probe binds specifically to the target and comprises an oligonucleotide tag comprising a unique identifier sequence associated with the target, andii) a plurality of adapters, each adapter being specifi c for an individual target or group of targets, wherein each adapter can specifically interact with a complex comprising one set of probes bound to their target,b) contacting the sample with:i) the plurality of sets of probes, thereby allowing the plurality of targets and the plurality of sets of probes to in teract to form a plurali ty of target-probe complexes, andii) the plurality of adapters, thereby allowing the plurality of adapters and the plurality of target-probe complexes to interact to form a plurality of target-probe-adapter complexes, wherein the plurality of adapters comprises a relatively low concentration of adapters specific for at least one higher-abundance target compared to the concentration of adapters specific for lower-abundance targets,c) performing an enzymatic reaction that connects probes of a set of probes directly or indirectly together, thereby producing a unified nucleic acid marker (UNAM) from each of the plurality of target-probe-adapter complexes, thereby producing a plurality of UNAMs, andd) detecting the plurality of UNAMs by parallel sequencing.

2. The method of claim 1, whereina) a first oligonucleotide tag and a second oligonucleotide tag of a set of probes that binds specifically to a target each comprise a double-stranded nucleic acid with an overhang,b) an adapter specific for the indivi dual target or group of targets i s an insert comprising a double-stranded nucleic acid with a first overhang on a first end and a second overhang on a second end,c) the first overhang of the adapter comprises a nucleotide sequence complementary to a nucleotide sequence of the overhang of the first oligonucleotide tag, d) the second overhang of the adapter comprises a nucleotide sequence complementary to a nucleotide sequence of the overhang of the second oligonucleotide tag, ande the enzymatic reaction is mediated by a ligase.

3. The method of claim 1, wherein:a) a first oligonucleotide tag and a second oligonucleotide tag of a set of probes that binds specifically to a target each comprise a nucleic acid with a single-stranded end, andb) an adapter specific for the individual target or group of targets comprises a linker comprising a nucleotide sequence complementary to the end of the first oligonucleotide tag and a nucleotide sequence complementary to the end of the second oligonucleotide tag, andc) the enzymatic reaction is mediated by a polymerase.

4. The method of claim 1, wherein:a) a first oligonucleotide tag and a second oligonucleotide tag of a set of probes that binds specifically to a target each comprise a nucleic acid with a single-stranded end,b) an adapter specific for the individual target or group of targets is a splint comprising a sequence complementary to a sequence of the first oligonucleotide tag and a sequence complementary to a sequence of the second oligonucleotide tag, andc) the enzymatic reaction is mediated by a ligase.

5. The method of claim 1, wherein:a) a first oligonucleotide tag and a second oligonucleotide tag of a set of probes that binds specifically to a target each comprise a nucleic acid with a single-stranded end,b) an adapter specific for the individual target or group of targets comprises a linker hybridized to a splint,c) a first end of the splint comprises a nucleotide sequence complementary to a nucleotide sequence of the overhang of the first oligonucleotide tag, d) a second end of the splint comprises a nucleotide sequence complementary to a nucleotide sequence of the overhang of the second oligonucleotide lag, and e) the enzymatic reaction is mediated by a ligase.

6. The method of claim 1, wherein:a) a first oligonuc leotide tag and a second oligonucleotide tag of a set of probes that binds specifically to a target each comprise a nucleic acid with a single-stranded end,b) the target comprises nucleic acid comprising a binding region,c) the single stranded end of the first oligonucleotide tag comprises a sequence complementary' to a first portion of the binding region of the target nucleic acid, d) the single stranded end of the second oligonucleotide tag comprises a sequence complementary to a second portion of the binding region of the target nucleic acid,e) the first and second portions of the binding region are separated by a gap, f) an adapter specific for the individual target or group of t argets is a linker comprising a nucleic acid sequence complementary to a nucleotide sequence of the gap, andg) the enzymatic reaction is mediated by a ligase.

7. The method of claim 6, wherein a single nucleic acid comprises the first probe and the second probe, and the UNAM produced from a target-probe-adapter complex comprising the two probes is circular.

8. The method of any one of claims 1 -7, wherein a target of the plurality of targets comprises a nucleic acid, a first probe for the target comprises a nucleic acid sequence complementary to a nucleotide sequence of a first portion of the target, and a second probe for the target comprises a nucleic acid sequence complementary' to a nucleotide sequence of a second portion of the target.

9. The method of any one of claims 1-5, wherein a target of the plurality' of targets comprises a protein and a probe that binds specifically to the target comprises an antibody, and antigen-binding fragment of an antibody, an aptamer, or a peptide.

10. The method of any one of claims 1-9, wherein each adapter of the plurality of adapters is specific for an individual target.

11. The method of any one of claims 1-10, wherein an oligonucleotide tag of at least one probe of the plurality of sets of probes comprises a locked nucleic acid base.

12. The method of any one of claims 1-11, wherein the method further comprises a step of amplifying the plurality of UNAMs before detecting the plurality of UN AMs by parallel sequencing.

13. The method of any one of claims 1-12, wherein a higher-abundance target is 5-fold to 500- fold, 500-fold - 50,000-fold, 50,000-fold - 1,000,000-fold higher in concentration than a lotver-abundance target.

14. The method of any one of claims 1-13, further comprising a step of enriching a lower- abundance target before or after the contacting.

15. The method of any one of claims 1-14, wherein:a) the targets are classified as the at least one higher-abundance target or the lower- abundance targets and the concentration of adapters for the at least one higher- abundance target is reduced by at least 50%, 90%, 95% or 98% compared to the concentration of adapters for the lower-abundance targets; orb) the targets are classified as the at least one higher-abundance target, mediumabundance targets, or lower-abundance targets, the concentration of adapters for the at least one higher-abundance target is reduced by at least 95%, 98% or 99% compared to the concentration of adapters for the lower-abundance targets, and the concentration of adapters for the medium abundance targets is reduced by' at least 50% or 90% compared to the concentration of adapters for the lower- abundance targets.

16. The method of claim 15, wherein the abundances of the targets are classified according to the percentages of UNAMs per target from a method identical to the method of claim 1 except wherein the plurality of adapters comprises identical or more similar concentrations of adapters for each of the plurality of targets.

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