Methods and kits designed for high-throughput testing of nucleic acid-containing samples with a high dynamic range.

Abstract

This application describes compositions, methods, and kits for the simultaneous multiplex detection and quantification of nucleic acid molecules. In particular, compressed PCR is disclosed for compressing the dynamic range of highly complex biological samples and improving the accuracy and efficiency of high-throughput sequencing analysis.

Description

[Technical Field]

[0001] Reference to related applications This application claims priority rights to U.S. Provisional Application No. 63 / 506,995 (filed on June 8, 2023). The entire contents of this document are incorporated herein by reference.

[0002] Statement on research funded by the federal government This invention was made possible with government support under grant number GM140211 from the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence listing reference This application includes an electronically submitted sequence listing XML, which is incorporated herein by reference in its entirety. The sequence listing XML was created on 7 June 2024, is named RICEP0139WO_ST26.xml, and is 40,755 bytes in size.

[0004] 1. Field This application relates in general to compositions, methods, kits, and apparatus for nucleic acid sequence amplification, and more specifically to compositions, methods, kits, and apparatus for detecting and / or quantifying polynucleotide sequences. [Background technology]

[0005] 2. Explanation of related technologies Nucleic acid samples with high or very high dynamic range can present problems with next-generation sequencing (NGS) or other quantification methods. Dynamic range refers to the concentration difference between the most abundant and least abundant molecular species in a sample. High dynamic range means that the level of a particular molecule is significantly higher than that of others, which can present challenges to accurate sequencing and data analysis. In NGS, DNA or RNA is fragmented and sequenced, and then the sequence reads are aligned to a reference genome or transcriptome. However, the accuracy and completeness of the analysis are limited because a small fraction of high-abundance molecular species make up the majority of the sequencing reads, while the majority of low-abundance species share only a small fraction of the sequencing reads in the sequencing data.

[0006] High dynamic range samples may also require deeper sequencing depth and coverage to accurately capture the entire range of expression levels. This can result in higher sequencing costs, as more reads are needed to ensure the data are reliable and representative of the sample. Furthermore, high dynamic range samples may also require more extensive data analysis (e.g., normalization and statistical modeling) to account for differences in expression levels and identify meaningful patterns and associations.

[0007] Therefore, there is a strong need for technologies that can mitigate the challenges of high dynamic range, improve the accuracy and efficiency of NGS analysis, ultimately save costs, and enable more comprehensive and meaningful insights into biological quantification (e.g., gene expression profiling and cell type analysis). [Overview of the Initiative]

[0008] In one embodiment, the present disclosure provides a method for sequencing a plurality of target sequences in a nucleic acid mixture sample, comprising: (a) (i) subjecting the nucleic acid mixture sample to an asymmetric PCR reaction in a multipleplex format for the plurality of target sequences, and (ii) for each individual target sequence, using an excess primer and a restriction primer, each containing at least a sequence portion capable of binding to a separate strand of the individual target sequence, wherein the concentration of the excess primer is at least 5 times higher than that of the restriction primer; and (b) preparing a high-throughput sequencing or hybridization library from nucleic acid products amplified from the asymmetric PCR reaction.

[0009] In another aspect, the Disclosure provides a method for sequencing multiple target sequences in a nucleic acid mixture sample, comprising: (a) subjecting the nucleic acid mixture sample to an asymmetric PCR reaction in a multipleplex format for multiple target sequences, wherein the asymmetric PCR reaction involves three primers for each target sequence: (i) a forward primer, (ii) a reverse primer, and (iii) an excess primer, where both the forward and reverse primers are restriction primers, and the excess primer is shared among all or part of the target sequences and is capable of binding to an amplicon generated by the forward and reverse primers; and (b) preparing a high-throughput sequencing or hybridization library from nucleic acid products amplified from the asymmetric PCR reaction.

[0010] In a further embodiment, the disclosure provides a kit comprising a panel of primer pairs, each pair comprising an excess primer and a restriction primer in a predetermined stock concentration configuration for asymmetric PCR amplification from a desired target amplicon.

[0011] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only. This is because various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description.

Brief Description of the Drawings

[0012] The following drawings form part of this specification and are included to further illustrate certain aspects of the present invention. The present invention can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

[0013] [Figure 1A] Provide a first typical schematic diagram of the workflow of multiplexed compression PCR (cPCR). In this typical workflow, for each target polynucleotide, the same excess primer is involved in both stage 1 (exponential amplification stage) and stage 2 (linear amplification stage). P_E1 (multiplexed to P_Ek) and P_L1 (multiplexed to P_Lk) represent lists of excess primers and limiting primers, respectively. T_1 (multiplexed to T_k) represents the amplified target sequence, which can be either ssDNA or dsDNA, and ssDNA is shown as an example. Pr_1 (multiplexed to Pr_k) is a Taqman probe used for reading the RT-PCR signal and is not required for a general cPCR reaction aimed at sequencing reads. The procedure (before library preparation) is performed in two separate reactions. Rxn1 is an asymmetric reaction using many primer pairs (and probes for RT-PCR, but not required for a sequencing-based workflow), and Rxn2 is a single-cycle primer extension reaction that converts the ssDNA product from Rxn1 to dsDNA. [Figure 1B]This paper compares the exponential amplification stage (conventional PCR) with the linear amplification stage, and shows the combination of the two stages in cPCR. [Figure 1C] This diagram shows a schematic of a two-step reaction in cPCR that occurs when the concentrations of the two primers are different. A typical schematic shows that the restriction primer (PL) is exhausted after the first exponential step, and a long linear amplification proceeds with only the excess primer (PE) remaining. [Figure 1D] The graph shows the expected results for conventional PCR and real-time monitored cPCR, along with the compressed dynamic range due to the linear amplification step that converts the original relative abundance of nucleic acid species to its logarithm. Ct is the number of cycles required to generate a detectable signal beyond the background. [Figure 1E] Further typical schematic diagrams of PCR versus cPCR are provided. These comparisons are shown through amplification curves and product versus input plots, demonstrating that only cPCR can achieve effective dynamic range compression. Here, we show that cPCR enables more accurate quantification of low-abundance genes. In the cPCR conversion formula, [PL], [T], and [Amp] represent the concentrations of the restriction primer, target, and cPCR product, respectively. [Figure 1F]A second typical schematic diagram of the multiplex compressed PCR (cPCR) workflow is provided. In this typical workflow, for each target polynucleotide, different primers are involved in Stage 1 (exponential amplification stage) and Stage 2 (linear amplification stage). Here, Stage 1 involves two primers for each target polynucleotide (PF1 and PR1 ~ PFk and PRk), each of which contains a target-specific region (PEi and PLi, respectively) and a common region (PE and PC, respectively). Stage 2 then performs linear amplification of all target polynucleotides using the common excess primer PE. PFA and PRA represent primers used to generate a high-throughput sequencing library via primer extension reactions. Various variations of PFA and PRA are conceivable, some of which may contain barcodes (PF-BC and PR-BC) and others may contain nested primers (PFInti and PRInti). In all cases, when using an Illumina sequencer, different combinations of Illumina sequencing primers (PF-Seq and PR-Seq) should be considered. [Figure 1G] This provides a typical cPCR-based single-cell sequencing workflow for measuring 3' gene fragment-based expression. In this example, both barcode (BC) and UMI (Universal Molecular Identifier) ​​are intended. Similar sequencing primer combinations (PF-Seq and PR-Seq) are intended, as in Figure 1D. [Figure 1H] This provides another typical cPCR-based single-cell sequencing workflow for measuring 5' gene fragment-based expression. [Figure 1I] It provides a more typical cPCR-based single-cell sequencing workflow for transcriptomics. It can be started before cDNA amplification and / or cleanup, or it can start from a GEM mixture. [Figure 1J]A schematic diagram and simulated effects of pseudo-excess primers are provided. Two typical strategies are shown here: one uses direct pseudo-primers (subpanel b, corresponding to Figure 1A), and the other applies pseudo-primers to an extended common primer sequence (subpanel c, corresponding to Figure 1D). Both strategies can reduce the product concentration relative to the primer concentration, and as shown in the figure, can reduce the total product concentration when scaling up the cPCR reaction to a high level of multiplexing (reductions of 85% and 86% are achieved based on simulations).

[0014] [Figure 2] Figure 2A shows qPCR amplification, and the two-step reaction exhibited by cPCR is confirmed by tracing. Figure 2B demonstrates that cPCR exhibits a large log-linear dynamic range (5log).

[0015] [Figure 3] This shows the real-time amplification trace of a 4-target multiplex cPCR. Each color indicates a different fluorescence channel.

[0016] [Figure 4] This figure shows the real-time PCR fluorescence signal as a function of input target concentration in a four-target multiplex cPCR. The horizontal axis applies only to the first two targets. A log-linear relationship was observed between signal amplitude and input target concentration.

[0017] [Figure 5] This study demonstrates that in four-target multiplex cPCR, the next-generation sequencing read count appears as a function of the input target concentration. The horizontal axis applies only to the first two targets. A log-linear relationship was observed between the normalized read count and the input target concentration.

[0018] [Figure 6]This shows real-time amplification traces of a 4-target multiplex cPCR. Each color represents a different dilution series sample.

[0019] [Figure 7] This study demonstrates that in four-target multiplex cPCR, the endpoint RT-PCR fluorescence signal appears as a log-linear function of the input target concentration. Linear signals were observed at R² = 0.997 and 0.987 in the low concentration range (5-log dynamic range).

[0020] [Figure 8] This study demonstrates that in four-target multiplex cPCR, the normalized read count from next-generation sequencing appears as a log-linear function of the input target concentration. A linear signal was observed in the low-concentration range, with R² = 0.988 and 0.92.f.

[0021] [Figure 9] This shows a 4-target multiplex cPCR test with varying concentrations for #1 and #2, and fixed concentrations for #3 and #4, along with qPCR amplification traces.

[0022] [Figure 10] This study shows a 4-target multiplex cPCR test with varying concentrations for #1 and #2, and fixed concentrations for #3 and #4, demonstrating highly linear qPCR endpoint signals.

[0023] [Figure 11] This study demonstrates that in 8-target multiplex cPCR, the read count (normalized) from next-generation sequencing appears as a log-linear function of the input target concentration. A linear signal was observed in the range of 10 pM to 100 aM (i.e., a 5-log dynamic range).

[0024] [Figure 12]This study demonstrates that in 8-target multiplex cPCR, the next-generation sequencing read count (normalized) appears as a log-linear function of the input target concentration. Under appropriately tuned experimental conditions, a linear signal was observed in the range of 10 pM to 100 aM (i.e., a 5-log dynamic range).

[0025] [Figure 13] Figure 13A shows that in 96-target multiplex cPCR, the next-generation sequencing read count (normalized) appears as a log-linear function of the input target concentration. A linear signal was observed for 76% (73 out of 96) of the targets in the range of 10 fM to 100 aM. The upper panel of Figure 13B shows the distribution of R2 values ​​from linear fits for all targets when forward primers were used as overprimers (PE=PF) or when reverse primers were used as overprimers (PE=PR). The lower panel of Figure 13B shows the distribution of better R2 values ​​(of the two choices) for all targets.

[0026] [Figure 14A] Figures 14A–14B show preliminary data for a highly multiplexed (96-target) cPCR test with high linearity and primer success rate. In Figure 14A, the cPCR test on serially diluted samples shows high linearity (R2 > 0.95 for 75% of primer pairs). [Figure 14B] Figure 14B shows partial accuracy (+ / -25%) using RNA-seq.

[0027] [Figure 15]Figures 15A–15B show that in a 30-target multiplex cPCR applied to a total human RNA sample, the next-generation sequencing read count (normalized) appears as a function of the input target concentration. Figure 15A shows the next-generation sequencing read count against the predicted value after performing a linear fit. Figure 15B shows that the difference in next-generation sequencing reads between two cPCR experiments exhibits excellent linearity with respect to the gene expression difference reported by RNA-seq observed over a 50,000-fold dynamic range (expression difference) in two human cell line samples (HeLa and Jurkat).

[0028] [Figure 16] Compared to a standard 10X single-cell dataset, cPCR demonstrates high barcode coverage.

[0029] [Figure 17A] Typical results from a multiplex single-cell compressed sequencing test (using PCR-validated primers) performed on hPBMC samples using a panel of 60 gene targets designed in three groups (high, medium, and low abundance) across a dynamic range of >4log, as reported in the 10X single-cell profiling test. [Figure 17B] cPCR sequencing shows nearly uniform read assignment across 60 gene targets compared to the 10X Genomics 3' mRNA assay, indicating that low-abundance genes are enriched 100-5,000 times despite the underlying broad dynamic range. [Figure 17C] cPCR sequencing showed a 100-5,000-fold improvement in reads compared to 10X Genomics3' for low abundances, achieving an overall dynamic range reduction of approximately 1,000-fold. [Figure 17D]In mutually detected single cells, cPCR detected significantly more mapped reads (reads mapped to the same 3' terminal mRNA position) compared to 10X sequencing, particularly for low-abundance genes. [Figure 17E] In mutually detected single cells, particularly for low-abundance genes, the map reads show that they cover up to 20 times more of the UMI molecule and 2 to 10 times more of the cell barcodes expressing this gene.

[0030] [Figure 18] This method exhibits 3–5 times higher barcode coverage compared to standard 10x sequencing datasets using cPCR for multiplex single-cell sequencing.

[0031] [Figure 19] This paper describes a typical design and experimental workflow for single-cell sequencing using cPCR.

[0032] [Figure 20A] The sequencing reads exhibit a relatively uniform distribution across a panel of 60 selected targets with a wide dynamic range (>3 × 10⁴). Roughly speaking, the abundance of target molecules (i.e., number of molecules or "#moles") decreases from target 1 to target 60, while the number of cPCR-seq (indicated as "lsPCR") reads (per 50k) remains relatively stable. [Figure 20B] This shows that cPCR assigned approximately 10 times fewer sequencing reads per gene to the most abundant gene group (right-hand group), 10 to 100 times more reads to genes with medium to low abundance (center group), and up to approximately 1000 times more reads to genes with the lowest abundance (leftmost group). [Figure 20C] This study demonstrates that cPCR can detect more moderately to low-abundance gene targets compared to standard 10X analysis. [Figure 20D]For each of the moderately to low-abundance gene targets, cPCR detected 10 to 100 times more reads compared to standard 10X analysis. [Figure 20E] For each of the moderately to low-abundance gene targets, cPCR detected 10 to 100 times more single cells expressing the target gene. [Modes for carrying out the invention]

[0033] Detailed explanation Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains.

[0034] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated as being incorporated by reference in whole.

[0035] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless the context clearly indicates otherwise. For example, “element” means at least one element, but may include multiple elements.

[0036] Where a range of values ​​is indicated, it is understood that each intermediate value between the upper and lower limits of that range, and any other indicated or intermediate values ​​within that indicated range, are included in this disclosure. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also included in this disclosure, subject to any particularly excluded restrictions within the indicated range. If the indicated range includes one or both of the limit values, the range excluding one or both of the included limit values ​​is also included in this disclosure.

[0037] When a grouping of options is presented, every possible combination of the members constituting that group is specifically envisioned. For example, if an item is selected from a group consisting of A, B, C, and D, the inventor not only specifically envisions each option individually (e.g., A only, B only, etc.), but also envisions combinations (e.g., A, B, and D, A and C, B and C, etc.).

[0038] When the term "and / or" is used in a list of two or more items, it means any one of the listed items, either alone or in combination with one or more of the other listed items. For example, the expression "A and / or B" is intended to mean either A or B, or both (i.e., A only, B only, or a combination of A and B). The expression "A, B, and / or C" is intended to mean A only, B only, C only, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0039] I. General Definition As used herein, the term “substantially” means, when used to alter quality, that generally allows for some degree of variation without loss of quality. For example, in certain embodiments, the degree of such variation could be less than 0.1%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, 1-2%, 2-3%, 3-4%, 4-5%, or more than 5% or 10%.

[0040] The terms “about,” “approximately,” or “nearly” refer to any variation in quantity that may occur when changing the quantity of a substance or composition (e.g., mg) or the value of a parameter characterizing a step in a method, for example, through general measurement, handling, and sampling procedures associated with the preparation, characterization, and / or use of a substance or composition, through careless errors in these procedures, through differences in the manufacture, source, or purity of the components used in the production or use of a composition or in the execution of a procedure. In certain embodiments, “about” may mean a variation of ±0.1%, ±0.5%, ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%.

[0041] As used herein, the term "dynamic range" refers to the concentration difference between the most abundant and least abundant molecular species in a nucleic acid-containing sample.

[0042] The dynamic range of nucleic acid samples is 10 6 The dynamic range can vary significantly, by 1 or more. Compressing the dynamic range of nucleic acid samples can be crucial for improving the sensitivity and accuracy of sequencing results in high-throughput sequencing applications. Various techniques have attempted to address the dynamic range problem of nucleic acid samples through targeted analysis. These include emulsion PCR, hybridization-based methods (e.g., molecular inversion probes (MIP) and capture probes), size or species selection (e.g., rRNA removal or poly(A) selection), and digital PCR. None of these methods are satisfactory, and are particularly unsatisfactory in compressing the dynamic range of samples in multiplex or high-throughput settings.

[0043] As used herein, “DNA” means deoxyribonucleic acid. DNA can be single-stranded or double-stranded. DNA typically contains four nucleotides: cytosine (C), guanine (G), adenine (A), and thymine (T). In one embodiment, the sequences of DNA molecules also provided herein contain one or more degenerate nucleotides. As used herein, “degenerate nucleotide” means a nucleotide that can perform the same function or produce the same output as structurally different nucleotides. Non-limiting examples of degenerate nucleotides include: C, G, or T nucleotide (B), A, G, or T nucleotide (D), A, C, or T nucleotide (H), G or T nucleotide (K), A or C nucleotide (M), any nucleotide (N), A or G nucleotide (R), G or C nucleotide (S), A, C, or G nucleotide (V), A or T nucleotide (W), and C or T nucleotide (Y).

[0044] II. Asymmetric PCR Asymmetric PCR is a variation of conventional PCR techniques that amplifies one strand of a double-stranded DNA template to produce a single-stranded DNA product. In conventional PCR, nearly equal amounts of forward and reverse primers are used, and amplification proceeds exponentially on both strands of the DNA template. In contrast, in asymmetric PCR, one primer is used at a lower concentration than the other. As a result, when the lower concentration primer is depleted, the exponential amplification stops, and linear amplification continues, with only one primer extending to produce a single-stranded product. See also: Gyllensten and Erlich, Proc. Natl. Acad. Sci. (USA) 85:7652-7656 (1988), and U.S. Patent No. 5,066,584.

[0045] As an improvement over asymmetric PCR, known as exponential post-linear PCR (LATE-PCR) (see US7,632,642 B2, US7,972,786 B2, US9,476,092 B2), maintains reaction efficiency by using restriction primers with higher melting temperatures than excess primers. This is because lower restriction primer concentrations result in higher primer melting temperatures (Tm). See also: Sanchez et al. (2004) Proc. Natl. Acad. Sci. (USA) 101:1933-1938, and also US20060057611A1.

[0046] However, neither asymmetric PCR nor late-PCR has ever been used as a quantitative assay method, nor has it ever been used in combination with high-throughput sequencing. Furthermore, both can lead to the formation of nonspecific amplification products, which should be minimized.

[0047] This specification provides methods and compositions for multiplex asymmetric PCR reactions that effectively reduce the dynamic range of highly complex nucleic acid samples and enable multiplex quantitative measurement of nucleic acid abundances in the samples. This specification also provides methods and compositions for improved primer designs and optimized reaction conditions that reduce nonspecific amplification products and enable such multiplex quantitative compression and reading.

[0048] III. Compressed PCR As used herein, “compressed PCR” or “cPCR” refers to a process for processing complex nucleic acid samples containing many target sequences (typically up to hundreds, thousands, or tens of thousands) and exhibiting a wide dynamic range (typically exceeding 100:1 (i.e., 2log) and up to 6–8log) by compressing the dynamic range through a multiplex asymmetric PCR reaction that performs a logarithmic transformation on the abundance of target sequences, thereby enabling efficient and economical detection and quantification of these targets downstream. The process of sequencing by high-throughput sequencing following cPCR processing is specifically referred to herein as cPCR-seq (also referred to herein as “lsPCR”).

[0049] Typically, asymmetric PCR conditions for cPCR are designed so that the exponential step of the reaction terminates before the linear step is reached. In some embodiments, this can be achieved by utilizing the limiting concentration of at least one of the primers underlying the exponential step (referred to as the "limiting primer"). Once the limiting primer is consumed by the exponential step, the synthesis of the double-stranded amplicon terminates. In some embodiments, the amount of the limiting primer can be adjusted so that a selected number of exponential amplicons are produced. For example, one of the primers may be diluted 5- to 100-fold so that it is present in a limited amount of 1-20 percent of the concentration of the excess primer.

[0050] Unlike conventional PCR, which involves the exponential increase of PCR products, compressed PCR (cPCR) exhibits a two-step amplification model. For example, in this model, after the first exponential step is completed, the primer with the lower initial concentration ("restriction primer," or P) is used. L ) is depleted, and the primer with the higher initial concentration ("excess primer", or P EOnly the ) remains, and the reaction enters the second linear amplification step, producing single-stranded DNA as the final product. Although not bound by any scientific theory, the transition point represents a logarithmic transformation of the initial target concentration (essentially the Ct value for the RT-PCR test). On the other hand, the final concentration of the linear amplified product (ssDNA product) is linearly related to the number of additional cycles after the transition point. Taken together, the final concentration of the ssDNA product is essentially a logarithmic transformation of the original target concentration, and this reaction operates in an unbiased, autonomous manner, enabling effective dynamic range compression of the original sample mixture.

[0051] cPCR offers advantages over conventional PCR-based targeted amplification for high-throughput sequencing or hybridization-based quantification when performing multiplex assays (e.g., gene expression analysis). For example, in conventional PCR, amplification primers are used at non-limiting concentrations. Therefore, if the dynamic range of the target sequence is wide (e.g., 4–8 log), highly expressed genes compete with less expressed genes for the PCR reagent. As a result, a small fraction of highly abundant genes may occupy the majority of the PCR amplicon, while most of the less abundant genes share only a small portion of the PCR amplicon and may not be properly detected during sequencing. In some embodiments, this is avoided by compressed PCR, which generates a similar number of double-stranded amplicons for each target sequence using limiting concentrations of at least one primer, and then performs linear amplification. Thus, overexpression of highly expressed genes is minimized because the exponential amplification steps are converted to linear amplification. Consequently, the dynamic range of the amplicons is compressed compared to the original sample, avoiding the problems of overexpression and underexpression in conventional PCR. Therefore, cPCR improves both the sensitivity and accuracy of rare sequence detection.

[0052] This specification discloses compositions and methods for cPCR. In one embodiment, a method for sequencing multiple target sequences in a nucleic acid mixture sample is disclosed. The method comprises (a) subjecting a nucleic acid mixture sample to an asymmetric PCR reaction using excess primers and restriction primers, each containing at least a sequence portion capable of binding to a separate strand of an individual target sequence in a multiplex format for the multiple target sequences; and (b) preparing a high-throughput sequencing or hybridization library from nucleic acid products amplified from the asymmetric PCR reaction.

[0053] In another embodiment, a method for sequencing multiple target sequences in a nucleic acid mixture sample is disclosed. The method includes (a) subjecting a nucleic acid mixture sample to an asymmetric PCR reaction in a multipleplex format for multiple target sequences, wherein the asymmetric PCR reaction involves three primers for each target sequence: (i) a forward primer, (ii) a reverse primer, and (iii) a common excess primer, where both the forward and reverse primers are restriction primers, and the common excess primer is shared among all or part of the target sequences and is capable of binding to the amplicons generated by the forward and reverse primers; and (b) preparing a high-throughput sequencing or hybridization library from nucleic acid products amplified from the asymmetric PCR reaction.

[0054] In another embodiment, the cPCR method disclosed herein further includes a step of converting single-stranded nucleic acids generated from an asymmetric PCR reaction into double-stranded nucleic acids.

[0055] In a further embodiment, the method disclosed herein further includes (c) obtaining sequencing read data from a high-throughput sequencing library, wherein the sequencing read data (i) reflects the transformed abundances of a plurality of target sequences in a nucleic acid mixture sample and (ii) exhibits a compressed dynamic range relative to the nucleic acid mixture sample; and (d) determining the original abundances of the plurality of target sequences in the nucleic acid mixture sample based on the transformed abundances.

[0056] In another embodiment, the cPCR method further includes the step of normalizing sequencing read data or abundance readings by a normalization coefficient determined based on at least one of the following four embodiments: (i) sequencing yield, i.e., the ratio between the number of sequencing reads and the cPCR product concentration (in one embodiment, this is done by spiking in a reference amplicon having a known sequence at the end of the cPCR reaction); (ii) sequence-specific amplification efficiency (in one embodiment, this is done by spiking in an internal calibration standard with the sample before initiating the cPCR reaction, or by pre-measuring and tabulating it); (iii) functional form of the cPCR transformation, i.e., the slope of the log-linear fit (in one embodiment, this is done by assuming (by the value of the formula), or by pre-measuring and recording it, or by spiking in an internal calibration standard); and (iv) linearity correction of the cPCR transformation. In one embodiment, this is done by using a pre-measuring transformation value or a spiked-in internal standard.

[0057] In one embodiment, in the method disclosed herein, the excess primer is at a higher concentration than the restriction primer, and the chain targeted by the excess primer is preferentially amplified over the chain targeted by the restriction primer.

[0058] In one embodiment, the length of the interval between the locations of two distinct primers, or the length of the interval between a primer location and a poly(A) site location, can vary. Herein, “interval” means the number of nucleotides between two approximate locations. Therefore, the term “interval” is used herein to define the length of the distance between the location of one primer and the location of another primer or a point or genomic location. Generally, for any given poly(A) site, there may be a specific point or genomic location following the primer where poly(A) is expected to begin. In one embodiment, this point or genomic location is more clearly defined. In another embodiment, this point or genomic location has, for example, a window of 5, 10, 20, 50, or 100 nucleotides.

[0059] In one embodiment, the distance between either the restriction primer location or the excess primer location and the poly(A) site location is at least 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 40, 30, or 20 nucleotides in length. In one embodiment, the distance between either the restriction primer location or the excess primer location and the poly(A) site location is at most 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 40, 30, or 20 nucleotides in length.

[0060] In one embodiment, the interval between either the restriction primer position or the excess primer position and the poly(A) site is 500-450, 500-400, 500-350, 500-300, 500-250, 500-200, 500-150, 500-100, 500-50, or 500-0 nucleotides in length. In one embodiment, the interval between either the restriction primer position or the excess primer position and the poly(A) site is 500-400, 450-350, 400-300, 350-250, 300-200, 250-150, 200-100, 150-50, or 100-0 nucleotides in length. In one embodiment, the interval between either the restriction primer position or the excess primer position and the poly(A) site is 500-20, 400-20, 300-20, 200-20, 150-20, 100-20, 90-20, 80-20, 70-20, 60-20, 50-20, 40-20, or 30-20 nucleotides in length.

[0061] In one embodiment, the interval between the restriction primer position and the excess primer position, or between the forward primer position and the reverse primer position, is at least 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 40, 30, or 20 nucleotides in length. In one embodiment, the interval between the restriction primer position and the excess primer position, or between the forward primer position and the reverse primer position, is at most 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 40, 30, or 20 nucleotides in length.

[0062] In one embodiment, the interval between the restriction primer position and the excess primer position, or between the forward primer position and the reverse primer position, is 500-450, 500-400, 500-350, 500-300, 500-250, 500-200, 500-150, 500-100, 500-50, or 500-0 nucleotides in length. In one embodiment, the interval between the restriction primer position and the excess primer position, or between the forward primer position and the reverse primer position, is 500-400, 450-350, 400-300, 350-250, 300-200, 250-150, 200-100, 150-50, or 100-0 nucleotides in length. In one embodiment, the interval between the restriction primer position and the excess primer position, or between the forward primer position and the reverse primer position, is 500-20, 400-20, 300-20, 200-20, 150-20, 100-20, 90-20, 80-20, 70-20, 60-20, 50-20, 40-20, or 30-20 nucleotides in length.

[0063] In one embodiment, the method disclosed herein further includes detecting nucleic acid products amplified from an asymmetric PCR reaction using sequencing, hybridization, microarray, digital PCR, or quantitative PCR.

[0064] In one embodiment, the method disclosed herein is for determining multiple target sequences in a nucleic acid mixture sample that include an original dynamic range of at least 50:1, 100:1, 1000:1, 10000:1, or 100000:1. In another embodiment, the multiple target sequences in a nucleic acid mixture sample include an original dynamic range of 50:1–100:1, 50:1–1000:1, 50:1–10000:1, 100:1–500:1, 500:1–1000:1, 500:1–5000:1, 500:1–10000:1, 1000:1–5000:1, or 1000:1–10000:1. The dynamic range of the nucleic acid sample may result from copy number variation and / or gene expression difference levels of a particular target gene.

[0065] In one embodiment, the method disclosed herein logarithmically transforms the original relative abundance into a compressed relative abundance. In another embodiment, the original abundance is determined based on the transformed abundance and the concentration of the excess primer used in the asymmetric PCR reaction.

[0066] In one embodiment, the compressed dynamic range is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 10:1, 25:1, 50:1, 100:1, or 250:1 or less. In another embodiment, the compressed dynamic range is 2:1-3:1, 2:1-4:1, 2:1-5:1, 5:1-10:1, 5:1-15:1, 5:1-20:1, 10:1-50:1, 10:1-100:1, 50:1-100:1, 50:1-250:1, or 100:1-250:1.

[0067] In one embodiment, the cPCR method disclosed herein is a multiplex format of at least 10plex, 20plex, 50plex, 100plex, 200plex, 300plex, 400plex, 500plex, 750plex, 1000plex, 2000plex, 5000plex, 10000plex, 20000plex, 50000plex, or 100000plex. In another context, the multiplex formats are 500plex to 1000plex, 500plex to 2000plex, 500plex to 3000plex, 500plex to 4000plex, 500plex to 5000plex, 500plex to 10000plex, 1000plex to 2000plex, 2000plex to 4000plex, 2000plex to 6000plex, 2000plex to 8000plex, 5000plex to 8000plex, 5000plex to 10000plex, 10000plex to 20000plex, 20000plex to 50000plex, and 50000plex to 100000plex.

[0068] In one embodiment, a cPCR reaction or cPCR kit provides a single-tube reaction. As used herein, a “single-tube” method means a sequence of at least two operations (e.g., sample preparation, amplification, or sequencing) that can be performed without transferring the sample from one container (a test tube, reaction well, chamber in a microfluidic device, glass slide, or any other device capable of holding the reaction mixture) to another container. In one embodiment, a cPCR reaction or cPCR kit requires a reaction in multiple tubes, with optional processing steps between them.

[0069] In one embodiment, cPCR can be used to compare or quantify polymorphic variants at a specific gene locus.

[0070] Many factors are considered to optimize cPCR conditions (e.g., the quantity of each target nucleic acid sequence, the relative amount of each target nucleic acid sequence, the number of different target nucleic acid sequences to be amplified in a single reaction, and the desired degree of precision). Further factors to consider include, for example, sequence design, the type and amount of polymerase used, and the polymerase buffer.

[0071] In one embodiment, the cPCR reaction includes a series of temperature cycles comprising a denaturation step, an annealing step, and an extension step. In another embodiment, the cPCR reaction includes a series of temperature cycles comprising a denaturation step and a step combining annealing and extension. In another embodiment, the annealing step is performed at approximately 55°C. In yet another embodiment, the annealing step is at 35–65°C, 40–60°C, 45–55°C, 40–65°C, 45–60°C, 50–60°C, 50–55°C, or 55–60°C.

[0072] In one embodiment, the elongation step is approximately 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 30, 40, 60, 75, 90, 105, or 120 minutes. In another embodiment, the elongation step is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 30, 40, 60, 75, 90, 105, or 120 minutes. In yet another embodiment, the elongation step is at most 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 30, 40, 60, 75, 90, 105, or 120 minutes.

[0073] In one embodiment, the annealing step is approximately 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 30, 40, 60, 75, 90, 105, or 120 minutes. In another embodiment, the annealing step is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 30, 40, 60, 75, 90, 105, or 120 minutes. In yet another embodiment, the annealing step is at most 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 30, 40, 60, 75, 90, 105, or 120 minutes.

[0074] In one embodiment, the combined annealing / stretching step is approximately 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 30, 40, 60, 75, 90, 105, or 120 minutes. In another embodiment, the combined annealing / stretching step is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 30, 40, 60, 75, 90, 105, or 120 minutes. In another embodiment, the annealing / extension combined step may be up to 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 30, 40, 60, 75, 90, 105, or 120 minutes.

[0075] In a further embodiment, the extension step is 0.5-20, 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 15-20, 1-18, 2-16, 3-14, 4-12, 5-10, 6-9, 7-8, 4-6, 6-8, 8-10, 9-12, or 10-12 minutes.

[0076] In one embodiment, the cPCR reaction comprises at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 cycles. In another embodiment, the cPCR reaction comprises about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 cycles. In a further embodiment, the cPCR reaction includes 15-90, 20-80, 30-70, 40-60, 25-90, 30-90, 40-90, 50-90, 60-90, 70-90, 25-80, 25-70, 25-60, 25-50, 25-40, 15-30, 25-50, or 35-50 cycles, or 45-60 cycles.

[0077] IV. Primers The primers of this disclosure, or primers useful in the methods and kits of this disclosure, are oligonucleotides in the broad sense, meaning that they may be DNA, RNA, or a mixture of DNA and RNA, and may contain non-natural nucleotides (e.g., 2'o-methylribonucleotides) and non-natural nucleotide bonds (e.g., phosphorothioate bonds). The primers function partially by hybridizing to a target sequence in the reaction mixture. In one embodiment, the primer is a single-stranded oligonucleotide that hybridizes to its complementary sequence at the primer annealing temperature of the amplification reaction and can be extended at its 3' end by DNA polymerase. In another embodiment, the primers of this disclosure may be primers that signal the hybridization of their priming sequence by an indirectly excitable phosphor.

[0078] In one embodiment, the primers of the present disclosure are short oligonucleotides generally less than 50 nucleotides in length that hybridize to a target chain and are extended by a suitable polymerase. The primers are generally linear oligonucleotides but may include secondary structures. Amplification often involves using one or more primer pairs, each consisting of a forward primer and a reverse primer. In some embodiments, in the methods, kits, and oligonucleotide sets according to the present disclosure, one or both primers of a pair may be labeled with a covalently bound phosphor that fluoresces when stimulated by a nearby fluorescent DNA dye. Using these primers, the synthesis of the product resulting from DNA polymerase extension (e.g., obtained from PCR and primer extension assays) can be monitored in real time or by endpoint detection, and / or product specificity can be evaluated by melting curve analysis. In one embodiment, the primers comprise one or more degenerate nucleotides.

[0079] Primers may be target sequence specific or designed to hybridize to sequences adjacent to the target sequence to be amplified. Therefore, the actual nucleotide sequence of each primer may depend on the target sequence and target polynucleotide, which is evident to those skilled in the art. Methods for designing primers suitable for amplifying a desired target sequence are known. See: Dieffenbach, CW and Dveksler, GS (Eds.) (2003) PCR Primer: A Laboratory Manual (2nd edition) Cold Spring Harbor Laboratory Press.

[0080] In one embodiment, one or more primer design principles are considered, essentially (i) designing a primer pool to minimize 3' terminal sequence interactions, and (ii) P during the design and optimization process. L (iii)P E and P LThe design includes using different Tm / binding energies.

[0081] Generally, the primer needs to be long enough to prime template - directed synthesis in the conditioning of the cPCR reaction. The exact length of the primer can depend on many factors (e.g., without limitation, the desired hybridization temperature between the primer and the template polynucleotide, the complexity of the different target polynucleotide sequences to be amplified, salt concentration, ionic strength, pH and other buffer conditions, and the sequences of the primer and the template). In one embodiment, the primer contains from about 15 to about 35 nucleotides, which is suitable for hybridizing to the target sequence and forms a substrate suitable for DNA synthesis, although the primer can contain more or fewer nucleotides. Generally, the shorter the primer, the lower the temperature required to form a sufficiently stable hybrid complex with the target sequence. The annealing ability of a polynucleotide can be determined by the melting temperature ( "T m m") of the hybrid complex. T m m is the temperature at which 50% of the polynucleotide strand and its perfect complement form a double - stranded polynucleotide. Thus, T m m for a selected polynucleotide varies depending on factors that affect or are affected by hybridization. In one embodiment where thermal cycling is performed, the amplification primer can be designed such that the melting temperature ( "T m m") is in the range of about 60 - 75 °C. When the melting temperature is in this range, there is a tendency to ensure that the primer remains annealed or hybridized to the target polynucleotide at the start of primer extension. The actual temperature used in the primer extension reaction can depend on other factors, among others, for example, the concentration of the primer used in the cPCR reaction. When amplification is performed using a thermostable polymerase (e.g., Taq DNA polymerase), in a typical embodiment, the amplification primer has a T mThe melting temperature can be designed to be in the range of approximately 60 to 78°C or approximately 55 to 70°C. The melting temperatures of different amplification primers may differ, but in alternative embodiments, they should all be approximately the same, i.e., the T of each amplification primer. m The temperature can be within a range of approximately 5°C or lower. Various primers m This can be determined empirically using melting techniques well known in the relevant field. Alternatively, the T of the primer can be determined. m It is possible to calculate the T of the primer. m Numerous references and aids for calculating primer T are available in the art. For example, without limitation, the following are listed: Bresslauer et al., 1986, Proc. Natl. Acad. Sci. USA 83: 8893-8897; Freier et al., 1986, Proc. Natl. Acad. Sci. USA 83: 9373-9377; Rychliket al., 1990, Nucleic Acids Res. 18: 6409-6412. Any of these methods can be used with primer T. m This can be used to determine the annealing ability of polynucleotides. In another embodiment, the annealing ability of polynucleotides can be determined by the binding free energy (ΔG) of the hybrid complex.

[0082] In one embodiment, only one excess primer is used for a single target nucleic acid sequence. In another embodiment, multiple excess primers are used for a single target nucleic acid sequence. In a further embodiment, one excess primer is used for multiple target sequences (for example, using a poly-T based primer to analyze an mRNA sample). In one embodiment, the excess primer or restriction primer is designed to be substantially complementary to the region of the target polynucleotide. In this specification, “substantially complementary” means that the primer sequence has sufficient complementarity to hybridize to the target polynucleotide and be extended by DNA polymerase under the concentrations, temperatures, and conditions used in the cPCR amplification reaction, but is not perfectly complementary.

[0083] In one embodiment, the cPCR reaction also uses one or more pseudo-excess primers along with their corresponding excess primers. A “pseudo-primer” is a modified primer that shares the same or substantially the same underlying target-binding sequence and contains additional modifications (e.g., a polymerase inhibitor at the 3' end). Typical polymerase inhibitors include non-extendable chemical modifications (e.g., inverted dT bases, carbon linkers, other non-extendable chemical groups, or non-complementary or non-specific sequences) (details can be found in U.S. Patent No. 11,208,676). The pseudo-excess primer competes with its corresponding normal excess primer for target binding but does not allow polymerase extension. Therefore, the use of pseudo-excess primers reduces the concentration of the cPCR product and contributes to a high level of multiplexing.

[0084] In one embodiment, a primer in cPCR may be perfectly complementary to the target polynucleotide. In another embodiment, it may be desirable to include one or more mismatched or non-complementary nucleotides in the primer. "Mismatched region" and "non-complementary" mean that at least one nucleotide in a polynucleotide sequence is not suitable for base pairing with another polynucleotide sequence. Thus, the term "mismatched region" is used when comparing sequences (e.g., primer sequence and target sequence, probe sequence and target sequence, primer sequence and amplicon sequence, etc.). In some embodiments, a primer sequence that is a mismatched region compared to the target sequence is substantially unique to that primer. In other embodiments, a primer sequence that is a mismatched region compared to the target sequence is also present in other primers or probes. Thus, in some embodiments, the mismatched region between the primer and the target sequence is a coding sequence. "Coded sequence" means a primer sequence of consecutive nucleotides that is substantially non-complementary to the target sequence but substantially unique to that primer. "Substantially unique" means that the sequence is suitable for identifying or distinguishing the primer and its amplification product from other primers and other amplification products. Primers and methods for amplifying sequences to include such coding sequences are known in the art (see, for example, U.S. Patents 6,090,552 and 6,355,431).

[0085] In some embodiments, the mismatch region between the primer and the target sequence is a sequence shared by multiple primer sequences. In various typical embodiments, the “shared sequence” may be common to each forward primer, each reverse primer, each excess primer, or each restriction primer.

[0086] Therefore, a “common over-primer” refers to a sequence of nucleotide primers that do not directly bind to the target sequence but are shared by one or more (or each) forward or reverse primers in a cPCR reaction. In one embodiment, multiple over-primers are used for multiple target sequences, and each over-primer has an extension that can be further amplified by a combined common over-primer. In another embodiment, a large number of different common over-primers are used, for example, each common over-primer corresponds to about 100 (which can be any number from 2 to 10,000) target sequences.

[0087] Therefore, "restriction primer" and "excess primer" can also mean "forward primer" and "reverse primer," which indicate a restriction primer and excess primer having a directional orientation.

[0088] Determining the number, type, length, and composition of mismatch regions, their positions within primers or probes, and their distribution or commonality among nucleic acids in a cPCR reaction is within the scope of a person skilled in the art. Generally, mismatch regions are designed to perform a user-selected function when incorporated into exponential or linear amplicons. In one embodiment, these mismatch regions function as excess primer binding sites. In another embodiment, the incorporated sequences provide sites useful for downstream hybridization or amplification reactions.

[0089] In one embodiment, the concentration of the excess primer is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or 500 times higher than the corresponding restriction primer. In another embodiment, the concentration of the excess primer is 2-5, 2-10, 5-10, 5-15, 10-16, 10-20, 15-30, 20-30, 25-50, 30-40, 30-50, 30-60, 30-70, 40-50, 40-60, 40-70, 40-80, 50-60, 50-100, 50-150, 70-200, or 100-200 times higher than the corresponding restriction primer.

[0090] In one embodiment, the concentration of the excess primer is 20-50, 30-60, 40-80, or 50-100 times higher than the corresponding limiting primer. In another embodiment, the concentration of the excess primer is approximately 1, 2, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nM. In yet another embodiment, the concentration of the excess primer is approximately 1, 2, 5, 10, 20, 30, 40, or 50 μM. In one embodiment, the concentration of the excess primer is at least 1, 2, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nM. In another embodiment, the concentration of the excess primer is at least 1, 2, 5, 10, 20, 30, 40, or 50 μM. In one embodiment, each of the excess primer concentrations or concentration ranges (or concentration multipliers relative to the limiting primer) referred to in this application (including this paragraph and all preceding and following paragraphs) represents the total concentration of both the excess primer and its corresponding pseudo-excess primer(s).

[0091] In another embodiment, the concentrations of excess primer are 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 100-1000, 150-1000, 250-1000, 350-1000, 450-1000, and 55 The ranges are 0-1000, 650-1000, 750-1000, 850-1000, 100-900, 150-800, 250-700, 350-600, 450-500, 150-250, 250-350, 350-450, 450-550, 550-650, 650-750, or 750-850 nM.

[0092] In another embodiment, the concentration of the excess primer is 0.1-0.5, 0.1-0.8, 0.1-1, 0.5-1, 1-5, 5-10, 10-15, 15-25, 25-35, 35-45, 45-55, 55-65, 65-75, 75-85, 95-105, 105-125, 125-155, 155-175, 175-195, or 195-225 nM.

[0093] In a further embodiment, the concentrations of the amplicon-specific excess primer are 1-1000, 1-750, 1-500, 1-250, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, The ranges are 5-20, 10-95, 15-85, 25-75, 35-65, 45-55, 15-100, 25-100, 35-100, 45-100, 55-100, 65-100, 75-100, 85-100, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000, 600-1000, 700-1000, 800-1000, and 900-1000 nM.

[0094] In a further embodiment, the concentration of the excess primer or common excess primer is 0.1–0.25, 0.25–0.5, 0.5–1, 1–1.5, 1.5–2.5, 2.5–3.5, 3.5–4.5, 4.5–5.5, 1–5, 5–10, 10–15, 15–25, 25–35, 35–45, or 45–55 μM.

[0095] In a further embodiment, the concentration of the excess primer or common excess primer is 0.1–1, 0.25–1, 0.5–5, 1–5, 1.5–5, 2.5–5, 3.5–5, 4.5–15, 1–15, 5–15, 10–25, 15–35, 25–45, or 35–55 μM.

[0096] In a further embodiment, the concentration of the excess primer or common excess primer is 0.1–5, 0.25–5, 0.75–5, 1–15, 1.5–15, 2.5–15, 3.5–15, 4.5–25, 10–25, 15–25, 10–35, 15–45, 25–55, or 15–55 μM.

[0097] In one embodiment, the concentration of the limiting primer is approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 20, 30, 40, or 50, 60, 70, 80, 90, or 100 nM. In another embodiment, the concentration of the limiting primer is at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nM.

[0098] In one embodiment, the concentrations of the limiting primers are 0.01-0.05, 0.01-0.1, 0.01-0.5, 0.01-1, 0.01-2.5, 0.01-5, 0.01-10, 0.01-20, 0.01-30, 0.01-40, 0.01-50, 0.01-60, 0.01-70, 0.01-80, 0.0 The ranges are 1-90, 0.01-100, 0.1-1, 0.1-2.5, 0.1-5, 0.1-10, 0.1-20, 0.1-50, 0.1-100, 1-2.5, 1-5, 1-10, 1-20, 1-50, 1-100, 2.5-5, 2.5-10, 2.5-20, 2.5-50, or 2.5-100 nM.

[0099] In one embodiment, the concentrations of the limiting primers are 0.01-45, 0.1-40, 0.5-35, 1-30, 2.5-25, 5-20, 7.5-17.5, 10-15, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-100, 15-100, and 25. These ranges are ~100, 35~100, 45~100, 55~100, 65~100, 75~100, 85~100, 10~90, 15~80, 25~70, 35~60, 45~50, 15~25, 25~35, 35~45, 45~55, 55~65, 65~75, 75~85, 85~95, or 95~100 nM.

[0100] In one embodiment, the concentration of the limiting primer is 2.5-25, 5-20, 7.5-17.5, 10-15, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-100, 15-100, 25-100, 35-100, 45-100, 55-100, 65-100, 75-100, 85-100, 10-90, 15-80, 25-70, 35-60, 45-50, 15-25, 25-35, 35-45, 45-55, 55-65, 65-75, 75-85, 85-95, or 95-100 nM.

[0101] In one embodiment, the concentration difference between the excess primer and the restriction primer is optimized for each target sequence.

[0102] In one embodiment, the excess primer and the restriction primer have different melting temperatures, and the annealing temperature used in the cPCR reaction is optimized for the excess primer. In another embodiment, the annealing temperature used in the cPCR reaction is optimized for both the excess primer and the restriction primer. In a further embodiment, the optimization of the cPCR conditions takes into account a lower concentration(s) of the restriction primer(s) that results in a different effective ΔG(or Tm) compared to normal PCR.

[0103] V. Target sequence As disclosed herein, cPCR is suitable for various types of nucleic acid samples and target sequences (e.g., genomic DNA, mRNA, cDNA, microRNA, chromatin immunoprecipitation samples, and bisulfite-treated DNA). In one embodiment, the nucleic acid sample and target sequence are essentially DNA (e.g., cDNA, genomic DNA, or extrachromosomal DNA) or RNA (e.g., mRNA, rRNA, or genomic RNA). The target nucleic acid can be derived or obtained from substantially any sample or source, and the sample may be deficient or limited in quantity as needed. For example, the sample may be one or a few cells taken from a crime scene, or a small amount of tissue taken by biopsy. In one embodiment, the target nucleic acid sample is derived from circulating tumor DNA (ctDNA). In another embodiment, the target nucleic acid may be a synthetic polynucleotide containing nucleotide analogs or mimetic compounds manufactured for purposes such as diagnosis, testing, or therapy.

[0104] In one embodiment, cPCR is used in a range of gene expression panels for various cancers (e.g., panels for breast cancer, lung cancer, colorectal cancer, liver cancer, kidney cancer, skin cancer, ovarian cancer, and prostate cancer). In another embodiment, cPCR is used in a range of gene expression panels for various immunological and / or neurological disorders. In one embodiment, nucleic acid mixture samples for cPCR are barcoded for their cellular origin. In another embodiment, high-throughput sequencing libraries of cPCR are barcoded by unique molecular identifiers (UMIs).

[0105] In one embodiment, the nucleic acid products amplified from an asymmetric PCR reaction have different lengths for each individual target sequence. In another embodiment, the nucleic acid products amplified from an asymmetric PCR reaction have substantially similar amplicon lengths. In one embodiment, the nucleic acid products amplified from an asymmetric PCR reaction have amplicon lengths with variability of 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less than 5%. In another embodiment, the nucleic acid products amplified from an asymmetric PCR reaction have at least 600, 500, 400, 300, 200, 100, 50, 40, 30, or 20 nucleotide lengths. In one embodiment, the nucleic acid products amplified from an asymmetric PCR reaction have a maximum length of 600, 500, 400, 300, 200, 100, 50, 40, 30, or 20 nucleotides.

[0106] In another embodiment, the nucleic acid product amplified from the asymmetric PCR reaction has a length of 600-500, 600-400, 600-300, 600-200, 600-100, 600-50, 600-40, 600-30, or 600-20 nucleotides. In one embodiment, the nucleic acid product amplified from the asymmetric PCR reaction has a length of 600-500, 500-400, 400-300, 300-200, 200-100, 100-50, 100-40, 100-30, or 100-20 nucleotides. In another embodiment, the nucleic acid product amplified from the asymmetric PCR reaction has a length of 500-20, 400-20, 300-20, 200-20, 150-20, 100-20, 90-20, 80-20, 70-20, 60-20, 50-20, 40-20, or 30-20 nucleotides.

[0107] VI. Polymerase As used herein, "DNA polymerase" refers to an enzyme capable of catalyzing the synthesis of DNA molecules from nucleoside triphosphates. DNA polymerase adds nucleotides to the 3' end of a DNA strand, one at a time, creating an antiparallel DNA strand compared to a template DNA strand. DNA polymerase cannot initiate a new DNA molecule from scratch; a primer capable of adding the first new nucleotide is required.

[0108] Compressed PCR can be performed using various different DNA polymerases (e.g., Taq, Phusion, Pfu, Q5, AccuPrime Taq, KOD, or Vent polymerase). In some embodiments, the DNA polymerase also has 5'-3' endonuclease activity. In some embodiments, the DNA polymerase is a thermostable polymerase. In some embodiments, the DNA polymerase has 5'-3' nuclease activity. Non-limiting examples of polymerases having 5'-3' nuclease activity include, but are not limited to, the following: AmpliTaq® DNA polymerase, Ampli-Taq® GOLD polymerase and Tth polymerase (Applied Biosystems, Foster City, Calif.), E. coli DNA polymerase I (New England Biolabs, Beverly, Mass.), rBst DNA polymerase (Epicenter®, Madison, Wis.), and Tfl DNA polymerase (Promega Corp., Madison, Wis.). Furthermore, cPCR amplification reactions can be carried out using a variety of different reverse transcriptases, but in some embodiments, thermostable reverse transcriptases are preferred. Suitable thermostable reverse transcriptases include, but are not limited to, AMV reverse transcriptase, MuLV, and Tth reverse transcriptase. Suitable temperatures for carrying out various denaturation, annealing, and primer extension reactions using polymerases and reverse transcriptases are well known in the art. Optional reagents commonly used in conventional PCR and RT-PCR amplification reactions (e.g., reagents designed to enhance PCR, modify Tm, or reduce primer dimer formation) can also be used in log-linear amplification reactions.

[0109] In one embodiment, the DNA polymerase is a heat-resistant DNA polymerase. As used herein, “heat-resistant DNA polymerase” refers to a DNA polymerase that can function at high temperatures (e.g., above 65°C) and survive at even higher temperatures (e.g., up to about 100°C). Heat-resistant DNA polymerases often exhibit maximum catalytic activity at temperatures between 70°C and 80°C. In one embodiment, the heat-resistant DNA polymerase is selected from the group consisting of Taq DNA polymerase, Phusion® DNA polymerase, Q5® DNA polymerase, and KAPA High Fidelity DNA polymerase.

[0110] In one embodiment, the DNA polymerase is a non-heat-stable DNA polymerase. As used herein, "non-heat-stable DNA polymerase" refers to a DNA polymerase that cannot function at high temperatures. In one embodiment, the non-heat-stable DNA polymerase is selected from the group consisting of phi29 DNA polymerase and Bst DNA polymerase.

[0111] VII. Preparation of NGS Libraries In one embodiment, the method includes high-throughput sequencing. In one embodiment, the method includes subjecting multiple amplicons to high-throughput sequencing. As used herein, “high-throughput sequencing” refers to any sequencing method that can sequence multiple (e.g., tens, hundreds, thousands, millions, hundreds of millions, tens of billions) DNA molecules in parallel. In one embodiment, Sanger sequencing is not high-throughput sequencing. In one embodiment, high-throughput DNA sequencing includes sequencing bisynthesis or nanopore-based sequencing. In one embodiment, high-throughput sequencing includes the use of a sequencing bisynthesis (SBS) flow cell. In one embodiment, the SBS flow cell is selected from the group consisting of Illumina SBS flow cells and Pacific Biosciences (PacBio) SBS flow cells. In one embodiment, high-throughput sequencing is performed by current measurement in combination with Oxford nanopores.

[0112] The typical preparation of a high-throughput sequencing library is a multi-step process that optionally includes sample preparation, fragmentation, end repair and A-tailing, adapter ligation, size selection and PCR amplification, and library quantification and quality control. In cPCR-seq, the ssDNA product from cPCR is first converted to dsDNA before the usual high-throughput sequencing library preparation workflow. In one embodiment, the preparation of a high-throughput sequencing library from nucleic acid products amplified from a cPCR reaction includes adapter ligation. In another embodiment, the appropriate adapter sequence is embedded (i) within an excess primer and / or restriction primer, or (ii) in a separate adapter sequence as part of the sequence library preparation workflow after the cPCR reaction.

[0113] Typically, high-throughput sequencing generates sequence files. As used herein, “sequence file” means a computer-readable text file containing the sequences of at least one next-generation sequencing (NGS) read. As used herein, “NGS read” means the nucleotide sequence of a single nucleic acid molecule generated by a high-throughput sequencing method. In one embodiment, the NGS read includes a UMI sequence. In one embodiment, the NGS read includes a cell barcode sequence. In one embodiment, the NGS read includes a gene sequence. In one embodiment, the NGS read includes both a UMI sequence and a gene sequence. In one embodiment, the NGS read includes at least 10 nucleotides. In one embodiment, the NGS read includes at least 25 nucleotides. In one embodiment, the NGS read includes at least 50 nucleotides. In one embodiment, the NGS read includes at least 100 nucleotides. In one embodiment, the NGS read includes at least 250 nucleotides. In one embodiment, the NGS read includes at least 500 nucleotides. In one embodiment, the NGS read includes at least 1000 nucleotides. In one embodiment, the NGS read contains 10 to 10,000 nucleotides. In another embodiment, the NGS read contains 10 to 1,000 nucleotides. In yet another embodiment, the NGS read contains 25 to 150 nucleotides.

[0114] In one embodiment, the cPCR NGS reads contain 600, 500, 400, 300, 200, 100, 50, 40, 30, or 20 nucleotides or less.

[0115] VIII. Kits and Reagents This disclosure also provides kits containing reagents necessary or important for performing cPCR. Furthermore, this specification provides a kit comprising a panel of primer pairs, each pair comprising an excess primer and a restriction primer in a predetermined stock concentration configuration for cPCR amplification from a desired target amplicon. In one embodiment, the panel comprises at least 10, 20, 50, 100, 200, 300, 400, 500, 750, 1000, 2000, 5000, 10000, 20000, or 100000 primer pairs. In one embodiment, the cPCR kit comprises a primer panel comprising one unique restriction primer for each target amplicon, while sharing excess primers across all or a subset of target amplicons. In another embodiment, the excess primer and restriction primer for each primer pair are packaged separately. In a further embodiment, the primer pairs are individually pre-mixed. In one embodiment, all restriction primers have similar concentrations, while in another embodiment, all restriction primers have different concentrations predetermined for each amplicon. In yet another embodiment, the cPCR kit further comprises one or more of the following: a set of dNTPs, polymerase, buffer, and a set of labeled probes.

[0116] As used herein, “kit” means an assembly of reagents for performing amplification or assay. A kit may be “complete,” that is, it may contain all the reagents necessary for all steps of amplification or amplification detection. Alternatively, a kit may be “partial,” and may omit certain reagents necessary for those operations. Both complete and partial kits of this disclosure may further include reagents for sample preparation (e.g., nucleic acid separation and reverse transcription). Sequencing may involve two kits (e.g., a complete cPCR amplification kit and a complete sequencing library preparation kit), or these two may be combined into a single kit.

[0117] In one embodiment, the present disclosure provides reagents and buffers necessary for cPCR. Non-limiting examples of necessary reagents and buffers include Tris-HCl, potassium chloride, magnesium chloride, oligonucleotide primers, deoxyribonucleotides (dNTPs), DNA polymerase, betaine, and dimethyl sulfoxide. As those skilled in the art will see, different DNA polymerases and different target sequences may require different groups of desired reagents and buffers.

[0118] IX. Typical Application Examples The compositions and methods disclosed herein are suitable for detecting gene copy number and / or chromosome copy number in multiplex reactions. Furthermore, the methods, assays, and kits described herein can be applied to the identification, diagnosis, and monitoring of disorders (e.g., cancer, developmental and degenerative diseases, neurological disorders, and stem cell diseases, without limitation).

[0119] As will be readily apparent to those skilled in the art, other suitable modifications and adaptations of the devices, systems, and methods described herein can be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. While specific embodiments have been described in detail so far, they will be better understood by referring to the following examples, which are included solely for illustrative purposes and are not intended to be limiting. All patents, patent applications, and references mentioned herein are incorporated by reference in their entirety for all purposes. [Examples]

[0120] X. Example The following examples are included to illustrate preferred embodiments of the present invention. As those skilled in the art will see, the techniques disclosed in the following examples represent techniques found by the inventors to function well in the implementation of the present invention and can therefore be considered to constitute preferred forms for implementation. However, as those skilled in the art will see from the consideration of this disclosure, many modifications can be made to the specific embodiments disclosed, and similar or comparable results can still be obtained without departing from the spirit and scope of the present invention.

[0121] Example 1 - cPCR workflow strategy and expected results Figure 1A shows a typical workflow example of compressed PCR. Figure 1B shows a comparison between the exponential amplification stage (conventional PCR) and the linear amplification stage, and combinations of the two stages in cPCR. Figure 1C shows a schematic diagram of the two-step reaction in cPCR that occurs when the concentrations of both primers are different. A typical schematic diagram shows restriction primers (P L ) is used up after the first exponential step, and excess primer (P E This shows that long-term linear amplification proceeds in the presence of only [P]. Figure 1D shows a graph of the expected results of conventional PCR and cPCR monitored in real time, and the compressed dynamic range due to the linear amplification step that converts the original relative abundance of the nucleic acid species to its logarithm. Figure 1E shows a further typical schematic of PCR vs cPCR. These comparisons are shown through amplification curves and product vs input plots, showing that only cPCR can achieve effective dynamic range compression. Here, we show that cPCR enables more accurate quantification of low-abundance genes. In the cPCR conversion formula, [P L [T], and [Amp] indicate the concentrations of the restriction primer, target, and cPCR product, respectively.

[0122] Figure 1F shows a second typical schematic diagram of the multiplex compressed PCR (cPCR) workflow. In this typical workflow, different primers are involved in Stage 1 (exponential amplification stage) and Stage 2 (linear amplification stage) for each target polynucleotide. Here, Stage 1 involves two primers (P) for each target polynucleotide. F1 and P R1~ P Fk and P Rk ) accompanied by each of the two primers, each with a target-specific region (P Ei and P Li ) and the common area (each P E and P C ) contains the following. Next, Stage 2 is the common excess primer P E Linear amplification of all target polynucleotides is performed using P. FA and P RA This represents the primers used to generate a high-throughput sequencing library via primer extension reactions. Various variations of P FA and P RA This was planned, and among them was a barcode (P F-BC and P R-BC ) may also contain nested primers (P FInti and P RInti ) may also contain. In all cases, when using an Illumina sequencer, a combination of different versions of Illumina sequencing primers (P F-Seq and P R-Seq ) is planned.

[0123] Figure 1G shows a typical cPCR-based single-cell sequencing workflow for measuring 3' gene fragment-based expression. In this example, both barcode (BC) and UMI (Universal Molecular Identifier) ​​are intended. Similar to Figure 1D, a similar sequencing primer combination (P F-Seq and P R-Seq ) is planned.

[0124] Figure 1H shows another typical cPCR-based single-cell sequencing workflow for measuring 5' gene fragment-based expression. Figure 1I shows a further typical cPCR-based single-cell sequencing workflow for transcriptomics. It can be started before cDNA amplification and / or cleanup, or it can start from a GEM mixture.

[0125] Figure 1J shows a schematic diagram of pseudo-excess primers and their simulated effects. Two typical strategies are shown here: one uses direct pseudo-primers (subpanel b, corresponding to Figure 1A), and the other applies pseudo-primers to an extended common primer sequence (subpanel c, corresponding to Figure 1D). Both strategies can reduce the product concentration relative to the primer concentration, and as shown in the figure, can reduce the total product concentration when scaling up the cPCR reaction to a high level of multiplexing (reductions of 85% and 86% are achieved based on simulations).

[0126] Figure 2A shows qPCR amplification, and the trace confirms the two-step reaction shown by cPCR. Figure 2B shows that cPCR exhibits a large log-linear dynamic range (5log).

[0127] Example 2 - 4-target multiplex cPCR reading by real-time PCR Multiplex cPCR is tested by mixing four sets of target templates and primer strands in the same reaction, following a typical workflow for compressed PCR (Figure 1A). Each set has been pre-validated with individual cPCR tests. The amplicon, primer, and probe sequences for each target are shown in Table 1. Table 1: Sequences used in 4-plex cPCR tests TIFF2026519816000001.tif157170

[0128] In this experiment, all four pairs of primers were included at 5 nM for each restriction primer and 250 nM for each excess primer. Taqman probes for real-time detection were also included at 250 nM each. Template amplicons were added in the range of 10 aM to 1 pM. To test the dynamic range, sequence crosstalk, and signal linearity of the multiplex cPCR, the concentrations of two of the four targets were varied in a 10-fold dilution series from 1 pM to 10 aM, while the concentrations of the other two were kept constant at 10 fM. The cPCR reaction was performed using a 60-cycle thermal cycling program, with the annealing step set to 55°C and 12.5 minutes per cycle.

[0129] Real-time amplification performance is monitored using a 4-channel qPCR readout (one channel per target) on an RT-PCR instrument (Bio-Rad CFX Opus). Due to fluorescence crosstalk between different channels, the signal is corrected for background by manually adjusting the cycle limit. After subtracting the background, the expected two-stage amplification signal is observed for all samples in all channels, and all channels show an exponential-to-linear transition at similar signal levels (Figure 3). For the two targets with varying concentrations, parallel and equally spaced amplification signals are observed, as expected for the dilution series samples, suggesting linear amplification of each signal regardless of the presence of other targets. For the two fixed targets, overlapping signals are observed as expected. These signal intensities are extracted at cycle 30, and a logarithmically decreasing signal intensity as a function of input target concentration is observed (Figure 4).

[0130] During the later stages of amplification, a higher signal was observed, which is thought to be due to nonspecific primer binding. However, amplification resulting from such nonspecific binding is expected to have a different sequence from the amplicon on the target.

[0131] Example 3 - 4-target multiplex cPCR reading by next-generation sequencing The performance of endpoint cPCR is also evaluated by combining different samples from Example 2 with pooled next-generation sequencing. After the cPCR reaction, the samples are first treated with a double-stranded control amplicon and converted to double-stranded DNA by a single-cycle PCR (including all four restriction primers). The samples are then barcoded and pooled using the NEB index primer set (NEBNext Multiplex Oligos for Illumina) following a protocol that is somewhat adapted from the manufacturer's recommendations. Briefly, the converted double-stranded DNA samples are first purified using AmPure XP magnetic beads. Next, the purified samples are ligated using the NEB Universal Adapter, treated with USER enzyme after endoprep, and purified again using magnetic beads. Library PCR is then performed using NEB index primers and purified a third time using magnetic beads. Finally, the barcoded samples are normalized on Qubit and mixed for high-throughput sequencing on a MiSeq machine. After sequencing, the reads were locally aligned to a library containing all target amplicon sequences using bowtie2, and full-length sequence matches with low sequencing error (edit distance ≤ 2) were selected and counted. The read counts were then normalized relative to the control amplicon, and a target-specific normalization factor was used to account for target-specific amplification bias.

[0132] The final normalized sequencing reads, as expected, show a linear relationship with respect to the input target concentration (Figure 5).

[0133] Example 4 - 4-target multiplex cPCR with updated conditions and expanded dynamic range The same 4-plex cPCR templates and primers from Examples 2 and 3 are reused and repeated under improved test conditions. In particular, the ratio of excess primer concentration to restriction primer concentration is maintained by using higher excess primer concentrations (1.25 μM or 500 nM) along with higher restriction primer concentrations (25 nM or 10 nM). Shorter RT-PCR reactions (40 cycles) with shorter extension times per cycle (5 minutes) are pre-formed. A wider dynamic range of the original target concentrations is also tested. In particular, the concentrations of the first two targets are varied from 100 pM to 10 aM (e.g., 7 log) in a 10-fold dilution series, while the other two are kept constant at 100 fM.

[0134] Real-time fluorescence tracing on RT-PCR reveals two distinct stages of cPCR reaction amplification (exponential and linear) (Figure 6). No manual background subtraction was performed. A second stage of signal increase is also observed, presumably due to nonspecific primer binding, beginning at cycles 25–35 depending on the original target concentration (Figure 6). In the range of 1 pM–10 aM, good linearity is observed between the endpoint fluorescence signal level and the logarithm (original target concentration), for both targets respectively. 2 The values ​​are 0.997 and 0.987 (Figure 7).

[0135] Next, the reaction product is converted from ssDNA to dsDNA. After adding two double-stranded control amplicons, the library is prepared for sequencing following the same procedure as in Example 3. Briefly, the samples are ligated using the NEBNext Universal Adapter, barcoded using the NEBNext Multiplex Index Oligos, and pooled before sequencing on the MiSeq instrument. After sequencing, the reads are analyzed using the same procedure as in Example 3. Briefly, the raw reads are locally aligned using bowtie2, filtered by full-length sequence matching and an edit distance threshold (≤2), normalized by control amplicons, and manually scaled by a target-specific normalization factor.

[0136] The final normalized sequencing reads show a monotonic decreasing relationship with respect to the input target concentration, and a linear relationship in the low concentration range of 100 fM to 10 aM (Figure 8).

[0137] Four-target multiplex cPCR was performed with varying target concentrations for #1 and #2 (Figure 9), and with fixed concentrations for #3 and #4 (Figure 9). The qPCR amplification traces are shown in Figure 9. As shown in Figure 10, the endpoint signals exhibit high linearity.

[0138] Examples 5-8 Targeted Multiplex cPCR An 8-target multiplex cPCR is performed using primers custom-designed for human genome targets. Specifically, 96 SNPs in the human genome with significant mutant allele frequencies are selected. A custom primer pool design algorithm is used to minimize crosstalk between any primer pairs while maintaining a strict binding energy distribution. Table 2 shows the sequences of the targets and primers used. Synthetic DNA (IDT ultramer) is used as a template for testing. In the cPCR reaction, restriction primers are used at 10 nM each, and excess primers are used at 500 nM. 40 cycles of PCR thermal cycling are performed, with 5 minutes of annealing and extension per cycle at 55°C. Of the eight targets, the concentrations of the first four targets are varied from 10 pM to 10 aM (e.g., 6 log) in a 10-fold dilution series, while the other two targets are kept constant at 10 fM. After the first PCR reaction, the reaction product is converted from ssDNA to dsDNA after the addition of two double-stranded control amplicons. Prepare an NGS sequencing library, and data analysis will follow the same procedure as in the 4-plex cPCR experiment in Example 4. Table 2: Sequences used in 8-plex cPCR tests TIFF2026519816000002.tif197170TIFF2026519816000003.tif64170

[0139] A clear linear decrease relationship is observed between the normalized sequencing read count and the logarithm of the input target concentration over the target concentration range from 10 pM to 100 aM (i.e., a 5-log dynamic range) (Figure 11). According to the linear fit, for the four targets, R 2 The coefficients are 0.982, 0.871, 0.994, and 0.989, respectively. The lowest concentration (10 aM) was not clearly detected, which is likely due to the insufficient sensitivity of this test design. The other four targets with constant input concentrations show fixed sequencing readings.

[0140] Examples 6-8 Targeted Multiplex cPCR Another 8-target multiplex experiment was performed to compare different experimental conditions. In the cPCR reaction, restriction primers were used at 10 nM each, and excess primers at 500 nM. 40 cycles of PCR thermal cycling were performed, with 5 minutes of annealing and extension per cycle at 55°C. Different enzyme amounts (1:5 ratio) were used for comparison. Figure 12 shows that, only when the enzyme amounts were appropriately adjusted, the cPCR reaction showed a linear response with respect to the logarithm of the input concentration and a constant signal for targets kept at a fixed concentration.

[0141] Examples 7-96 Targeted Multiplex cPCR 96-target multiplex cPCR is performed using primers custom-designed for human genomic SNP targets with significant mutant allele frequencies. A custom primer pool design algorithm is used to minimize crosstalk between any primer pairs while maintaining a precise binding energy distribution. The test is performed using a human genomic DNA sample (sigma) across a dilution series from 10 fM to 0.1 fM. In the cPCR reaction, restriction primers are used at 5 nM each, and excess primers are used at 150 nM. 40 cycles of PCR thermal cycling are performed, with annealing and extension for 12.5 minutes per cycle at 55°C. After the first PCR reaction, two double-stranded control amplicons are added, and the reaction product is converted from ssDNA to dsDNA. An NGS sequencing library is prepared, and data analysis follows the same procedure as in Example 4. Figures 13A-B show that 76% (73 out of 96) of the targets showed a linear response (R) to the logarithm of the input gDNA concentration. 2 Figure 14A shows that the cPCR test on serially diluted samples exhibits high linearity (R for 75% of primer pairs). 2 This indicates that it has >0.95. Figure 14B shows partial accuracy (+ / -25%) using RNA-seq.

[0142] Example 8 - 30-target multiplex cPCR on total RNA sample Eight-target multiplex cPCR was performed using primers custom-designed for human transcriptome targets. In particular, 30 human mRNA targets were designed to span the full dynamic range of gene expression and, based on previously reported RNA-seq datasets, demonstrated significant gene expression differences between HeLa and Jurkat cell lines. Total RNA samples (BiChain) from HeLa and Jurkat were reverse transcribed using polydT primers and purified using magnetic beads before the cPCR reaction. In the cPCR reaction, restriction primers were used at 5 nM each, and excess primers at 250 nM. 40 cycles of PCR thermal cycling were performed, with annealing and extension for 12.5 minutes per cycle at 55°C. The cDNA samples were diluted to an effective abundance of approximately 100 cells. After the initial PCR reaction, two double-stranded control amplicons were added, and the reaction product was converted from ssDNA to dsDNA. An NGS sequencing library was prepared, and data analysis followed the same procedure as in Example 4.

[0143] Figure 15A shows that after cPCR and high-throughput sequencing, genes were detected across all 4 log gene expressions (from the reported RNA-seq dataset), although not all targets were detected, which may be due to sample variability. The data show good agreement with expected gene abundances after linear fitting and correction for sequence-specific amplification bias. Figure 15B further shows the linear correlation (R²=0.94) of gene expression differences compared to previous RNA-seq reports.

[0144] Example 9 - Single-cell multiplex cPCR sequencing using hPBMC samples Single-cell multiplex cPCR sequencing tests were performed on human peripheral blood mononuclear cell (hPBMC, Lonza) samples on a panel of 60 gene targets. hPBMC samples were prepared according to a standard 10X Genomics 3' mRNA profiling workflow (MD Anderson Advanced Technology Genomics Core) and analyzed using the cellranger pipeline (10X). Following sequencing library preparation using the 10X Genomics workflow, cPCR was performed on 121 gene targets using custom-designed primers. These targets were selected from 10X sequencing results and were 3 × 10⁶ 4 The dynamic range spanned a range of (or more, but limited by sequencing depth). PCR testing showed that 60 of the designed primers produced well-amplified sequences that closely matched the human genome reference database at the expected loci. Multiplex cPCR reactions were performed using 2 nM restriction primers and common primers (7.2 μM) that overlapped with the Illumina read 1 primers as excess primers for all targets (see Figure 1E). Sample preparation and high-throughput sequencing were performed as in Example 8. There were high, medium, and low abundance groups spanning a dynamic range of >4 log, as reported in 10X single-cell profiling tests (Figure 17A).

[0145] According to bulk sequencing analysis (i.e., without cell barcoding and UMI), within a set of 60 well-designed targets, cPCR showed a wider overall dynamic range (3 × 10) of the tested gene compared to the 10X Genomics 3' mRNA assay. 4The target read depth was nearly uniform across the range, and low-abundance genes were enriched 100 to 5,000 times (Figures 17B and 17C), achieving an overall dynamic range reduction of approximately 1,000 times. When normalized to the same total number of sequencing reads (50,000), cPCR assigned approximately 10 times fewer sequencing reads per gene to the most abundant gene group, 10 to 100 times more reads to medium to low-abundance genes, and up to approximately 1,000 times more reads to the lowest-abundance genes tested (Figures 20A and 20B). After inverse multiplexing of cell barcodes and UMI analysis, cPCR (total number of reads 74,000) detected 26,897 distinct cell barcodes. This represents >99% barcode coverage, with 4058 duplicates out of 4074 total cell barcodes detected by the 10X single-cell dataset (with 10,000,000 randomly subsampled reads). Specifically, 4015 of the detected barcodes showed at least five distinct reads, of which 3757 were common with the 10X dataset (92% coverage, Figure 16). In mutually detected single cells, cPCR detected significantly more mapped reads (reads mapped to the same 3' terminal mRNA position) compared to 10X sequencing, particularly for low-abundance genes (Figure 17D). Of the 4058 mutually detected single cells and 46 medium-to-low-abundance gene targets, cPCR detected up to >20 genes within single cells (mean 7.21, standard deviation 4.13). This is approximately four times higher compared to ≤5 genes (mean 1.83, standard deviation 1.45, Figure 20C) for a standard 10X analysis. Similarly, for low-abundance genes, particularly in mutually detected single cells, map reads covered up to 20 times more molecular UMIs and 2–10 times more cellular barcodes expressing these genes (Figure 17E). For each of the medium-to-low-abundance gene targets, cPCR detected 10–100 times more reads compared to the 10X dataset (Figure 20D) and 10–100 times more single cells expressing the target gene (Figure 20E).These results suggest that cPCR enables >100 times more effective sequencing depth compared to standard 10X single-cell 3' mRNA analysis, allowing for very deep molecular profiling of the transcriptome (more molecules, more cells expressing target genes) at single-cell resolution. Furthermore, cPCR has 3–5 times higher barcode coverage compared to standard 10X sequencing datasets for multiplex single-cell sequencing (Figure 18). Figure 19 shows a typical design and experimental workflow for single-cell sequencing using cPCR.

[0146] All methods disclosed and claimed herein can be prepared and performed without excessive experimentation in light of this disclosure. While the compositions and methods of the present invention are described based on preferred embodiments, as will be apparent to those skilled in the art, the methods described herein and the steps or order of steps thereof can be modified without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain chemically and physiologically relevant agents can be used in place of the agents described herein, and the same or similar results can be obtained. All similar substitutes and modifications that will be apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

Claims

1. A method for sequencing multiple target sequences in a nucleic acid mixture sample, a. The nucleic acid mixture sample, (i) For the multiple target sequences, in a multiplex format, (ii) For each individual target sequence, using an excess primer and a restriction primer, each containing at least a sequence portion capable of binding to a separate strand of the individual target sequence, A step of subjecting an asymmetric PCR reaction, wherein the concentration of the excess primer is at least five times higher than that of the restriction primer, b. A step of preparing a high-throughput sequencing or hybridization library from nucleic acid products amplified from the asymmetric PCR reaction. Methods that include...

2. The method according to claim 1, wherein the excess primer is shared among all or part of the target sequences and is capable of binding to a common primer binding site shared among all or part of the target sequences.

3. The method according to claim 2, wherein the concentration of the excess primer is determined to be at least five times higher for each target sequence.

4. A method for sequencing multiple target sequences in a nucleic acid mixture sample, a. A step of subjecting the nucleic acid mixture sample to an asymmetric PCR reaction in a multiplex format for the plurality of target sequences, wherein the asymmetric PCR reaction involves three primers for each target sequence: (i) a forward primer, (ii) a reverse primer, and (iii) an excess primer, wherein both the forward primer and the reverse primer are restriction primers, and the excess primer is shared among all or part of the target sequences and is capable of binding to the amplicons generated by the forward primer and the reverse primer. b. A step of preparing a high-throughput sequencing or hybridization library from nucleic acid products amplified from the asymmetric PCR reaction. Methods that include...

5. The method according to any one of claims 1 to 4, further comprising the step of converting single-stranded nucleic acids generated from the asymmetric PCR reaction into double-stranded nucleic acids.

6. The method according to any one of claims 1 to 5, wherein the concentration of the excess primer is at least 10 times higher than that of the limiting primer, and as a result, the chain targeted by the excess primer is preferentially amplified.

7. The method according to any one of claims 1 to 6, further comprising the step of detecting the nucleic acid product amplified from the asymmetric PCR reaction using a sequencing method, a hybridization method, a microarray method, or a quantitative PCR method.

8. The method according to any one of claims 1 to 7, wherein the preparation of the high-throughput sequencing or hybridization library includes a ligation reaction.

9. The method according to any one of claims 1 to 7, wherein the preparation of the high-throughput sequencing or hybridization library comprises a polymerase-based reaction.

10. c. A step of obtaining sequencing read data from the high-throughput sequencing library, wherein the sequencing read data (i) reflects the transformed abundance of the plurality of target sequences in the nucleic acid mixture sample, and (ii) exhibits a compressed dynamic range relative to the nucleic acid mixture sample. d. A step of determining the original abundance of the plurality of target sequences in the nucleic acid mixture sample based on the converted abundance. The method according to any one of claims 1 to 9, further comprising:

11. c. A step of obtaining abundance readings from the high-throughput hybridization library, wherein the abundance readings (i) reflect the transformed abundances of the plurality of target sequences in the nucleic acid mixture sample, and (ii) exhibit a compressed dynamic range relative to the nucleic acid mixture sample. d. A step of determining the original abundance of the plurality of target sequences in the nucleic acid mixture sample based on the converted abundance. The method according to any one of claims 1 to 9, further comprising:

12. The method according to any one of claims 1 to 11, wherein the plurality of target sequences in the nucleic acid mixture sample include an original dynamic range of at least 50:1, 100:1, 1000:1, 10000:1, or 100000:

1.

13. The method according to any one of claims 1 to 11, wherein the plurality of target sequences in the nucleic acid mixture sample include the original dynamic ranges of 50:1 to 100:1, 50:1 to 1000:1, 50:1 to 10000:1, 100:1 to 500:1, 500:1 to 1000:1, 500:1 to 5000:1, 500:1 to 10000:1, 1000:1 to 5000:1, or 1000:1 to 10000:

1.

14. The method according to any one of claims 11 to 13, wherein the original abundance is logarithmically transformed into the transformed abundance by an arbitrarily selected linearity correction.

15. The method according to any one of claims 11 to 13, wherein the original abundance is determined based on (i) the converted abundance and (ii) the concentration of the restriction primer used in the asymmetric PCR reaction.

16. The method according to any one of claims 11 to 13, wherein the compressed dynamic range is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 10:1, 25:1, 50:1, 100:1, or 250:1 or less.

17. The method according to any one of claims 11 to 13, wherein the compressed dynamic range is 2:1 to 3:1, 2:1 to 4:1, 2:1 to 5:1, 5:1 to 10:1, 5:1 to 15:1, 5:1 to 20:1, 10:1 to 50:1, 10:1 to 100:1, 50:1 to 100:1, 50:1 to 250:1, or 100:1 to 250:

1.

18. The method according to any one of claims 1 to 17, wherein the multiplex format is at least 10plex, 20plex, 50plex, 100plex, 200plex, 300plex, 400plex, 500plex, 750plex, 1000plex, 2000plex, 5000plex, 10000plex, 20000plex, 50000plex, or 100000plex.

19. The above multiplex formats include 500plex to 1000plex, 500plex to 2000plex, 500plex to 3000plex, 500plex to 4000plex, 500plex to 5000plex, 500plex to 10000plex, 1000plex to 2000plex, 2000plex to 4000plex, 2000plex to 6000plex, 2000plex to 8000plex, 5000plex to 8000plex, 5000plex to 10000plex, 5000plex to 20000plex, 5000plex to 30000plex, 5000plex to 40000plex, 5000plex to 50000plex, and 50 The method according to any one of claims 1 to 17, wherein the plexiplex range is 00 to 60000, 15000 to 20000, 15000 to 30000, 15000 to 40000, 7000 to 100000, 10000 to 100000, 15000 to 100000, 25000 to 100000, 35000 to 100000, 45000 to 100000, 55000 to 100000, 65000 to 100000, 75000 to 100000, or 85000 to 100000.

20. The method according to any one of claims 1 to 19, wherein the concentration of the excess primer is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or 500 times higher than that of the limiting primer.

21. The method according to any one of claims 1 to 19, wherein the concentration of the excess primer is 2 to 5, 2 to 10, 5 to 10, 5 to 15, 10 to 16, 10 to 20, 15 to 30, 20 to 30, 25 to 50, 30 to 70, 40 to 60, 40 to 70, 40 to 80, 50 to 100, 50 to 150, 70 to 200, or 100 to 200 times higher than that of the limiting primer.

22. The method according to any one of claims 1 to 19, wherein the concentration of the excess primer is about 20 to 50 times higher than that of the limiting primer.

23. The method according to any one of claims 1 to 19, wherein the concentration of the excess primer is (i) about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nM, or (ii) about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 μM.

24. The method according to any one of claims 1 to 19, wherein the concentration of the excess primer is (i) at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nM, or (ii) at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 μM.

25. The concentration of the excess primer is (i) 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 100-1000, 150-1000, 250-1000, 350-1000, 450-1000, 550-1000, 650-1000, 750-1000, 850-1000, 100-900, 150-800, 250-700, 350-600, 450-500, 150-250, 250-350, 350-450, 450- The method according to any one of claims 1 to 19, wherein the M6 ​​is 550, 550-650, 650-750, or 750-850 nM, or (ii) 2-50, 3-45, 4-40, 5-35, 6-30, 7-25, 8-20, 9-15, 3-50, 4-50, 5-50, 6-50, 7-50, 8-50, 9-50, 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, and 2-5 μM.

26. The method according to any one of claims 1 to 19, wherein the concentration of the limiting primer is about 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 20, 30, 40, or 50 nM.

27. The method according to any one of claims 1 to 19, wherein the concentration of the limiting primer is at least 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 20, 30, 40, or 50 nM.

28. The concentrations of the limiting primers are (i) 2.5-25, 5-20, 7.5-17.5, 10-15, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-100, 15-100, 25-100, 35-100, 45-100 , 55-100, 65-100, 75-100, 85-100, 10-90, 15-80, 25-70, 35-60, 45-50, 15-25, 25-35, 35-45, 45-55, 55-65, 65-75, or 75-85 nM, or (ii) 0.01-2, 0.02-2, 0. The method according to any one of claims 1 to 19, wherein the nM is 0.5-2, 0.1-2, 0.2-2, 0.3-2, 0.4-2, 0.5-2, 0.75-2, 1-2, 1.25-2, 1.5-2, 1.75-2, 0.02-1.75, 0.05-1.5, 0.1-1.25, 0.2-1, 0.3-0.75, 0.4-0.5, 0.02-1.5, 0.02-1.25, 0.02-1, 0.02-0.75, 0.02-0.5, 0.02-0.4, 0.02-0.3, 0.02-0.2, 0.02-0.1, 0.1-0.2, 0.2-0.3, 0.3-0.4, or 0.2-0.5 nM.

29. The method according to any one of claims 1 to 28, wherein the concentration difference between the excess primer and the limiting primer is optimized for each target sequence.

30. The method according to any one of claims 1 to 29, wherein the nucleic acid product amplified from the asymmetric PCR reaction has a different length for each individual target sequence.

31. The method according to any one of claims 1 to 29, wherein the nucleic acid product amplified from the asymmetric PCR reaction has substantially similar amplicon lengths.

32. The method according to any one of claims 1 to 29, wherein the nucleic acid product amplified from the asymmetric PCR reaction has an amplicon length with a variation of less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%.

33. The method according to any one of claims 1 to 29, wherein the nucleic acid product amplified from the asymmetric PCR reaction has a length of at least 100,000, 75,000, 50,000, 25,000, 10,000, 750, 5,000, 2,500, 2,000, 1,500, 1,000, 750, 600, 500, 400, 300, 200, 100, 50, 40, 30, or 20 nucleotides.

34. The method according to any one of claims 1 to 29, wherein the nucleic acid product amplified from the asymmetric PCR reaction has a maximum length of 100,000, 75,000, 50,000, 25,000, 10,000, 7,500, 5,000, 2,500, 2,000, 1,500, 1,000, 750, 600, 500, 400, 300, 200, 100, 50, 40, 30, or 20 nucleotides.

35. The method according to any one of claims 1 to 29, wherein the nucleic acid product amplified from the asymmetric PCR reaction has a length of 600 to 500, 600 to 400, 600 to 300, 600 to 200, 600 to 100, 600 to 50, 600 to 40, 600 to 30, or 600 to 20 nucleotides.

36. The method according to any one of claims 1 to 29, wherein the nucleic acid product amplified from the asymmetric PCR reaction has a length of 600 to 500, 500 to 400, 400 to 300, 300 to 200, 200 to 100, 100 to 50, 100 to 40, 100 to 30, or 100 to 20 nucleotides.

37. The method according to any one of claims 1 to 29, wherein the nucleic acid product amplified from the asymmetric PCR reaction has a nucleotide length of 500-20, 400-20, 300-20, 200-20, 150-20, 100-20, 90-20, 80-20, 70-20, 60-20, 50-20, 40-20, or 30-20.

38. The method according to any one of claims 1 to 37, wherein the asymmetric PCR reaction comprises (i) a series of temperature cycles comprising a denaturation step, an annealing step, and an extension step, or (ii) a series of temperature cycles comprising a denaturation step, and a step combining annealing and extension.

39. The method according to claim 38, wherein the annealing step is at approximately 55°C or approximately 60°C.

40. The method according to claim 38, wherein the annealing step is at 35-65, 40-60, 45-55, 40-65, 45-60, 50-60, 50-55, 55-60, or 60-65°C.

41. The method according to any one of claims 38 to 40, wherein the extension step is approximately 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160, or 180 minutes.

42. The method according to any one of claims 38 to 40, wherein the extension step is for at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160, or 180 minutes.

43. The method according to any one of claims 38 to 40, wherein the extension step is a maximum of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160, or 180 minutes.

44. The method according to any one of claims 38 to 40, wherein the extension step is for 0.5 to 20, 1 to 20, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 11 to 20, 12 to 20, 13 to 20, 15 to 20, 1 to 18, 2 to 16, 3 to 14, 4 to 12, 5 to 10, 6 to 9, 7 to 8, 4 to 6, 6 to 8, 8 to 10, 9 to 12, or 10 to 12 minutes.

45. The method according to any one of claims 1 to 44, wherein the asymmetric PCR reaction comprises at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 cycles.

46. The method according to any one of claims 1 to 44, wherein the asymmetric PCR reaction comprises about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 cycles.

47. The method according to any one of claims 1 to 44, wherein the asymmetric PCR reaction comprises 15 to 90, 20 to 80, 30 to 70, 40 to 60, 25 to 90, 30 to 90, 40 to 90, 50 to 90, 60 to 90, 70 to 90, 25 to 80, 25 to 70, 25 to 60, 25 to 50, 25 to 40, 15 to 30, 25 to 50, or 35 to 50 cycles.

48. The method according to any one of claims 1 to 47, wherein the excess primer and the limiting primer have different melting temperatures, and the annealing temperature used in the asymmetric PCR reaction is optimized for the excess primer.

49. The method according to any one of claims 1 to 48, further comprising the step of normalizing the sequencing read data or abundance readings by a normalization coefficient determined based on one or more elements selected from the group consisting of (i) sequencing yield, (ii) sequence-dependent amplification efficiency, (iii) transformation function, and (iv) linearity correction.

50. The method according to any one of claims 1 to 49, wherein the nucleic acid mixture sample is a cDNA sample.

51. The method according to any one of claims 1 to 50, wherein the nucleic acid mixture sample is barcoded according to its cellular origin.

52. The method according to any one of claims 1 to 51, wherein the high-throughput sequencing library is barcoded by a unique molecular identifier (UMI).

53. A kit comprising a panel of primer pairs, each pair containing an excess primer and a restriction primer in a predetermined stock concentration configuration for asymmetric PCR amplification from a desired target amplicon.

54. The kit according to claim 53, wherein the panel comprises at least 10, 20, 50, 100, 200, 300, 400, 500, 750, 1000, 2000, 5000, 10000, 20000, or 100000 primer pairs.

55. The kit according to claim 54, wherein a subset or all of the primer pairs share the same excess primer.

56. The kit according to claim 53 or 54, wherein the excess primer and the limiting primer of each primer pair are packaged separately.

57. The kit according to claim 53 or 54, wherein the primer pairs are individually pre-mixed.

58. The kit according to any one of claims 53 to 57, further comprising one or more sets of dNTPs, polymerase, buffer, and labeled probes.

Images