Compositions and methods for oncology assays

Abstract

Methods and compositions for preparing a library of target nucleic acid sequences are provided that can be used to assess gene mutations for tumor biomarker profiling of a sample. In particular, a target-specific primer set is provided that allows for selective amplification of a target sequence of a biomarker of tumor in a sample. In one aspect, the invention relates to target-specific primers that can be used to selectively amplify one or more target sequences associated with neoplastic biomarkers from two or more sample types. In some aspects, the amplified target sequences obtained using the disclosed methods and compositions can be used in various processes, including nucleic acid sequencing, and for detecting the presence of genetic variants of one or more targeting sequences associated with oncology.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 342,867, filed on May 17, 2022, under 35 U.S.C. §119(e), the entirety of which is incorporated herein by reference.

[0003] Sequence Listing

[0004] This application is hereby incorporated by reference into the electronic sequence listing materials submitted at the same time. As defined in 37 C.FR1.822, the nucleic acid sequences and amino acid sequences listed in the attached sequence listing are shown using standard letter abbreviations for nucleotide bases and single-letter codes for amino acids. Only one chain is shown for each nucleic acid sequence, but the complementary chain is understood to be contained in the displayed chain. The material in the electronic sequence list is submitted in the form of an extensible markup language (.xml) file named "TP109605WO1_ST26" created on May 8, 2023 (whose file size is ~8,088,321 bytes) and is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates to compositions and methods for preparing target nucleic acid libraries and uses thereof. Background Art

[0006] Advances in cancer therapy have begun to provide promising results in oncology. Compared with conventional chemotherapy, targeted therapy, immune checkpoint inhibitors, cancer vaccines and T cell therapy show sustainable results in responsive populations. However, effectively identifying responsive candidates and / or monitoring responses have proven challenging. There is an urgent need to better understand tumor microenvironment, tumor evolution and drug response biomarkers. High-throughput, systematic and standardized assay solutions that can effectively and efficiently detect a variety of related biomarkers in various sample types are desirable. Summary of the invention

[0007] In one aspect of the present invention, a composition for performing a single-stream multiplex determination of executable oncology biomarkers in a sample is provided. In some embodiments, the composition is composed of a variety of primer reagents, and the various primer reagents are directed to multiple target sequences to quickly and efficiently detect low-level targets in a sample. The composition provided targets oncology gene sequences, wherein multiple gene sequences are selected from targets in DNA hotspot mutation genes, copy number variation (CNV) genes, intergenic fusion genes, and intragenic fusion genes. In certain embodiments, the target gene is selected from the genes of Table 1. In a specific embodiment, the target gene is composed of the genes of Table 1. The composition provided is integrated and automated in a workflow, and detects key biomarkers from various samples (e.g., FFPE tissues, plasma) to the maximum extent within one day, such as EGFR, ALK, BRAF, ROS1, HER2, MET, NTRK, and RET.

[0008] In some embodiments, multiple executable target genes in a sample determine changes in oncological activity in the sample that indicate potential diagnosis, prognosis, candidate treatment options and / or adverse events. In specific embodiments, provided compositions comprise a plurality of primer reagents selected from Table A. In some embodiments, multiple assays comprising the compositions of the invention are provided. In some embodiments, test kits comprising the compositions of the invention are provided.

[0009] In another aspect of the present invention, a method for determining executable oncology biomarkers in a biological sample is provided. Such methods include multiple amplification of multiple target sequences from a biological sample containing a target sequence. Amplification includes contacting at least a portion of a sample including multiple target sequences of interest using multiple target-specific primers under amplification conditions in the presence of a polymerase to produce multiple amplified target sequences. The method further includes detecting the presence of each target sequence in the multiple target oncology sequences, wherein the detection of one or more executable oncology biomarkers determines the change in oncology activity indicating potential diagnosis, prognosis, candidate treatment regimens and / or adverse events in the sample compared to a control sample. The method described herein uses the composition of the present invention provided herein. In some embodiments, the target gene is selected from the group consisting of: DNA hotspot mutation genes, copy number variation (CNV) genes, intergenic fusion genes, and intragenic fusion genes. In certain embodiments, the target gene is selected from the genes of Table 1. In a specific embodiment, the target gene is composed of the genes of Table 1.

[0010] In addition, using the provided compositions and the kit comprising the provided compositions to analyze the sequence of nucleic acid libraries is other aspects of the present invention. In some embodiments, analysis of the sequence of the resulting library is provided to detect low-frequency alleles, improve the detection of gene fusions and novel fusions and / or detect the gene mutation in the sample of interest and / or the multiple samples of interest. In certain embodiments, manual, partially automated and fully automated embodiments of the provided compositions and methods are considered. In a specific embodiment, the purposes of the compositions are provided to be implemented in the fully integrated library preparation, templated and sequencing systems for sample genetic analysis. In certain embodiments, the purposes of the provided compositions and methods of the present invention provide benefits for research and clinical applications, including the first-line test of tissue and / or plasma samples and the continuous monitoring of samples for the recurrence and / or drug resistance detection of biomarkers.

[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0012] For various nucleic acid analyses, it is desirable to use an efficient method for generating a target library covering executable oncology biomarkers from a complex sample. The present invention particularly provides a method for preparing a library of target nucleic acid sequences, allowing rapid generation of highly multiplexed target libraries, comprising unique tag sequences; and the resulting library composition can be used for a variety of applications, including sequencing applications. The provided composition is designed to detect mutations, copy number variations (CNVs), and gene fusions in tissue and plasma-derived samples. The provided composition includes a targeted primer set and reagents for high-throughput samples to produce a next-generation workflow for genetic analysis. In a specific embodiment, the use is implemented on a fully integrated sample-to-analysis system. The novel features of the present invention are specifically set forth in the appended claims. And by reference to the following detailed description of illustrative embodiments and the accompanying drawings, a complete understanding of these features and advantages of the present invention will be obtained, in which the principles of the present invention are utilized. DETAILED DESCRIPTION

[0013] The section titles used herein are only for organizational purposes and should not be understood as limiting the described subject matter in any way. All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers and Internet web pages, are explicitly incorporated as a whole by reference for any purpose. When the definition of the term in the incorporated reference is presented as different from the definition provided in the present invention, the definition provided in the present invention should be used as the standard. It should be understood that there is an implied "about" before the temperature, concentration, time, etc. discussed in the present invention, so that small and insubstantial deviations are within the scope of the present invention in this article. In this application, unless otherwise specifically specified, the use of the singular includes the plural. It should be noted that unless explicitly and positively limited to one indicator, the singular form "one" and "the" and the singular use of any word as used in this specification include multiple indicators. In addition, the use of "including", "containing" and "including" is not intended to be restrictive. It should be understood that the general description is only exemplary and explanatory, and does not limit the present invention.

[0014] Unless otherwise defined, the scientific and technical terms used in conjunction with the present invention described herein should have the meanings commonly understood by those skilled in the art. In addition, unless the context otherwise requires, singular terms should include plural and plural terms should include singular. In general, the nomenclature used in conjunction with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization used herein is well-known and commonly used nomenclature in the art. Unless otherwise noted, the practice of this theme can adopt the conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant technology), cell biology and biochemistry within the skills of the art. Such conventional techniques include, but are not limited to, the preparation of synthetic polynucleotides, polymerization techniques, chemical and physical analysis of polymer particles, preparation of nucleic acid libraries, nucleic acid sequencing and analysis, etc. The specific description of suitable technology can be used with reference to the embodiments provided herein. Other equivalent routine procedures can also be used. Such routine techniques and instructions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Volumes I-IV), PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Hermanson, Bioconjugate Techniques, 2nd Edition (Academic Press, 2008); Merkus, Particle Size Measurements (Springer, 2009); Rubinstein and Colby, Polymer Physics (Oxford University Press, 2003); etc. As used in accordance with the examples provided herein, unless otherwise indicated, the following terms should be understood to have the following meanings:

[0015] As used herein, "amplification" or "amplification reaction" and its derivatives generally refer to the action or process for copying or replicating at least a portion of a nucleic acid molecule (referred to as a template nucleic acid molecule) into at least one other nucleic acid molecule. The additional nucleic acid molecules optionally include sequences that are substantially identical or substantially complementary to at least some portions of the template nucleic acid molecule. The template target nucleic acid molecule can be single-stranded or double-stranded. Other resulting replicated nucleic acid molecules can be single-stranded or double-stranded independently. In some embodiments, amplification comprises a template-dependent in vitro enzyme-catalyzed reaction for producing at least one copy of at least a portion of the target nucleic acid molecule, or producing at least one copy of a target nucleic acid sequence that is complementary to at least a portion of the target nucleic acid molecule. Amplification optionally comprises linear or exponential replication of nucleic acid molecules. In some embodiments, such amplification is performed using isothermal conditions; in other embodiments, such amplification may comprise thermal cycling. In some embodiments, amplification is multiplex amplification, which comprises simultaneously amplifying multiple target sequences in a single amplification reaction. At least some of the target sequences may be located on the same nucleic acid molecule or different target nucleic acid molecules contained in the single amplification reaction. In some embodiments, "amplification" comprises the amplification of at least a portion of a nucleic acid (alone or in combination) based on DNA and / or RNA. The amplification reaction can comprise a single-stranded or double-stranded nucleic acid substrate and can further comprise any amplification method known to those of ordinary skill in the art. In some embodiments, the amplification reaction comprises a polymerase chain reaction (PCR). In some embodiments, the amplification reaction comprises isothermal amplification.

[0016] As used herein, "amplification conditions" and derivatives (e.g., conditions for amplification, etc.) generally refer to conditions suitable for amplifying one or more nucleic acid sequences. Amplification can be linear or exponential. In some embodiments, amplification conditions include isothermal conditions or alternatively include thermal cycling conditions, or a combination of isothermal conditions and thermal cycling conditions. In some embodiments, conditions suitable for amplifying one or more target nucleic acid sequences include polymerase chain reaction (PCR) conditions. Typically, amplification conditions refer to a reaction mixture sufficient to amplify nucleic acids such as one or more target sequences or to amplify amplified target sequences joined to one or more adapters (e.g., amplified target sequences joined by adapters). Typically, amplification conditions include catalysts for amplification or for nucleic acid synthesis, such as polymerases; primers having a certain degree of complementarity with the nucleic acid to be amplified; and nucleotides, such as deoxyribonucleoside triphosphates (dNTPs), to promote extension of primers once hybridized with nucleic acids. Amplification conditions require hybridization or annealing of primers to nucleic acids, extension and denaturation steps of primers, wherein the extended primers are separated from the nucleic acid sequences undergoing amplification. Typically, but not necessarily, amplification conditions may include thermal cycling. In some embodiments, the amplification conditions comprise multiple cycles in which the steps of annealing, extension, and separation are repeated. Typically, the amplification conditions comprise cations such as Mg ++or Mn ++ (e.g. MgCl2, etc.), and may also optionally contain a variety of ionic strength adjusters.

[0017] As used herein, "target sequence," "target nucleic acid sequence," or "target sequence of interest" and derivatives thereof generally refer to any single-stranded or double-stranded nucleic acid sequence that can be amplified or synthesized according to the present disclosure, including any nucleic acid sequence suspected or expected to be present in a sample. In some embodiments, the target sequence exists in double-stranded form and comprises at least a portion of a specific nucleotide sequence to be amplified or synthesized, or its complement, prior to the addition of a target-specific primer or an attached adapter. The target sequence may comprise a nucleic acid that can hybridize with a primer suitable for an amplification or synthesis reaction prior to polymerase extension. In some embodiments, the term refers to a nucleic acid sequence whose sequence identity, order, or position of nucleotides is determined by one or more of the methods of the present disclosure.

[0018] As used herein, the term "portion" and variations thereof, when used in reference to a given nucleic acid molecule, such as a primer or template nucleic acid molecule, includes any number of contiguous nucleotides within the length of the nucleic acid molecule, including a portion or the entire length of the nucleic acid molecule.

[0019] As used herein, when used with respect to two or more components, "contact" and its derivatives generally refer to any process for promoting or achieving the proximity, proximity, mixture or blending of reference components without necessarily requiring physical contact of such components, and include the mixing of solutions containing any one or more of the reference components with each other. The components mentioned can be contacted in any particular order or combination and the specific order of the component citation is not restrictive. For example, "contacting A with B and C" covers embodiments in which A is first contacted with B and then with C, and embodiments in which C is contacted with A and then with B, and embodiments in which a mixture of A and C is contacted with B, etc. In addition, such contact does not necessarily require that the final result of the contact process is a mixture including all the mentioned components, as long as at some point in time during the contact process, all the mentioned components are present at the same time or are included in the same mixture or solution at the same time. For example, "contacting A with B and C" may include embodiments in which C is first contacted with A to form a first mixture, which is then contacted with B to form a second mixture, and then C is removed from the second mixture; optionally, A may also be removed, leaving only B. When one or more of the referenced components to be contacted comprises a plurality (e.g., "the target sequence is contacted with a plurality of target-specific primers and a polymerase"), each member of the plurality can be considered an individual component of the contacting process, such that contacting can comprise contacting any one or more members of the plurality with any other member of the plurality and / or with any other referenced components in any order or combination (e.g., some but not all of the plurality of target-specific primers can contact the target sequence, followed by the polymerase, and then contacted with other members of the plurality of target-specific primers).

[0020] As used herein, the term "primer" and its derivatives generally refer to any polynucleotide that can hybridize with a target sequence of interest. In some embodiments, primers can also be used to initiate nucleic acid synthesis. Typically, primers can be used as substrates to which nucleotides can be polymerized by polymerase; however, in some schemes, primers can be incorporated into a synthetic nucleic acid chain and provide a site to which another primer can hybridize to initiate the synthesis of a new chain complementary to the synthetic nucleic acid molecule. A primer can include any combination of nucleotides or their analogs, which can optionally be connected to form a linear polymer of any suitable length. In some embodiments, a primer is a single-stranded oligonucleotide or a polynucleotide. (In the present disclosure, the terms "polynucleotide" and "oligonucleotide" can be used interchangeably herein and do not necessarily indicate any difference in length between two nucleotides). In some embodiments, a primer is double-stranded. If double-stranded, then before being used to prepare an extension product, the primer is first treated to separate its chains. Preferably, a primer is an oligodeoxynucleotide. The primer must be long enough to initiate the synthesis of an extension product. The length of primer will depend on many factors, including temperature, the source of primer and the method of use. In some embodiments, when exposed to amplification conditions or synthesis conditions, primer serves as a starting point for amplification or synthesis; Such amplification or synthesis can be carried out in a template-dependent manner and optionally form a primer extension product complementary to at least a portion of the target sequence. Exemplary amplification conditions or synthesis conditions may include contacting primers with polynucleotide templates (e.g., templates comprising target sequences), nucleotides and inducing agents (such as polymerases) at a suitable temperature and pH to induce polymerization of nucleotides on the ends of target-specific primers. If double-stranded, then before being used to prepare primer extension products, primers may be optionally treated to separate their chains. In some embodiments, primers are oligodeoxynucleotides or oligoribonucleotides. In some embodiments, primers may include one or more nucleotide analogs. The precise length and / or composition (comprising sequence) of target-specific primers can affect many characteristics, comprising melting temperature (Tm), GC content, formation of secondary structure, repeating nucleotide main structure, the length of predicted primer extension product, the degree of coverage across the nucleic acid molecule of interest, the number of primers in a single amplification or synthesis reaction, whether there are nucleotide analogs or modified nucleotides in the primer, etc. In some embodiments, primers can be paired with compatible primers in amplification or synthesis reaction to form a primer pair consisting of a forward primer and a reverse primer. In some embodiments, the forward primer of the primer pair comprises a sequence substantially complementary to at least a portion of a chain of the nucleic acid molecule, and the reverse primer of the primer pair primer comprises a sequence substantially consistent with at least a portion of the chain. In some embodiments, the forward primer and the reverse primer can hybridize with the relative chains of the nucleic acid double helix.Optionally, the forward primer initiates the synthesis of the first nucleic acid chain, and the reverse primer initiates the synthesis of the second nucleic acid chain, wherein the first chain and the second chain are substantially complementary to each other, or can hybridize to form a double-stranded nucleic acid molecule. In some embodiments, one end of amplification or synthetic product is defined by the forward primer and the other end of amplification or synthetic product is defined by the reverse primer. In some embodiments, when it is necessary to amplify or synthesize lengthy primer extension products (such as amplified exons, coding regions or genes), it is possible to produce several primer pairs rather than spanning the required length to achieve enough amplification in the region. In some embodiments, primers can include one or more cleavable groups. In some embodiments, primer length is within the range of about 10 to about 60 nucleotides, about 12 to about 50 nucleotides and about 15 to about 40 nucleotide lengths. Typically, when exposed to amplification conditions in the presence of dNTPs and polymerase, primers can hybridize with corresponding target sequences and carry out primer extension. In some cases, a part of a specific nucleotide sequence or primer is known or can be determined by one or more of the methods disclosed herein when the amplification reaction begins. In some embodiments, a primer comprises one or more cleavable groups at one or more positions within the primer.

[0021] As used herein, "target-specific primer" and its derivatives generally refer to a single-stranded nucleotide or a double-stranded polynucleotide, typically an oligonucleotide, which comprises at least one sequence that is at least 50% complementary to at least a portion of a nucleic acid molecule comprising a target sequence, typically at least 75% complementary or at least 85% complementary, more typically at least 90% complementary, more typically at least 95% complementary, more typically at least 98% or at least 99% complementary or identical. In such cases, the target-specific primer and the target sequence are described as "corresponding" to each other. In some embodiments, the target-specific primer is capable of hybridizing with at least a portion of its corresponding target sequence (or the complement of the target sequence); such hybridization can be optionally performed under standard hybridization conditions or under stringent hybridization conditions. In some embodiments, the target-specific primer cannot hybridize with the target sequence or its complement, but can hybridize with a portion of the nucleic acid chain comprising the target sequence or its complement. In some embodiments, the target-specific primer comprises at least one sequence that is at least 75% complementary to at least a portion of the target sequence itself, typically at least 85% complementary, more typically at least 90% complementary, more typically at least 95% complementary, more typically at least 98% complementary, or more typically at least 99% complementary; in other embodiments, the target-specific primer comprises at least one sequence that is at least 75% complementary to at least a portion of the target sequence itself, typically at least 85% complementary, more typically at least 90% complementary, more typically at least 95% complementary, more typically at least 98% complementary, or more typically at least 99% complementary; in some embodiments, the target-specific primer is not substantially complementary to other target sequences present in the sample; optionally, the target-specific primer is not substantially complementary to other nucleic acid molecules present in the sample. In some embodiments, nucleic acid molecules present in the sample that do not contain or correspond to the target sequence (or the complement of the target sequence) are referred to as "non-specific" sequences or "non-specific nucleic acids". In some embodiments, the target-specific primer is designed to comprise a nucleotide sequence that is substantially complementary to at least a portion of its corresponding target sequence. In some embodiments, the target-specific primer is at least 95% complementary or at least 99% complementary or identical (across its entire length) to at least a portion of a nucleic acid molecule comprising its corresponding target sequence. In some embodiments, the target-specific primer may be at least 90%, at least 95% complementary, at least 98% complementary, or at least 99% complementary or identical (across its entire length) to at least a portion of its corresponding target sequence. In some embodiments, the forward target-specific primer and the reverse target-specific primer define a target-specific primer pair, which can be used to amplify the target sequence via template-dependent primer extension. Typically, each primer of the target-specific primer pair includes at least one sequence that is substantially complementary to at least a portion of a nucleic acid molecule comprising a corresponding target sequence, but is less than 50% complementary to at least one other target sequence in the sample.In some embodiments, amplification can be performed using multiple target-specific primers in a single amplification reaction, wherein each primer pair comprises a forward target-specific primer and a reverse target-specific primer, each comprising at least one sequence substantially complementary or substantially consistent with the corresponding target sequence in the sample, and each primer pair has different corresponding target sequences. In some embodiments, the target-specific primer can be substantially non-complementary at its 3' end or its 5' end with any other target-specific primer in the amplification reaction. In some embodiments, the target-specific primer can include the minimum cross hybridization with other target-specific primers in the amplification reaction. In some embodiments, the target-specific primer is included in the minimum cross hybridization with non-specific sequences in the amplification reaction mixture. In some embodiments, the target-specific primer includes minimum self-complementarity. In some embodiments, the target-specific primer can include one or more cleavable groups located at the 3' end. In some embodiments, the target-specific primer can include one or more cleavable groups located near or around the central nucleotide of the target-specific primer. In some embodiments, one of more target-specific primers is only included in the non-cleavable nucleotides at the 5' end of the target-specific primer. In some embodiments, a target-specific primer comprises minimal nucleotide sequence overlap at the 3' end or 5' end of the primer compared to one or more different target-specific primers, optionally in the same amplification reaction. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target-specific primers in a single reaction mixture comprise one or more of the above embodiments. In some embodiments, substantially all of the multiple target-specific primers in a single reaction mixture comprise one or more of the above embodiments.

[0022] As used herein, the term "adapter" means a nucleic acid molecule that can be used for the operation of related polynucleotides. In some embodiments, the adaptor is used for the amplification of one or more target nucleic acids. In some embodiments, the adaptor is used in sequencing reactions. In some embodiments, the adaptor has one or more ends without 5' phosphate residues. In some embodiments, the adaptor comprises at least one priming site, is composed of at least one priming site, or is mainly composed of at least one priming site. This type of priming site containing an adaptor can be called a "primer" adaptor. In some embodiments, the adaptor priming site can be applied to a PCR process. In some embodiments, the adaptor comprises a nucleic acid sequence that is substantially complementary to the 3' end or 5' end of at least one target sequence (referred to herein as a gene-specific target sequence, a target-specific sequence, or a target-specific primer) in a sample. In some embodiments, the adaptor comprises a nucleic acid sequence that is substantially non-complementary to the 3' end or 5' end of any target sequence present in the sample. In some embodiments, the adaptor comprises a single-stranded or double-stranded linear oligonucleotide that is substantially non-complementary to a target nucleic acid sequence. In some embodiments, the adaptor comprises a nucleic acid sequence that is substantially non-complementary to at least one and preferably some or all of the nucleic acid molecules in the sample. In some embodiments, suitable adapter length is in the range of about 10-75 nucleotides, about 12-50 nucleotides and about 15-40 nucleotides in length. Generally, adapters can include any combination of nucleotides and / or nucleic acids. In some aspects, adapters include one or more cleavable groups at one or more positions. In some embodiments, adapters include a sequence that is substantially consistent or substantially complementary to at least a portion of a primer (e.g., a universal primer). In some embodiments, adapters include a tag sequence to help classification, identification or sequencing. In some embodiments, adapters serve as substrates for amplification of target sequences, especially at suitable temperature and pH, in the presence of polymerase and dNTPs.

[0023] As used herein, "polymerase" and its derivatives generally refer to any enzyme that can catalyze the polymerization of nucleotides (including its analogs) into nucleic acid chains. Typically, but not necessarily, such nucleotide polymerization can be carried out in a template-dependent manner. Such polymerases may include, but are not limited to, naturally occurring polymerases and any subunits thereof and truncated, mutant polymerases, variant polymerases, recombinant, fused or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or components, and any analogs, derivatives, or fragments thereof that retain the ability to catalyze such polymerization. Optionally, the polymerase may be a mutant polymerase comprising one or more mutations, the one or more mutations involving replacement of one or more amino acids with other amino acids, insertion or deletion of one or more amino acids from the polymerase, or connection of two or more parts of the polymerase. Typically, the polymerase includes one or more active sites that can perform nucleotide binding and / or nucleotide polymerization catalysis. Some exemplary polymerases include, but are not limited to, DNA polymerases and RNA polymerases. As used herein, the term "polymerase" and its variants also refer to a fusion protein comprising at least two parts connected to each other, wherein the first part comprises a peptide that can catalyze the polymerization of nucleotides into a nucleic acid chain and the first part is connected to a second part comprising a second polypeptide. In some embodiments, the second polypeptide may comprise a reporter enzyme or a domain that enhances continuous synthesis. Optionally, the polymerase may have 5' exonuclease activity or terminal transferase activity. In some embodiments, the polymerase may optionally be reactivated, for example, by using heat, chemicals or by adding a new amount of the polymerase to the reaction mixture. In some embodiments, the polymerase may comprise a hot start polymerase that may optionally be reactivated and / or an aptamer-based polymerase.

[0024] As used herein, the terms "identity" and "identical" and variations thereof, when used to refer to two or more nucleic acid sequences or peptide sequences, refer to the sequence similarity of two or more sequences (e.g., nucleotide sequences or polypeptide sequences). In the case of two or more homologous sequences, the identity or homology percentage of a sequence or its subsequence indicates the percentage of all monomeric units (e.g., nucleotides or amino acids) that are identical (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 95%, 98% or 99% identity). The identity percentage can be in a specified region, when compared and aligned for maximum correspondence in a comparison window, or in a specified region, as measured using BLAST or BLAST 2.0 sequence comparison algorithms with the default parameters described below or by manual alignment and visual inspection. When there is at least 85% identity at the amino acid level or nucleotide level, the sequence is referred to as "substantially identical". Preferably, identity is present in a region of at least about 25, 50 or 100 residues long or over the entire length of at least one compared sequence. Typical algorithms for determining percentages of sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977). Other methods include Smith and Waterman, Adv. Appl. Math. 2:482 (1981) and Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), etc. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent hybridization conditions.

[0025] As used herein, the terms "complementary" and "complement" and variations thereof refer to any two or more nucleic acid sequences (e.g., part or all of a template nucleic acid molecule, a target sequence, and / or a primer) that undergo cumulative base pairing at two or more independent corresponding positions in an antiparallel orientation (e.g., in a hybridized double helix). Such base pairing can be performed according to any existing set of rules, such as according to the Watson-Crick base pairing rules or according to some other base pairing paradigm. Optionally, there may be "complete" or "overall" complementarity between the first and second nucleic acid sequences, wherein each nucleotide in the first nucleic acid sequence may perform a stabilizing base pairing interaction with a nucleotide in a corresponding antiparallel position on the second nucleic acid sequence. "Partial" complementarity describes a nucleic acid sequence in which at least 20% but less than 100% of the residues of a nucleic acid sequence are complementary to the residues in another nucleic acid sequence. In some embodiments, at least 50% but less than 100% of the residues of a nucleic acid sequence are complementary to the residues in another nucleic acid sequence. In some embodiments, at least 70%, 80%, 90%, 95% or 98% but less than 100% of the residues of a nucleic acid sequence are complementary to the residues in another nucleic acid sequence. When at least 85% of the residues in a nucleic acid sequence are complementary to the residues in another nucleic acid sequence, the sequence is referred to as "substantially complementary". In some embodiments, two complementary or substantially complementary sequences can hybridize with each other under standard or stringent hybridization conditions. "Non-complementary" describes a nucleic acid sequence in which less than 20% of the residues of one nucleic acid sequence are complementary to the residues in another nucleic acid sequence. When less than 15% of the residues in a nucleic acid sequence are complementary to the residues in another nucleic acid sequence, the sequence is referred to as "substantially non-complementary". In some embodiments, two non-complementary or substantially non-complementary sequences cannot hybridize with each other under standard or stringent hybridization conditions. "Mismatch" is present in any position of two non-complementary relative nucleotides. Complementary nucleotides are included in nucleotides effectively incorporated by relative DNA polymerases during DNA replication under physiological conditions. In a typical embodiment, between the core bases of nucleotides and / or polynucleotides that are located in antiparallel positions to each other, complementary nucleotides can form base pairs with each other, such as AT / U and GC base pairs formed by specific Watson-Crick type hydrogen bonds, or base pairs formed by some other type of base pairing paradigm. The complementarity of other artificial base pairs can be based on other types of hydrogen bonds and / or the hydrophobicity of the bases and / or shape complementarity between the bases.

[0026] As used herein, "amplified target sequence" and its derivatives generally refer to a nucleic acid sequence generated by amplification of a target sequence / amplification of a target sequence using target-specific primers and methods provided herein. The amplified target sequence can be either synonymous (positive strands produced in the second and subsequent even-numbered rounds of amplification) or antisense (i.e., negative strands produced during the first and subsequent odd-numbered rounds of amplification) relative to the target sequence. For the purposes of this disclosure, an amplified target sequence is generally less than 50% complementary to any portion of another amplified target sequence in the reaction.

[0027] As used herein, the term "joining" and its derivatives generally refer to an action or method for covalently linking two or more molecules together (for example, making two or more nucleic acid molecules covalently linked to each other). In some embodiments, the joining is included in the connection nick between the adjacent nucleotides of the nucleic acid. In some embodiments, the joining comprises a covalent bond formed between the end of the first nucleic acid molecule and the end of the second nucleic acid molecule. In some embodiments, such as embodiments in which the nucleic acid molecules to be joined include conventional nucleotide residues, the joining may include a covalent bond formed between the 5' phosphate group of a nucleic acid and the 3' hydroxyl of the second nucleic acid, thereby forming a joined nucleic acid molecule. In some embodiments, any method for connecting a 5' phosphate to a 3' hydroxyl or a combination between adjacent nucleotides can be used. In an exemplary embodiment, an enzyme such as a ligase can be used.

[0028] As used herein, "ligase" and its derivatives generally refer to any agent capable of catalyzing the joining of two substrate molecules. In some embodiments, the ligase comprises an enzyme capable of catalyzing the joining of nicks between adjacent nucleotides of a nucleic acid. In some embodiments, the ligase comprises an enzyme capable of catalyzing the formation of a covalent bond between a 5' phosphate of one nucleic acid molecule and a 3' hydroxyl of another nucleic acid molecule, thereby forming a joined nucleic acid molecule. Suitable ligases may include, but are not limited to, T4 DNA ligase; T7 DNA ligase; Taq DNA ligase and E. coli DNA ligase.

[0029] As defined herein, "cleavable group" generally refers to any part that can be cracked under appropriate conditions after being incorporated into nucleic acid. For example, the cleavable group can be incorporated into the nucleic acid molecules of target-specific primers, amplified sequences, adapters or samples. In an exemplary embodiment, the target-specific primer can include a cleavable group, which becomes incorporated into the amplified product and then cracks after amplification, thereby removing a part or all of the target-specific primers from the amplified product. The cleavable group can be cracked or removed from the nucleic acid molecules of target-specific primers, amplified sequences, adapters or samples by any acceptable means. For example, the cleavable group can be removed from the target-specific primers, amplified sequences, adapters or nucleic acid molecules of samples by enzymatic, thermal, photo-oxidation or chemical treatment. In one aspect, the cleavable group can include non-naturally occurring core bases. For example, oligoribonucleotides can include one or more RNA core bases, such as uracil that can be removed by uracil glycosylase. In some embodiments, the cleavable group may include one or more modified nucleobases (such as 7-methylguanine, 8-oxo-guanine, xanthine, hypoxanthine, 5,6-dihydrouracil or 5-methylcytosine) or one or more modified nucleosides (i.e., 7-methylguanosine, 8-oxo-deoxyguanosine, xanthine nucleoside, inosine, dihydrouridine or 5-methylcytosine). The modified nucleobase or nucleotide may be removed from the nucleic acid by enzymatic, chemical or thermal means. In one embodiment, the cleavable group may be included in a portion that can be removed from the primer after amplification (or synthesis) when exposed to ultraviolet light (i.e., bromodeoxyuridine). In another embodiment, the cleavable group may include methylated cytosine. Typically, methylated cytosine can be cleaved by the primer, such as after induced amplification (or synthesis), when treated with sodium bisulfite. In some embodiments, the cleavable portion may include a restriction site. For example, a primer or target sequence can comprise a nucleic acid sequence that is specific to one or more restriction enzymes, and after amplification (or synthesis), the primer or target sequence can be treated with one or more restriction enzymes to remove the cleavable group. Typically, nucleic acid molecules that can comprise one or more cleavable groups and target-specific primers, amplified sequences, adapters, or samples at one or more positions.

[0030] As used herein, "digestion," "digestion step," and derivatives thereof generally refer to any method for cleaving or otherwise removing a cleavable group from a target-specific primer, an amplified sequence, an adapter, or a nucleic acid molecule of a sample. In some embodiments, the digestion step involves a chemical, thermal, photo-oxidative, or digestion process.

[0031] As used herein, the term "hybridization" is consistent with its use in the art and generally refers to a method for causing two nucleic acid molecules to undergo base pairing interactions. When any portion of one nucleic acid molecule is base paired with any portion of another nucleic acid molecule, the two nucleic acid molecules are called hybridized; it is not necessary to hybridize the two nucleic acid molecules over their entire corresponding lengths, and in some embodiments, at least one of the nucleic acid molecules may include a portion that does not hybridize with another nucleic acid molecule. The phrase "hybridization under stringent conditions" and its variants generally refer to conditions under which hybridization of a target-specific primer to a target sequence can be performed under conditions of high hybridization temperature and low ionic strength. As used herein, the phrase "standard hybridization conditions" and its variants generally refer to conditions under which hybridization of a primer to an oligonucleotide (i.e., a target sequence) can be performed in the presence of low hybridization temperature and high ionic strength. In an exemplary embodiment, standard hybridization conditions include an aqueous environment containing about 100 mM magnesium sulfate, about 500 mM Tris-sulfate (pH 8.9) and about 200 mM ammonium sulfate at about 50°C-55°C, or its equivalent.

[0032] As used herein, when used with respect to nucleic acid molecules (e.g., target sequences or amplified target sequences), the term "end" and its variants can include the terminal 30 nucleotides, terminal 20 and even more usually terminal 15 nucleotides of the nucleic acid molecule. A linear nucleic acid molecule comprising a series of adjacent nucleotides generally includes at least two ends. In some embodiments, one end of a nucleic acid molecule may include a 3' hydroxyl or its equivalent, and may be referred to as "3' end" and its derivatives. Optionally, the 3' end includes the 3' hydroxyl of the 5' phosphate group that is not connected to the mononucleotide pentose ring. Generally, the 3' end includes one or more 5' nucleotides that are positioned adjacent to the nucleotides that include the unconnected 3' hydroxyl, generally 30 nucleotides that are adjacent to the 3' hydroxyl, generally 20 and even more generally 15 nucleotides at the end. Generally, one or more connected nucleotides can be expressed as the percentage of the nucleotides present in the oligonucleotide, or can be provided in the form of many connected nucleotides adjacent to the unconnected 3' hydroxyl. For example, the 3' end can include less than 50% of the nucleotide length of the oligonucleotide. In some embodiments, the 3' end does not include any unconnected 3' hydroxyl, but may include any part that can serve as the site of connection of nucleotides via primer extension and / or nucleotide polymerization. In some embodiments, for example, when referring to a target-specific primer, the term "3' end" may include 10 nucleotides at the end of the 3' end, 5 nucleotides at the end, 4, 3, 2 or fewer nucleotides at the end. In some embodiments, when referring to a target-specific primer, the term "3' end" may include nucleotides located at nucleotide position 10 or fewer nucleotides from the 3' end. As used herein, "5' end" and its derivatives generally refer to the end of a nucleic acid molecule (for example, a target sequence or amplified target sequence), which includes free 5' phosphate groups or their equivalents. In some embodiments, the 5' end includes the 5' phosphate group of the 3' hydroxyl that is not connected to an adjacent mononucleotide pentose ring. Typically, the 5' end includes one or more nucleotides connected to the 5' phosphate adjacent to the 5' phosphate, typically 30 nucleotides adjacent to the nucleotides comprising the 5' phosphate, typically 20 at the end and even more typically 15 nucleotides at the end. Generally, the nucleotide of one or more connections can be expressed as the percentage of the nucleotide present in the oligonucleotide, or can be provided in the form of the nucleotides of many connections adjacent to 5' phosphate.For example, 5' end can be less than 50% of the nucleotide length of the oligonucleotide.In another exemplary embodiment, 5' end can include about 15 nucleotides adjacent to the nucleotides that include terminal 5' phosphate.In some embodiments, 5' end does not include any unconnected 5' phosphate groups, but can include any part that can serve as the connection site with 3' hydroxyl or 3' end of another nucleic acid molecule.In some embodiments, for example when with reference to target-specific primers, term " 5' end " can include terminal 10 nucleotides, terminal 5 nucleotides, terminal 4,3,2 or less nucleotides at 5' end.In some embodiments, when referring to a target-specific primer, the term "5' end" may include nucleotides located at position 10 or fewer nucleotides from the 5' end. In some embodiments, the 5' end of the target-specific primer may include only non-cleavable nucleotides, such as nucleotides that do not contain one or more cleavable groups as disclosed herein, or cleavable nucleotides that can be easily determined by a person of ordinary skill in the art. The "first end" and "second end" of a polynucleotide refer to the 5' end or the 3' end of the polynucleotide. The first end or the second end of a polynucleotide may be the 5' end or the 3' end of the polynucleotide; the terms "first" and "second" are not meant to indicate that the end is specifically the 5' end or the 3' end.

[0033] As used herein, "tag", "barcode", "unique tag" or "tag sequence" and its derivatives generally refer to a unique short (6-14 nucleotides) nucleic acid sequence within an adaptor or primer that can act as a "key" for distinguishing or separating multiple amplified target sequences in a sample. For the purposes of the present disclosure, a barcode or unique tag sequence is incorporated into the nucleotide sequence of an adaptor or primer. As used herein, a "barcode sequence" refers to a nucleic acid fixed sequence that is sufficient to identify a sample or source of a related nucleic acid sequence. The barcode sequence may, but is not necessarily, be a small segment of the original nucleic acid sequence that can be used to achieve identification. In some embodiments, the length of the barcode is 5-20 nucleic acids. In some embodiments, the barcode comprises similar nucleotides, such as L-DNA, LNA, PNA, etc. As used herein, a "unique tag sequence" refers to a nucleic acid sequence having at least one random sequence and at least one fixed sequence. The unique tag sequence (alone or in combination with a second unique tag sequence) is sufficient to achieve identification of a single target nucleic acid molecule in a sample. The unique tag sequence may, but is not necessarily, include a small segment of the original target nucleic acid sequence. In some embodiments, the length of the unique tag sequence is 2-50 nucleotides or base pairs, or 2-25 nucleotides or base pairs, or 2-10 nucleotides or base pairs.The unique tag sequence may include at least one random sequence interspersed with a fixed sequence.

[0034] As used herein, "comparable maximum and minimum melting temperatures" and its derivatives generally refer to the melting temperature (Tm) of each nucleic acid fragment of a single adapter or target-specific primer after digestion of a cleavable group. The hybridization temperature of each nucleic acid fragment generated by the adapter or target-specific primer is compared to determine the maximum and minimum temperatures required to prevent the nucleic acid sequence from the target-specific primer or adapter or its fragment or portion from hybridizing with the corresponding target sequence. After the maximum hybridization temperature is known, it is possible to operate the adapter or target-specific primer, for example, by moving the position of one or more cleavable groups along the length of the primer, to obtain a comparable maximum and minimum melting temperature relative to each nucleic acid fragment, thereby optimizing the digestion step and repair step of the library preparation.

[0035] As used herein, "addition only" and its derivatives generally refer to a series of steps in which reagents and components are added to a first or single reaction mixture. Typically, the series of steps does not include moving the reaction mixture from a first container to a second container to complete the series of steps. Typically, addition only methods do not include manipulation of the reaction mixture outside of the container containing the reaction mixture. Typically, addition only methods are suitable for automation and high throughput.

[0036] As used herein, "polymerization conditions" and its derivatives generally refer to conditions applicable to nucleotide polymerization. In typical embodiments, such nucleotide polymerization is catalyzed by a polymerase. In some embodiments, polymerization conditions include primer extension for optionally in a template-dependent manner, thereby resulting in the production of a synthetic nucleic acid sequence. In some embodiments, polymerization conditions include polymerase chain reaction (PCR). Typically, polymerization conditions include using a reaction mixture sufficient to synthesize nucleic acids and comprising a polymerase and nucleotides. Polymerization conditions may include conditions for annealing a target-specific primer to a target sequence and extending the primer in a template-dependent manner in the presence of a polymerase. In some embodiments, polymerization conditions may be implemented using thermal cycling. In addition, polymerization conditions may include multiple cycles, wherein annealing, extension and separation steps of two nucleic acid chains are repeated. Typically, polymerization conditions include cations such as MgCl2. Typically, one or more nucleotides are polymerized to form a nucleic acid chain comprising nucleotides connected to each other via a phosphodiester bond, however, in the context of a specific nucleotide analog, there may be an alternative connection.

[0037] As used herein, the term "nucleic acid" refers to natural nucleic acids, artificial nucleic acids, analogs thereof, or combinations thereof, including polynucleotides and oligonucleotides. As used herein, the terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to single-stranded and double-stranded polymers of nucleotides, including but not limited to 2'-deoxyribonucleotides (nucleic acids) and ribonucleotides (RNA) connected by internucleotide phosphodiester bonds such as 3'-5' and 2'-5', reverse connections such as 3'-3' and 5'-5', branched structures or analog nucleic acids. Polynucleotides have associated counterions, such as H + NH 4+ , trialkylammonium, Mg 2+ 、Na +Etc. Oligonucleotides can be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof. Oligonucleotides can include nucleobases and sugar analogs. The size of polynucleotides is generally in the range of several, such as 5-40 monomer units (when it is more commonly referred to as oligonucleotides in the art) to thousands of monomer nucleotide units (when it is more commonly referred to as polynucleotides in the art); however, for the purposes of this disclosure, both oligonucleotides and polynucleotides can have any suitable length. Unless otherwise indicated, whenever an oligonucleotide sequence is represented, it is understood that nucleotides are in 5' to 3' order from left to right, and "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, "T" represents thymidine, and "U" represents deoxyuridine. As discussed herein and known in the art, oligonucleotides and polynucleotides are referred to as having a "5' end" and a "3' end" because a single nucleotide is typically reacted to form an oligonucleotide via the 5' phosphate or equivalent group of one nucleotide to the 3' hydroxyl or equivalent group of its adjacent nucleotide (optionally via a phosphodiester or other suitable linkage).

[0038] As used herein, the term "polymerase chain reaction" ("PCR") refers to the methods of U.S. Patents Nos. 4,683,195 and 4,683,202 to KBMullis, which are incorporated herein by reference, describing a method for increasing the concentration of a fragment of a polynucleotide of interest in a genomic DNA mixture without cloning or purification. This process for amplifying a polynucleotide of interest consists of introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired polynucleotide of interest, followed by a precise sequence of thermal cycles in the presence of a DNA polymerase. The two primers are complementary to their corresponding chains of the double-stranded polynucleotide of interest. In order to achieve amplification, the mixture is denatured, and then the primers are annealed to their complementary sequences within the polynucleotide molecule of interest. After annealing, the primers are extended with a polymerase to form a pair of new complementary chains. The steps of denaturation, primer annealing, and polymerase extension can be repeated multiple times (i.e., denaturation, annealing, and extension constitute one "cycle"; multiple "cycles" can exist) to obtain a high concentration of amplified fragments of the desired polynucleotide of interest. The length of the amplified segment (amplicon) of the desired polynucleotide of interest is determined by the relative position of the primers relative to each other, and therefore this length is a controllable parameter. By means of repeating this process, the method is called "polymerase chain reaction" (hereinafter referred to as "PCR"). Because the desired amplified segment of the polynucleotide of interest becomes the main nucleic acid sequence in the mixture (in terms of concentration), it is called "PCR amplified". As defined herein, the target nucleic acid molecules in the sample containing a variety of target nucleic acid molecules are amplified via PCR. In an improvement of the method discussed above, the target nucleic acid molecules can be PCR amplified using a plurality of different primer pairs, and in some cases, one or more primer pairs for each target nucleic acid molecule of interest, thereby forming a multiple PCR reaction. Using multiple PCR, it is possible to simultaneously amplify a variety of nucleic acid molecules of interest from a sample to form an amplified target sequence. The amplified target sequence can also be detected by several different methods (e.g., quantified with a bioanalyzer or qPCR, hybridized with a labeled probe; incorporated with a biotinylated primer, followed by avidin-enzyme conjugate detection; 32 P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, are incorporated into the amplified target sequence. Any oligonucleotide sequence can be amplified with an appropriate primer set to achieve amplification of target nucleic acid molecules from genomic DNA, cDNA, formalin-fixed paraffin-embedded DNA, fine-needle biopsies, and a variety of other sources. In particular, the amplified target sequences generated by the multiplex PCR process as disclosed herein are themselves effective substrates for subsequent PCR amplification or a variety of downstream assays or operations.

[0039] As defined herein, "multiplex amplification" refers to the selective and non-random amplification of two or more target sequences in a sample using at least one target-specific primer. In some embodiments, multiplex amplification is performed so that some or all of the target sequences are amplified in a single reaction vessel. The "multiplicity" or "heavyness" of a given multiplex amplification generally refers to the number of different target-specific sequences amplified during the single multiplex amplification. In some embodiments, the multiplicity can be about 12-fold, 24-fold, 48-fold, 96-fold, 192-fold, 384-fold, 768-fold, 1536-fold, 3072-fold, 6144-fold or more.

[0040] Composition

[0041] A single-stream multiplexed next-generation sequencing workflow has been developed for determining actionable oncology tumor biomarkers in a sample in order to determine the oncology status in the sample. The oncology precision assay compositions and methods of the present invention provide specific and robust solutions for biomarker screening to understand mechanisms associated with tumor immune responses. Therefore, compositions for multiplexed library preparation and use in conjunction with next-generation sequencing technologies and workflow solutions (e.g., Ion TorrentTMNGS workflows) (manual or automated) are provided to evaluate low-level biomarker targets in various sample types to evaluate oncology status.

[0042] Therefore, a composition for performing a single-stream multiplex determination of executable oncology biomarkers in a sample is provided. In some embodiments, the composition is composed of multiple sets of primer pairs reagents, which are directed to multiple target sequences to detect low-level targets in the sample, wherein the target gene is selected from the oncology response gene composed of the following functions: DNA hotspot mutation genes, copy number variation (CNV) genes, intergenic fusion genes, and intragenic fusion genes. In some embodiments, the target gene is selected from the oncology gene composed of one or more functions of Table 1. In some embodiments, the target gene is selected from one or more executable target genes in the sample, which determines the change in oncology activity indicating potential diagnosis, prognosis, candidate treatment options, and / or adverse event possibilities in the sample. In short, the various functions of the genes provided by the multiple groups provided by the present invention provide a comprehensive picture of the executable method for recommending cancer therapy.

[0043] In certain embodiments, the target oncology sequence is directed to a sequence with a mutation associated with cancer. In some embodiments, the target sequence or amplified target sequence is directed to a sequence with a mutation associated with one or more solid tumor cancers, the one or more solid tumor cancers selected from the group consisting of: head and neck cancer (e.g., HNSCC, nasopharyngeal, salivary gland), brain cancer (e.g., glioblastoma, glioma, gliosarcoma, glioblastoma multiforme, neuroblastoma), breast cancer (e.g., TNBC, trastuzumab-resistant HER2+ breast cancer, ER+ / HER- breast cancer), gynecology (e.g., uterine, ovarian cancer, cervical cancer, endometrial cancer, fallopian tube cancer), Colorectal cancer, gallbladder cancer, esophageal cancer, gastrointestinal cancer, gastric cancer, bladder cancer, prostate cancer, testicular cancer, urothelial cancer, liver cancer (e.g., hepatocellular carcinoma, HCC), lung cancer (e.g., non-small cell lung, small cell lung), kidney (renal cell) cancer, pancreatic cancer (e.g., adenocarcinoma, ductal), thyroid cancer, bile duct cancer, pituitary tumor, Wilms' tumor, Kaposi's sarcoma, hairy cell carcinoma, osteosarcoma, thymic cancer, skin cancer, melanoma, heart cancer, oral cancer and laryngeal cancer, neuroblastoma, mesothelioma and other solid tumors (thymus, bone, soft tissue, oral SCC, myelofibrosis, synovial sarcoma). In one embodiment, the mutation can comprise substitution, insertion, inversion, point mutation, deletion, mispairing and translocation. In some embodiments, the target sequence or amplified target sequence is directed to a sequence having a mutation associated with one or more blood / blood cancers selected from multiple myeloma, diffuse large B-cell lymphoma (DLBCL), lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular lymphoma, leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), myelodysplastic syndrome. In one embodiment, the mutation biomarker associated with cancer is located in at least one of the genes provided in Table 1.

[0044] In some embodiments, the one or more mutant oncology sequences are mapped to at least one gene selected from the genes in Table 1. In some embodiments, the one or more mutant sequences are indicative of cancer activity.

[0045] In some embodiments, one or more mutant sequences indicate the likelihood that a patient will respond to a therapeutic agent. In some embodiments, one or more mutant oncology biomarker sequences indicate the likelihood that a patient will not respond to a therapeutic agent. In certain embodiments, the relevant therapeutic agent may be an oncology therapy, including but not limited to kinase inhibitors, cell signaling inhibitors, checkpoint blockers, T cell therapy, and therapeutic vaccines.

[0046] In some embodiments, the target sequence or mutant target sequence is directed to a mutation associated with cancer. In some embodiments, the target sequence or mutant target sequence is directed to a mutation associated with one or more solid tumor cancers, the one or more solid tumor cancers are selected from the group consisting of: head and neck cancer (e.g., HNSCC, nasopharyngeal, salivary gland), brain cancer (e.g., glioblastoma, glioma, gliosarcoma, glioblastoma multiforme, neuroblastoma), breast cancer (e.g., TNBC, trastuzumab-resistant HER2+ breast cancer, ER+ / HER- breast cancer), gynecology (e.g., uterine, ovarian cancer, cervical cancer, endometrial cancer, fallopian tube cancer), colorectal cancer, In one embodiment, mutation can include replacement, insertion, inversion, point mutation, deletion, mispairing and translocation. In one embodiment, mutation can include the change of copy number. In one embodiment, mutation can include germline or somatic mutation. In some embodiments, the target sequence or amplified target sequence is directed to a sequence having a mutation associated with one or more blood / blood cancers selected from multiple myeloma, diffuse large B-cell lymphoma (DLBCL), lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, follicular lymphoma, leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), myelodysplastic syndrome.

[0047] In one embodiment, the mutation associated with cancer is located in at least one gene provided in Table 1. In some embodiments, the mutant target sequence is directed to any one or more of the genes provided in Table 1. In some embodiments, the mutant target sequence comprises any one or more amplicon sequences of the genes provided in Table 1. In some embodiments, the mutant target sequence consists of any one or more amplicon sequences of the genes provided in Table 1. In some embodiments, the mutant target sequence comprises an amplicon sequence of each of the genes provided in Table 1.

[0048] In some embodiments, the composition includes any one or more of the oncology target-specific primer pairs provided in Table A. In some embodiments, the composition includes all of the oncology target-specific primer pairs provided in Table A. In some embodiments, any one or more of the oncology target-specific primer pairs provided in Table A can be used to amplify a target sequence present in a sample, as disclosed by the methods described herein.

[0049] In some embodiments, the oncology target-specific primers from Table A include 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or more target-specific primer pairs. In some embodiments, the amplified target sequence may include any one or more of the amplified target sequences generated using the target-specific primers provided in Table A. In some embodiments, at least one of the target-specific primers associated with cancer has at least 90% identity with at least one nucleic acid sequence generated using a target-specific primer selected from SEQ ID NO: 1-1559. In some embodiments, at least one of the target-specific primers associated with oncology is complementary to at least one target sequence in the sample over its entire length. In some embodiments, at least one of the target-specific primers includes a non-cleavable nucleotide at the 3' end. In some embodiments, the non-cleavable nucleotide at the 3' end includes the terminal 3' nucleotide. In one embodiment, the amplified target sequence is directed to one or more single exons having a mutation associated with cancer. In one embodiment, the amplified target sequence is directed to individual exons having a mutation associated with cancer.

[0050] method

[0051] The methods provided by the present invention include efficient procedures for rapid preparation of highly multiplexed libraries suitable for downstream analysis. The methods optionally allow for incorporation of one or more unique tag sequences. Certain methods include streamlined, additive-only procedures for extremely rapid library generation.

[0052] Provided herein is a method for determining oncological activity in a sample. In some embodiments, the method includes multiple amplification of multiple oncological sequences from a biological sample, wherein the amplification includes contacting at least a portion of the sample with multiple sets of primer pairs for multiple target sequences and a polymerase under amplification conditions, thereby producing an amplified target expression sequence. The method further includes detecting the presence of a mutation in one or more target sequences in the sample, wherein the mutation of one or more oncological markers determines the change in oncological activity in the sample compared to the control. In some embodiments, the oncological sequences of these methods are selected from the oncological response genes composed of the following functions: DNA hotspot mutation genes, copy number variation (CNV) genes, intergenic fusion genes, and intragenic fusion genes. In some embodiments, the target gene is selected from the oncological genes composed of one or more functions of Table 1. In some embodiments, the target gene is selected from one or more executable target genes in the sample, which determines the change in oncological activity indicating potential diagnosis, prognosis, candidate treatment options, and / or adverse events in the sample. In summary, the various functions of the genes provided by the multiple groups of the present invention provide a comprehensive picture of executable methods for recommending cancer therapy.

[0053] In certain embodiments, the target oncology sequence of the method is directed to a sequence with a mutation associated with cancer. In some embodiments, the target sequence or amplified target sequence is directed to a sequence with a mutation associated with one or more solid tumor cancers, the one or more solid tumor cancers selected from the group consisting of: head and neck cancer (e.g., HNSCC, nasopharyngeal, salivary gland), brain cancer (e.g., glioblastoma, glioma, gliosarcoma, glioblastoma multiforme, neuroblastoma), breast cancer (e.g., TNBC, trastuzumab-resistant HER2+ breast cancer, ER+ / HER- breast cancer), gynecology (e.g., uterine, ovarian cancer, cervical cancer, endometrial cancer, fallopian tube cancer), Colorectal cancer, gallbladder cancer, esophageal cancer, gastrointestinal cancer, gastric cancer, bladder cancer, prostate cancer, testicular cancer, urothelial cancer, liver cancer (e.g., hepatocellular carcinoma, HCC), lung cancer (e.g., non-small cell lung, small cell lung), kidney (renal cell) cancer, pancreatic cancer (e.g., adenocarcinoma, ductal), thyroid cancer, bile duct cancer, pituitary tumor, Wilms' tumor, Kaposi's sarcoma, hairy cell carcinoma, osteosarcoma, thymic cancer, skin cancer, melanoma, heart cancer, oral cancer and laryngeal cancer, neuroblastoma, mesothelioma and other solid tumors (thymus, bone, soft tissue, oral SCC, myelofibrosis, synovial sarcoma). In one embodiment, the mutation can comprise substitution, insertion, inversion, point mutation, deletion, mispairing and translocation. In some embodiments, the target sequence or amplified target sequence is directed to a sequence having a mutation associated with one or more blood / blood cancers selected from multiple myeloma, diffuse large B-cell lymphoma (DLBCL), lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular lymphoma, leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), myelodysplastic syndrome. In one embodiment, the mutation biomarker associated with cancer is located in at least one of the genes provided in Table 1.

[0054] In some embodiments, the one or more mutated oncology sequences of these methods are mapped to at least one gene selected from the genes in Table 1. In some embodiments, the one or more mutated sequences are indicative of cancer activity.

[0055] In some embodiments, one or more mutant sequences of these methods indicate the likelihood that a patient will respond to a therapeutic agent. In some embodiments, one or more mutant oncology biomarker sequences indicate the likelihood that a patient will not respond to a therapeutic agent. In certain embodiments, the relevant therapeutic agent may be an oncology therapy, including but not limited to kinase inhibitors, cell signaling inhibitors, checkpoint blockers, T cell therapy, and therapeutic vaccines.

[0056] In some embodiments, the target sequence or mutant target sequence of the method is directed to a mutation associated with cancer. In some embodiments, the target sequence or mutant target sequence of these methods is directed to a mutation associated with one or more solid tumor cancers, the one or more solid tumor cancers selected from the group consisting of: head and neck cancer (e.g., HNSCC, nasopharyngeal, salivary gland), selected from the group consisting of: head and neck cancer (e.g., HNSCC, nasopharyngeal, salivary gland), brain cancer (e.g., glioblastoma, glioma, gliosarcoma, glioblastoma multiforme, neuroblastoma), breast cancer (e.g., TNBC, trastuzumab-resistant HER2+ breast cancer, ER+ / HER- breast cancer), gynecology (e.g., uterine, ovarian cancer, cervical cancer), In one embodiment, the mutation may comprise a substitution, an insertion, an inversion, a point mutation, a deletion, a mismatch, and a translocation. In one embodiment, the mutation may comprise a change in copy number. In one embodiment, the mutation may comprise a germline or somatic mutation. In some embodiments, the target sequence or amplified target sequence is directed to a sequence having a mutation associated with one or more blood / blood cancers selected from multiple myeloma, diffuse large B-cell lymphoma (DLBCL), lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, follicular lymphoma, leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), myelodysplastic syndrome.

[0057] In one embodiment, the mutation associated with cancer is located in at least one gene provided in Table 1. In some embodiments, the mutant target sequence is directed to any one or more of the genes provided in Table 1. In some embodiments, the mutant target sequence comprises any one or more amplicon sequences of the genes provided in Table 1. In some embodiments, the mutant target sequence consists of any one or more amplicon sequences of the genes provided in Table 1. In some embodiments, the mutant target sequence comprises an amplicon sequence of each of the genes provided in Table 1.

[0058] In some embodiments, the methods include using any one or more of the oncology target-specific primer pairs provided in Table A. In some embodiments, the methods include using all of the oncology target-specific primer pairs provided in Table A. In some embodiments, using any one or more of the oncology target-specific primer pairs provided in Table A can be used to amplify a target sequence present in a sample, as disclosed by the methods described herein.

[0059] In some embodiments, the method comprises using an oncology target-specific primer from Table A comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or more target-specific primer pairs. In some embodiments, the method comprising detecting an amplified target sequence may comprise any one or more of the amplified target sequences generated using the target-specific primers provided in Table A. In some embodiments, the method comprises using at least one of the target-specific primers associated with cancer, having at least 90% identity to at least one nucleic acid sequence generated using a target-specific primer selected from SEQ ID NO: 1-1559. In some embodiments, at least one of the target-specific primers associated with oncology is complementary to at least one target sequence in the sample over its entire length. In some embodiments, at least one of the target-specific primers comprises a non-cleavable nucleotide at the 3' end. In some embodiments, the non-cleavable nucleotide at the 3' end comprises a terminal 3' nucleotide. In one embodiment, the amplified target sequence is directed to one or more single exons having a mutation associated with cancer. In one embodiment, the amplified target sequence is directed to a single exon having a mutation associated with cancer.

[0060] In some embodiments, the method includes detecting and optionally identifying clinically feasible markers. As defined herein, the term "clinically feasible marker" includes known or clinically feasible mutations and / or clinically feasible expression patterns that can be associated with (but not limited to) the prognosis of cancer treatment by a person of ordinary skill in the art. In one embodiment, the prognosis of cancer treatment includes the identification of mutations and / or expression patterns associated with cancer responding to or not responding to drugs, drug combinations, or treatment regimens. In one embodiment, the method includes amplification of multiple target sequences from a nucleic acid molecule population related to or associated with the onset, progression, or relief of cancer. In some embodiments, the method provided includes selective amplification of more than one target sequence in a sample and detection and / or identification of mutations associated with cancer. In some embodiments, the amplified target sequence includes two or more nucleotide sequences of the gene provided in Table 1. In some embodiments, the amplified target sequence may include any one or more amplified target sequences generated using the target-specific primers provided in Table A. In one embodiment, the amplified target sequence comprises 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or more amplicons from the genes in Table 1.

[0061] In one aspect of the present invention, a method for preparing a library of target nucleic acid sequences is provided. In some embodiments, the method includes contacting a nucleic acid sample with a plurality of adapters capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid undergoes a first amplification; digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and preparing partially digested amplicons, thereby producing double-stranded amplicons with gaps. These methods further include repairing the partially digested target amplicons; then, amplifying the repaired target amplicons in a second amplification using universal primers, thereby producing a library of target nucleic acid sequences. Each of the plurality of adapters used in the methods herein includes a universal handle sequence and a target nucleic acid sequence and a cleavable portion and optionally one or more tag sequences. The provided methods include at least two and up to one hundred thousand target-specific adapter pairs, wherein the target nucleic acid sequence of each adapter includes at least one cleavable portion and the universal handle sequence does not include a cleavable portion. In some embodiments in which at least one adapter includes an optional tag sequence, a cleavable portion is included in the adapter sequence at either end of the flanking tag sequence.

[0062] In one aspect of the present invention, a method for preparing a labeled library of target nucleic acid sequences is provided. In some embodiments, the method includes contacting a nucleic acid sample with a plurality of adapters capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid undergoes a first amplification; digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and preparing partially digested amplicons, thereby producing double-stranded amplicons with gaps. These methods further include repairing the partially digested target amplicons; then, amplifying the repaired target amplicons in a second amplification using universal primers, thereby producing a library of target nucleic acid sequences. Each of the plurality of adapters used in the methods herein includes a universal handle sequence and a target nucleic acid sequence and a cleavable portion and one or more tag sequences. The provided method includes at least two and up to one hundred thousand target-specific adapter pairs, wherein the target nucleic acid sequence of each adapter includes at least one cleavable portion, the universal handle sequence does not include a cleavable portion, and the cleavable portion is flanked by either end of the tag sequence.

[0063] In certain embodiments, the comparable maximum and minimum melting temperatures of each universal sequence are higher than the comparable maximum and minimum melting temperatures of each target nucleic acid sequence and each tag sequence present in the adaptor.

[0064] In some embodiments, each of these adaptors comprises a unique tag sequence as further described herein, and each further comprises a cleavable group flanking either end of the tag sequence in each adaptor. In some embodiments where unique tag sequences are used, each generated target-specific amplicon sequence comprises at least 1 different sequence and up to 10 7 In certain embodiments, each target-specific pair of the plurality of adaptors includes up to 16,777,216 different adaptor combinations comprising different tag sequences.

[0065] In some embodiments, the method comprises contacting a plurality of gapped polynucleotide products with a digestion reagent and a repair reagent simultaneously. In some embodiments, the method comprises contacting a plurality of gapped polynucleotide products with a digestion reagent and a repair reagent sequentially.

[0066] Digestion reagents suitable for use in the methods provided herein include any reagent capable of cleaving a cleavable site in a linker, and in some embodiments, include, but are not limited to, one or a combination of the following: uracil DNA glycosylase (UDG). apurinic endonuclease (e.g., APE1), RecJf, formamidopyrimidine [fapy]-DNA glycosylase (fpg), Nth endonuclease III, endonuclease VIII, polynucleotide kinase (PNK), Taq DNA polymerase, DNA polymerase I and / or human DNA polymerase β.

[0067] Repair reagents suitable for use in the methods provided herein include any reagent capable of repairing gapped amplicons, and in some embodiments, include, but are not limited to, one or a combination of the following: Phusion DNA polymerase, Phusion U DNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, human DNA polymerase β, T4 DNA polymerase and / or T7 DNA polymerase, SuperFiU DNA polymerase, Escherichia coli DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq DNA ligase, and / or 9°N DNA ligase.

[0068] Thus, in certain embodiments, the digestion and repair reagents include any one or a combination of the following: uracil DNA glycosylase (UDG), apurinic endonuclease (e.g., APE1), RecJf, formamidopyrimidine [fapy]-DNA glycosylase (fpg), Nth endonuclease III, endonuclease VIII, polynucleotide kinase (PNK), Taq DNA polymerase, DNA polymerase I and / or human DNA polymerase β; and any one or a combination of the following: Phusion DNA polymerase, PhusionU DNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, human DNA polymerase β, T4 DNA polymerase and / or T7 DNA polymerase, SuperFiU DNA polymerase, Escherichia coli DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq DNA ligase and / or 9°N DNA ligase. In certain embodiments, the digestion and repair reagents include any one or a combination of the following: uracil DNA glycosylase (UDG), apurinic endonuclease (e.g., APE1), Taq DNA polymerase, Phusion U DNA polymerase, SuperFiU DNA polymerase, T7 DNA ligase. In certain embodiments, the digestion and repair reagents include any one or a combination of the following: uracil DNA glycosylase (UDG), formamidopyrimidine [fapy]-DNA glycosylase (fpg), Phusion U DNA polymerase, Taq DNA polymerase, SuperFiU DNA polymerase, T4 PNK, and T7 DNA ligase.

[0069] In some embodiments, the method comprises performing the digestion step and the repair step in a single step. In other embodiments, the method comprises performing the digestion and repair steps at different temperatures in a short-time separated manner.

[0070] In some embodiments, the method of the present invention is carried out, wherein one or more steps of the method are carried out in manual mode. In a specific embodiment, the method of the present invention is carried out, wherein each step of the method is carried out in a manual mode. In some embodiments, the method of the present invention is carried out, wherein one or more steps of the method are carried out in an automatic mode. In a specific embodiment, the method of the present invention is carried out, wherein each step of the method is carried out automatically. In some embodiments, the method of the present invention is carried out, wherein one or more steps of the method are carried out in a combination of manual mode and automatic mode.

[0071] In some embodiments, the methods of the invention include at least one purification step. For example, in certain embodiments, a purification step is performed only after the second amplification of the repaired amplicon. In some embodiments, two purification steps are utilized, wherein a first purification step is performed after digestion and repair, and a second purification step is performed after the second amplification of the repaired amplicon.

[0072] In some embodiments, the purification step comprises performing a solid phase adhesion reaction, a solid phase immobilization reaction, or gel electrophoresis. In certain embodiments, the purification step comprises separation using solid phase reversible immobilization (SPRI) beads. In specific embodiments, the purification step comprises separation using SPRI beads, wherein the SPRI beads comprise paramagnetic beads.

[0073] In some embodiments, the method includes contacting a nucleic acid sample with a plurality of adapters capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid undergoes a first amplification; digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and preparing partially digested amplicons, thereby producing a double-stranded amplicon with a gap. These methods further include repairing the partially digested target amplicon; then, purifying the repaired amplicon; then, amplifying the repaired target amplicon in a second amplification using universal primers, thereby producing a library of target nucleic acid sequences; then purifying the resulting library. Each of the plurality of adapters used in the methods herein includes a universal handle sequence and a target nucleic acid sequence and a cleavable portion and optionally one or more tag sequences. The provided methods include at least two and up to one hundred thousand target-specific adapter pairs, wherein the target nucleic acid sequence of each adapter includes at least one cleavable portion and the universal handle sequence does not include a cleavable portion. In some embodiments in which at least one adapter includes an optional tag sequence, a cleavable portion is included in the adapter sequence at either end of the flanking tag sequence.

[0074] In some embodiments, the method includes contacting a nucleic acid sample with a plurality of adapters capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid undergoes a first amplification; digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and preparing partially digested amplicons, thereby producing a double-stranded amplicon with a gap. These methods further include repairing the partially digested target amplicon; then, and purifying the repaired amplicon; then, amplifying the repaired target amplicon in a second amplification using universal primers, thereby producing a library of target nucleic acid sequences; then purifying the resulting library. Each of the plurality of adapters used in the methods herein includes a universal handle sequence and a target nucleic acid sequence and a cleavable portion and one or more tag sequences. The provided methods include at least two and up to one hundred thousand target-specific adapter pairs, wherein the target nucleic acid sequence of each adapter includes at least one cleavable portion, the universal handle sequence does not include a cleavable portion, and the cleavable portion is included at either end of the flanking tag sequence.

[0075] In some embodiments, the method includes contacting a nucleic acid sample with a plurality of adapters capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid undergoes a first amplification; digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and preparing partially digested amplicons, thereby producing a double-stranded amplicon with a gap. These methods further include repairing the partially digested target amplicon; then, purifying the repaired amplicon; then, amplifying the repaired target amplicon in a second amplification using universal primers, thereby producing a library of target nucleic acid sequences; then purifying the resulting library. Each of the plurality of adapters used in the methods herein includes a universal handle sequence and a target nucleic acid sequence and a cleavable portion and optionally one or more tag sequences. The provided methods include at least two and up to one hundred thousand target-specific adapter pairs, wherein the target nucleic acid sequence of each adapter includes at least one cleavable portion and the universal handle sequence does not include a cleavable portion. In some embodiments in which at least one adapter includes an optional tag sequence, a cleavable portion is included in the adapter sequence at either end of the flanking tag sequence. In some embodiments, the digestion and repair reagents include any one or a combination of the following: uracil DNA glycosylase (UDG), apurinic endonuclease (e.g., APE1), RecJf, formamidopyrimidine [fapy]-DNA glycosylase (fpg), Nth endonuclease III, endonuclease VIII, polynucleotide kinase (PNK), Taq DNA polymerase, DNA polymerase I and / or human DNA polymerase β; and any one or a combination of the following: Phusion DNA polymerase, Phusion U DNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, human DNA polymerase β, T4 DNA polymerase and / or T7 DNA polymerase, SuperFiU DNA polymerase, Escherichia coli DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq DNA ligase and / or 9°N DNA ligase. In certain embodiments, the digestion and repair reagents include any one or a combination of the following: uracil DNA glycosylase (UDG), apurinic endonuclease (e.g., APE1), Taq DNA polymerase, Phusion U DNA polymerase, SuperFiU DNA polymerase, T7 DNA ligase. In certain embodiments, the digestion and repair reagents include any one or a combination of the following: uracil DNA glycosylase (UDG), formamidopyrimidine [fapy]-DNA glycosylase (fpg), Phusion U DNA polymerase, Taq DNA polymerase, SuperFiU DNA polymerase, T4 PNK, and T7 DNA ligase.

[0076] In some embodiments, the method includes contacting a nucleic acid sample with a plurality of adapters capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid undergoes a first amplification; digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and preparing partially digested amplicons, thereby producing a double-stranded amplicon with a gap. These methods further include repairing the partially digested target amplicon; then, and purifying the repaired amplicon; then, amplifying the repaired target amplicon in a second amplification using universal primers, thereby producing a library of target nucleic acid sequences; then purifying the resulting library. Each of the plurality of adapters used in the methods herein includes a universal handle sequence and a target nucleic acid sequence and a cleavable portion and one or more tag sequences. The provided methods include at least two and up to one hundred thousand target-specific adapter pairs, wherein the target nucleic acid sequence of each adapter includes at least one cleavable portion, the universal handle sequence does not include a cleavable portion, and the cleavable portion is included at either end of the flanking tag sequence. In some embodiments, the digestion and repair reagents include any one or a combination of the following: any one or a combination of the following: uracil DNA glycosylase (UDG), apurinic endonuclease (e.g., APE1), RecJf, formamidopyrimidine [fapy]-DNA glycosylase (fpg), Nth endonuclease III, endonuclease VIII, polynucleotide kinase (PNK), Taq DNA polymerase, DNA polymerase I and / or human DNA polymerase β; and any one or a combination of the following: Phusion DNA polymerase, Phusion U DNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, human DNA polymerase β, T4 DNA polymerase and / or T7 DNA polymerase, SuperFiU DNA polymerase, Escherichia coli DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq DNA ligase and / or 9°N DNA ligase. In certain embodiments, the digestion and repair reagents include any one or a combination of the following: uracil DNA glycosylase (UDG), apurinic endonuclease (e.g., APE1), Taq DNA polymerase, Phusion U DNA polymerase, SuperFiU DNA polymerase, T7 DNA ligase. In certain embodiments, the digestion and repair reagents include any one or a combination of the following: uracil DNA glycosylase (UDG), formamidopyrimidine [fapy]-DNA glycosylase (fpg), Phusion U DNA polymerase, Taq DNA polymerase, SuperFiU DNA polymerase, T4 PNK, and T7 DNA ligase.

[0077] In certain embodiments, the methods of the present invention are performed in a single, addition-only workflow reaction, enabling rapid generation of highly multiplexed target libraries. For example, in one embodiment, a method for preparing a library of target nucleic acid sequences comprises contacting a nucleic acid sample with a plurality of adapters capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid undergoes a first amplification; digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and preparing partially digested amplicons, thereby producing a double-stranded amplicon with a gap. These methods further include repairing the partially digested target amplicons; then, amplifying the repaired target amplicons in a second amplification using universal primers, thereby producing a library of target nucleic acid sequences, and purifying the resulting library. In certain embodiments, purification comprises a single or repeated separation step; the step is performed after the library is produced after the second amplification; and the other steps of the method are performed in a single reaction vessel without transferring a portion (aliquot) of any product produced in the step to another reaction vessel. Each of the plurality of adapters used in the methods herein comprises a universal handle sequence and a target nucleic acid sequence and a cleavable portion and optionally one or more tag sequences. The methods provided include at least two and up to one hundred thousand target-specific adapter pairs, wherein the target nucleic acid sequence of each adapter comprises at least one cleavable moiety and the universal handle sequence does not comprise a cleavable moiety. In some embodiments in which at least one adapter comprises an optional tag sequence, the adapter sequence flanking either end of the tag sequence comprises a cleavable moiety.

[0078] In another embodiment, methods for preparing a labeled library of target nucleic acid sequences are provided, the methods comprising contacting a nucleic acid sample with a plurality of adapters capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid undergoes a first amplification; digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and preparing partially digested amplicons, thereby producing double-stranded amplicons with gaps. The methods further include repairing the partially digested target amplicons; then, amplifying the repaired target amplicons in a second amplification using universal primers, thereby producing a library of target nucleic acid sequences, and purifying the resulting library. In certain embodiments, purification comprises a single or repeated separation step; and wherein the other steps of the method are optionally performed in a single reaction vessel without transferring a portion of any product produced in the step to another reaction vessel. Each of the plurality of adapters used in the methods herein comprises a universal handle sequence and a target nucleic acid sequence as well as a cleavable portion and one or more tag sequences. The provided methods include at least two and up to one hundred thousand target-specific adaptor pairs, wherein the target nucleic acid sequence of each adaptor comprises at least one cleavable portion, the universal handle sequence does not comprise a cleavable portion, and the cleavable portions flank either end of the tag sequence.

[0079] In one embodiment, a method for preparing a library of target nucleic acid sequences includes contacting a nucleic acid sample with a plurality of adapters capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid undergoes a first amplification; digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and preparing partially digested amplicons, thereby generating gapped double-stranded amplicons. These methods further include repairing the partially digested target amplicons; then, amplifying the repaired target amplicons in a second amplification using universal primers, thereby generating a library of target nucleic acid sequences, and purifying the resulting library.

[0080] In some embodiments, the digestion reagent includes any one or any combination of the following: uracil DNA glycosylase (UDG), AP endonuclease (APE1), RecJf, formamidopyrimidine [fapy]-DNA glycosylase (fpg), Nth endonuclease III, endonuclease VIII, polynucleotide kinase, Taq DNA polymerase, DNA polymerase I and / or human DNA polymerase β; in certain embodiments, the digestion reagent includes any one or any combination of the following: uracil DNA glycosylase (UDG), AP endonuclease (APE1), RecJf, formamidopyrimidine [fapy]-DNA glycosylase (fpg), Nth endonuclease III, endonuclease VIII, polynucleotide kinase, Taq DNA polymerase, DNA polymerase I and / or human DNA polymerase β, wherein the digestion reagent does not contain formamidopyrimidine [fapy]-DNA glycosylase (fpg).

[0081] In some embodiments, the digestion reagent comprises a single-stranded DNA exonuclease that degrades in the 5'-3' direction. In some embodiments, the cleavage reagent comprises a single-stranded DNA exonuclease that degrades abasic sites. In some embodiments herein, the digestion reagent comprises a RecJf exonuclease. In specific embodiments, the digestion reagent comprises APE1 and RecJf, wherein the cleavage reagent comprises an apurinic / apyrimidinic endonuclease. In certain embodiments, the digestion reagent comprises an AP endonuclease (APE1).

[0082] In some embodiments, the repair reagent comprises at least one DNA polymerase; wherein the gap filling reagent comprises: any one or any combination of the following: Phusion DNA polymerase, Phusion UDNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, human DNA polymerase β, T4 DNA polymerase and / or T7 DNA polymerase and / or SuperFi UDNA polymerase. In some embodiments, the repair reagent further comprises a plurality of nucleotides.

[0083] In some embodiments, the repair reagent comprises an ATP-dependent or ATP-independent ligase; wherein the repair reagent comprises any one or any combination of the following: Escherichia coli DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq DNA ligase, 9°N DNA ligase,

[0084] In certain embodiments, the digestion and repair reagents include any one or a combination of the following: uracil DNA glycosylase (UDG), apurinic endonuclease (e.g., APE1), RecJf, formamidopyrimidine [fapy]-DNA glycosylase (fpg), Nth endonuclease III, endonuclease VIII, polynucleotide kinase (PNK), Taq DNA polymerase, DNA polymerase I and / or human DNA polymerase β; and any one or a combination of the following: Phusion DNA polymerase, Phusion UDNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, human DNA polymerase β, T4 DNA polymerase and / or T7DNA polymerase, SuperFiU DNA polymerase, Escherichia coli DNA ligase, T3 DNA ligase, T4 DNA ligase, T7DNA ligase, Taq DNA ligase and / or 9°N DNA ligase. In certain embodiments, the digestion and repair reagents include any one or a combination of the following: uracil DNA glycosylase (UDG), apurinic endonuclease (e.g., APE1), Taq DNA polymerase, Phusion U DNA polymerase, SuperFiU DNA polymerase, T7 DNA ligase. In certain embodiments, purification includes a single or repeated separation step; the step is performed after the library is generated after the second amplification; and wherein the method steps are performed in a single reaction vessel without transferring a portion of any product produced in the step to another reaction vessel until the first purification. Each of the multiple adapters used in the methods herein includes a universal handle sequence and a target nucleic acid sequence and a cleavable portion and optionally one or more tag sequences. The provided method includes at least two and up to one hundred thousand target-specific adapter pairs, wherein the target nucleic acid sequence of each adapter includes at least one cleavable portion and the universal handle sequence does not include a cleavable portion. In some embodiments in which at least one adapter includes an optional tag sequence, a cleavable portion is included in the adapter sequence at either end of the flanking tag sequence.

[0085] In another embodiment, methods for preparing a labeled library of target nucleic acid sequences are provided, the methods comprising contacting a nucleic acid sample with a plurality of adapters capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid undergoes a first amplification; digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and preparing partially digested amplicons, thereby producing a gapped double-stranded amplicon. The methods further comprise repairing the partially digested target amplicons; then, amplifying the repaired target amplicons in a second amplification using universal primers, thereby producing a library of target nucleic acid sequences, and purifying the resulting library. In certain embodiments, the digestion and repair reagents include any one or a combination of the following: uracil DNA glycosylase (UDG), apurinic endonuclease (e.g., APE1), RecJf, formamidopyrimidine [fapy]-DNA glycosylase (fpg), Nth endonuclease III, endonuclease VIII, polynucleotide kinase (PNK), Taq DNA polymerase, DNA polymerase I and / or human DNA polymerase β; and any one or a combination of the following: Phusion DNA polymerase, Phusion U DNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, human DNA polymerase β, T4 DNA polymerase and / or T7 DNA polymerase, SuperFiU DNA polymerase, Escherichia coli DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq DNA ligase and / or 9°N DNA ligase. In certain embodiments, the digestion and repair reagents include any one or a combination of the following: uracil DNA glycosylase (UDG), apurinic endonuclease (e.g., APE1), Taq DNA polymerase, Phusion U DNA polymerase, SuperFiU DNA polymerase, T7 DNA ligase. In certain embodiments, purification includes a single or repeated separation step; the step is performed after the library is generated after the second amplification; and the steps of the method are performed in a single reaction vessel without transferring a portion (aliquot) of any product produced in the step to another reaction vessel. Each of the multiple adapters used in the method herein includes a universal handle sequence and a target nucleic acid sequence and a cleavable portion and one or more tag sequences. The provided method includes at least two and up to one hundred thousand target-specific adapter pairs, wherein the target nucleic acid sequence of each adapter includes at least one cleavable portion, the universal handle sequence does not include a cleavable portion, and the cleavable portion is flanked by either end of the tag sequence.

[0086] In some embodiments, the adapter-dimer byproduct produced by the first amplification step in the method is substantially removed from the resulting library. In certain embodiments, the enriched body of amplified target nucleic acid contains a reduced amount of adapter-dimer byproducts. In specific embodiments, the adapter-dimer byproduct is eliminated.

[0087] In some embodiments, the library is prepared in less than 4 hours. In some embodiments, the library is prepared, enriched, and sequenced in less than 3 hours. In some embodiments, the library is prepared, enriched, and sequenced in 2 to 3 hours. In some embodiments, the library is prepared in about 2.5 hours. In some embodiments, the library is prepared in about 2.75 hours. In some embodiments, the library is prepared in about 3 hours.

[0088] Composition

[0089] Other aspects of the invention include compositions comprising a plurality of nucleic acid adaptors, and library compositions prepared according to the methods of the invention. The provided compositions are suitable for use in conjunction with the methods described herein and for other analyses and applications known in the art.

[0090] Thus, a composition comprising a plurality of nucleic acid adaptors is provided, wherein each adaptor of the plurality of adaptors comprises a 5' universal handle sequence, optionally one or more tag sequences, and a 3' target nucleic acid sequence, wherein each adaptor comprises a cleavable portion, wherein the target nucleic acid sequence of the adaptor comprises at least one cleavable portion, and when a tag sequence is present, comprises a cleavable portion flanking either end of the tag sequence, and wherein the universal handle sequence does not comprise a cleavable portion. The provided composition comprises at least two and up to one hundred thousand target-specific adaptor pairs. The provided composition enables rapid generation of highly multiplexed target libraries.

[0091] In some embodiments, the provided compositions include a plurality of nucleic acid adaptors, wherein each adaptor in the plurality of adaptors includes a 5' universal handle sequence, one or more tag sequences, and a 3' target nucleic acid sequence, wherein each adaptor includes a cleavable portion; wherein the target nucleic acid sequence of the adaptor includes at least one cleavable portion, the cleavable portion flanking either end of the tag sequence, and the universal fixed structure sequence does not include the cleavable portion. The provided compositions include at least two and up to one hundred thousand target-specific adaptor pairs. The provided compositions enable rapid generation of highly multiplexed, labeled target libraries.

[0092] The primer / adapter composition can be single-stranded or double-stranded. In some embodiments, the adaptor composition includes a single-stranded adaptor. In some embodiments, the adaptor composition includes a double-stranded adaptor. In some embodiments, the adaptor composition includes a mixture of single-stranded adaptors and double-stranded adaptors.

[0093] In some embodiments, the composition comprises a plurality of adaptors capable of amplifying one or more target nucleic acid sequences, including a plurality of adaptor pairs capable of amplifying at least two different target nucleic acid sequences, wherein the target-specific primer sequence is substantially non-complementary to other target-specific primer sequences in the composition. In some embodiments, the composition comprises at least 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4500, 5000, 5500, 6000, 7000, 8000, 9000, 10,000, 11,000 or 12,000 or more target-specific adaptor pairs. In some embodiments, the length of the target-specific adapter pair includes about 15 nucleotides to about 40 nucleotides, wherein at least one nucleotide is replaced by a cleavable group. In some embodiments, the cleavable group is a uridine nucleotide. In some embodiments, the target-specific adapter pair is designed to amplify the exons, genes, exon groups or regions of the genome associated with clinical or pathological conditions, such as amplifying one or more mutations (such as driver mutations) associated with cancer (such as lung cancer, colon cancer, breast cancer, etc.), or amplifying mutations associated with genetic diseases (such as cystic fibrosis, muscular dystrophy, etc.). In some embodiments, when hybridized with the target sequence and amplified as provided herein, the target-specific adapter pair produces a library of amplified target sequences of adapters of about 100 to about 600 base pairs in length. In some embodiments, compared with the rest of the amplified target sequences of other adapters in the library, the amplified target sequences of no adapter are overexpressed in the library by more than 30%. In some embodiments, the adaptor-ligated amplified target sequence library is substantially homogenous in terms of GC content, length of the amplified target sequences, or melting temperature (Tm) of the respective target sequences.

[0094] In some embodiments, the target-specific primer sequence of the adapter pair in the composition of the present invention is a target-specific sequence that can amplify a specific region of a nucleic acid molecule. In some embodiments, the target-specific adapter can amplify genomic DNA or cDNA. In some embodiments, the target-specific adapter can amplify mammalian nucleic acids, such as (but not limited to) human DNA or RNA, murine DNA or RNA, bovine DNA or RNA, canine DNA or RNA, horse DNA or RNA, or any other related mammal. In other embodiments, the target-specific adapter comprises a sequence associated with amplifying a plant nucleic acid of interest. In other embodiments, the target-specific adapter comprises a sequence associated with amplifying an infectious agent (e.g., bacterial and / or viral nucleic acid). In some embodiments, the amount of nucleic acid required for selective amplification is about 1 ng to 1 microgram. In some embodiments, the amount of nucleic acid required for selective amplification of one or more target sequences is about 1 ng, about 5 ng, or about 10 ng. In some embodiments, the amount of nucleic acid required for selective amplification of a target sequence is about 10 ng to about 200 ng.

[0095] As described herein, each adapter in a plurality of adapters includes a 5' universal handle sequence. In some embodiments, the universal handle sequence includes any one or any combination of an amplification primer binding sequence, a sequencing primer binding sequence, and / or a capture primer binding sequence. In some embodiments, the comparable maximum minimum melting temperature of each adapter universal handle sequence is higher than the comparable maximum minimum melting temperature of each target nucleic acid sequence and each tag sequence present in the same adapter. Preferably, the universal handle sequence of the adapter provided does not present significant complementarity and / or hybridization with any part of the unique tag sequence and / or target nucleic acid sequence of interest. In some embodiments, the first universal handle sequence includes any one or any combination of an amplification primer binding sequence, a sequencing primer binding sequence, and / or a capture primer binding sequence. In some embodiments, the second universal handle sequence includes any one or any combination of an amplification primer binding sequence, a sequencing primer binding sequence, and / or a capture primer binding sequence. In certain embodiments, the first universal handle sequence and the second universal handle sequence correspond to forward and reverse universal handle sequences and in certain embodiments, each target-specific adapter pair in a plurality of target-specific adapter pairs comprises the same first universal handle sequence and the second universal handle sequence. In the generation of libraries according to the methods of the present invention, such forward and reverse universal handle sequences and universal primers are targeted for a second amplification of the repaired amplicons. In certain embodiments, the first 5' universal handle sequence comprises two universal handle sequences (e.g., a combination of an amplification primer binding sequence, a sequencing primer binding sequence, and / or a capture primer binding sequence); and the second 5' universal sequence comprises two universal handle sequences (e.g., a combination of an amplification primer binding sequence, a sequencing primer binding sequence, and / or a capture primer binding sequence), wherein the 5' first and second universal handle sequences do not exhibit significant hybridization with any portion of the target nucleic acid sequence of interest.

[0096] The structures and properties of universal amplification primers or universal primers are well known to those skilled in the art and can be implemented for use with the provided methods and compositions to suit specific analytical platforms. The universal handle sequences of the adapters provided herein are adjusted accordingly to accommodate the preferred universal primer sequences. For example, as described herein, universal P1 and A primers with optional barcode sequences have been described in the art and used for the Ion Torrent sequencing platform (IonXpress TMAdapter, Thermo Fisher Scientific (Thermo Fisher Scientific). Similarly, additional and other universal adapter / primer sequences described and known in the art (e.g., Illumina universal adapter / primer sequences can be found in, e.g., support.illumina.com / content / dam / illumina-support / documents / documentation / chemistry_documentation / experiment-design / illumina-adapter-sequences_1000000002694-01.pdf; PacBio universal adapter / primer sequences can be found in, e.g., s3.amazonaws.com / files.pacb.com / pdf / Guide_Pacific_Biosciences_Template_Preparation_and_Sequencing.pdf; etc.) can be used in conjunction with the methods and compositions provided herein. Suitable universal primers for nucleotide sequences suitable for use with the adapters of the present invention can be readily prepared using standard automated nucleic acid synthesis instruments and reagents commonly used in the art. A single type of universal primer or two different universal primers (or even a mixture) of separate types (e.g., a pair of universal amplification primers suitable for amplification of the repaired amplicon in the second amplification) is used in the method of the present invention. The universal primers optionally contain different tag (barcode) sequences, wherein the tag (barcode) sequence does not hybridize with the adapter. The barcode sequence incorporated into the amplicon in the second universal amplification can, for example, be used for effective identification of the sample source.

[0097] In some embodiments, the adapter further comprises a unique tag sequence located between the 5' first universal handle sequence and the 3' target-specific sequence, and wherein the unique tag sequence does not exhibit significant complementarity and / or hybridization with any portion of the unique tag sequence and / or target nucleic acid sequence of interest. 4 -10 9 Thus, in certain embodiments, each target-specific adapter pair generated comprises 10 4 -10 9 In some embodiments, the plurality of primer adapters includes each target-specific adapter including at least one different unique tag sequence and up to 10 5 In some embodiments, the plurality of primer adapters includes each target-specific adapter including at least one different unique tag sequence and up to 105 In certain embodiments, each target-specific amplicon generated includes at least two and up to 10 9 In some embodiments, the plurality of primer adapters comprises a plurality of different adapter combinations comprising different tag sequences, each of which has two different unique tag sequences. In some embodiments, the plurality of primer adapters comprises each target-specific adapter, which comprises 4096 different tag sequences. In some embodiments, each generated target-specific amplicon comprises up to 16,777,216 different adapter combinations comprising different tag sequences, each of which has two different unique tag sequences.

[0098] In some embodiments, a separate primer adapter in a plurality of adapters comprises a unique tag sequence (e.g., contained in a tag adapter) comprising different random tag sequences alternating with fixed tag sequences. In some embodiments, at least one unique tag sequence comprises at least one random sequence and at least one fixed sequence, or comprises a random sequence flanked by a fixed sequence on both sides, or comprises a fixed sequence flanked by a random sequence on both sides. In some embodiments, the unique tag sequence comprises a fixed sequence, the length of which is 2-2000 nucleotides or base pairs. In some embodiments, the unique tag sequence comprises a random sequence, the length of which is 2-2000 nucleotides or base pairs.

[0099] In some embodiments, the unique tag sequence comprises a sequence having at least one random sequence interspersed with a fixed sequence. In some embodiments, the individual tag sequences in the plurality of unique tags have the structure (N) n (X) x (M) m (Y) y, wherein "N" represents a random tag sequence generated from A, G, C, T, U or I, and wherein "N" is 2-10, indicating the nucleotide length of the "N" random tag sequence; wherein "X" represents a fixed tag sequence, and wherein "X" is 2-10, indicating the nucleotide length of the "X" random tag sequence; wherein "M" represents a random tag sequence generated from A, G, C, T, U or I, wherein the random tag sequence "M" is different or the same as the random tag sequence "N", and wherein "M" is 2-10, indicating the nucleotide length of the "M" random tag sequence; and wherein "Y" represents a fixed tag sequence, wherein the fixed tag sequence of "Y" is the same or different from the fixed tag sequence of "X", and wherein "Y" is 2-10, indicating the nucleotide length of the "Y" random tag sequence. In some embodiments, the fixed tag sequence "X" is the same in multiple tags. In some embodiments, the fixed tag sequence "X" is different in multiple tags. In some embodiments, the fixed tag sequence "Y" is the same in multiple tags. In some embodiments, the fixed tag sequence "Y" is different in the plurality of tags. In some embodiments, the fixed tag sequence "(X)" within the plurality of single-stranded adaptors is different in the plurality of single-stranded adaptors. x ” and “(Y) y ” is the sequence alignment anchor.

[0100] In some embodiments, the random sequence within the unique tag sequence is represented by "N", and the fixed sequence is represented by "X". Therefore, the unique tag sequence is represented by N1N2N3X1X2X3 or N1N2N3X1X2X3N4N5N6X4X5X6. Optionally, the unique tag sequence can have a random sequence, in which some or all of the nucleotide positions are randomly selected from the group consisting of: A, G, C, T, U and I. For example, the nucleotides at each position within the random sequence are independently selected from any one of A, G, C, T, U or I, or from a subset of these six different types of nucleotides. Optionally, the nucleotides at each position within the random sequence are independently selected from any one of A, G, C or T. In some embodiments, the first fixed tag sequence "X1X2X3" is the same or different sequence in multiple tags. In some embodiments, the second fixed tag sequence "X4X5X6" is the same or different sequence in multiple tags. In some embodiments, a first fixed tag sequence "X1X2X3" and a second fixed tag sequence "X4X5X6" within a plurality of adaptors are sequence alignment anchors.

[0101] In some embodiments, the unique tag sequence comprises the sequence 5'-NNNACTNNNTGA-3', where "N" represents a position within the random sequence randomly generated from A, G, C, or T, calculated as 4 6(or 4^6) is about 4096 possible different random labels, and the possible different combinations of two unique labels are 4 12 (or 4^12) is about 16.78 million. In some embodiments, 5'- NNN ACT NNN The underlined portion in TGA-3' is the sequence alignment anchor.

[0102] In some embodiments, a fixed sequence within a unique tag sequence is a sequence alignment anchor that can be used to generate error-corrected sequencing data. In some embodiments, a fixed sequence within a unique tag sequence is a sequence alignment anchor that can be used to generate error-corrected sequencing read families.

[0103] The adapters provided herein include at least one cleavable portion. In some embodiments, the cleavable portion is within the 3' target-specific sequence. In some embodiments, the cleavable portion is located at or near the junction between the 5' first universal handle sequence and the 3' target-specific sequence. In some embodiments, the cleavable portion is located at or near the junction between the 5' first universal handle sequence and the unique tag sequence, and at or near the junction between the unique tag sequence and the 3' target-specific sequence. The cleavable portion may be present in a modified nucleotide, nucleoside, or nucleobase. In some embodiments, the cleavable portion may comprise a nucleobase that does not naturally occur in the target sequence of interest.

[0104] In some embodiments, at least one cleavable portion of a plurality of adapters is a uracil base, a uridine or a deoxyuridine nucleotide. In some embodiments, the cleavable portion is within the 3' target-specific sequence and is located at the junction between the 5' universal handle sequence and the unique tag sequence and / or the 3' target-specific sequence, wherein at least one cleavable portion of a plurality of adapters is capable of being cleaved by uracil DNA glycosylase (UDG). In some embodiments, the cleavable portion is cleaved to produce a sensitive abasic site, wherein at least one enzyme capable of reacting at the abasic site produces a gap including an extendable 3' end. In certain embodiments, the resulting gap includes a 5'-deoxyribose phosphate group. In certain embodiments, the resulting gap includes an extendable 3' end and a 5' engageable phosphate group.

[0105] In another embodiment, inosine can be incorporated into a DNA-based nucleic acid as a cleavable group. In an exemplary embodiment, EndoV can be used to cleave near an inosine residue. In another exemplary embodiment, the enzyme hAAG can be used to cleave an inosine residue from a nucleic acid that produces an abasic site.

[0106] When there is a cleavable portion, the position of at least one cleavable portion in the adapter does not significantly change the melting temperature (Tm) of any given double-stranded adapter in the multiple double-stranded adapters. The melting temperatures (Tm) of any two given double-stranded adapters from the multiple double-stranded adapters are substantially the same, wherein the melting temperatures (Tm) of any two given double-stranded adapters differ from each other by no more than 10°C. However, within each adapter in the multiple adapters, the melting temperatures of the sequence regions are different, such that, for example, the comparable maximum and minimum melting temperatures of the universal handle sequence are higher than the comparable maximum and minimum melting temperatures of the unique tag sequence and / or target-specific sequence of any adapter. This local difference in the comparable maximum and minimum melting temperatures can be adjusted to optimize the digestion and repair of the amplicons, and ultimately improve the effectiveness of the methods provided herein.

[0107] Also provided are compositions comprising nucleic acid libraries produced by the methods of the present invention. Thus, compositions comprising a plurality of amplified target nucleic acid amplicons are provided, wherein each amplicon in the plurality of amplicons comprises a 5' universal handle sequence, an optional first unique tag sequence, an intermediate target nucleic acid sequence, an optional second unique tag sequence, and a 3' universal handle sequence. At least two and up to one hundred thousand target-specific amplicons are contained in the provided compositions. The provided compositions comprise highly multiplexed target libraries. In some embodiments, the provided compositions comprise a plurality of nucleic acid amplicons, wherein each amplicon in the plurality of amplicons comprises a 5' universal handle sequence, a first unique tag sequence, an intermediate target nucleic acid sequence, a second unique tag sequence, and a 3' universal handle sequence. At least two and up to one hundred thousand target-specific labeled amplicons are contained in the provided compositions. The provided compositions comprise highly multiplexed labeled target libraries.

[0108] In some embodiments, the library composition comprises a plurality of target-specific amplicons comprising a multiplex of at least two different target nucleic acid sequences. In some embodiments, the composition comprises at least 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4500, 5000, 5500, 6000, 7000, 8000, 9000, 10,000, 11,000 or 12,000 or more target-specific amplicons. In some embodiments, the target-specific amplicons include one or more exons, genes, exomes, or regions of a genome associated with a clinical or pathological condition, such as amplicons comprising one or more loci comprising one or more mutations (e.g., driver mutations) associated with cancer (e.g., lung cancer, colon cancer, breast cancer, etc.), or amplicons comprising mutations associated with a genetic disease (e.g., cystic fibrosis, muscular dystrophy, etc.). In some embodiments, the target-specific amplicons comprise a library of adapter-ligated amplicon target sequences that are about 100 to about 750 base pairs in length.

[0109] As described herein, each amplicon in a plurality of amplicons includes a 5' universal handle sequence. In some embodiments, the universal handle sequence includes any one or any combination of an amplification primer binding sequence, a sequencing primer binding sequence, and / or a capture primer binding sequence. Preferably, the universal handle sequence of the adapter provided does not present significant complementarity and / or hybridization with any portion of the unique tag sequence and / or target nucleic acid sequence of interest. In some embodiments, the first universal handle sequence includes any one or any combination of an amplification primer binding sequence, a sequencing primer binding sequence, and / or a capture primer binding sequence. In some embodiments, the second universal handle sequence includes any one or any combination of an amplification primer binding sequence, a sequencing primer binding sequence, and / or a capture primer binding sequence. In certain embodiments, the first universal handle sequence and the second universal handle sequence correspond to forward and reverse universal handle sequences and in certain embodiments, each target-specific amplicon in a plurality of target-specific amplicons includes the same first universal handle sequence and the second universal handle sequence. In the production of the resulting amplified product according to the method of the present invention, such forward and reverse universal handle sequences and universal primers are targeted to perform a second amplification of the primary library composition. In certain embodiments, the first 5' universal handle sequence comprises two universal handle sequences (e.g., a combination of an amplification primer binding sequence, a sequencing primer binding sequence, and / or a capture primer binding sequence); and the second 5' universal sequence comprises two universal handle sequences (e.g., a combination of an amplification primer binding sequence, a sequencing primer binding sequence, and / or a capture primer binding sequence), wherein the 5' first and second universal handle sequences do not exhibit significant hybridization with any portion of the target nucleic acid sequence of interest.

[0110] The structure and properties of universal amplification primers or universal primers are well known to those skilled in the art and can be implemented for use with the provided methods and compositions to suit specific analytical platforms. The universal handle sequences of the adapters and amplicons provided herein are adjusted accordingly to accommodate preferred universal primer sequences. For example, as described herein, universal P1 and A primers with optional barcode sequences have been described in the art and are used for the Ion Torrent sequencing platform (Ion Xpress TMAdapter, Thermo Fisher Scientific). Similarly, additional and other universal adapter / primer sequences described and known in the art (e.g., Illumina universal adapter / primer sequences can be found in, e.g., support.illumina.com / content / dam / illumina-support / documents / documentation / chemistry_documentation / experiment-design / illumina-adapter-sequences_1000000002694-01.pdf; PacBio universal adapter / primer sequences can be found in, e.g., s3.amazonaws.com / files.pacb.com / pdf / Guide_Pacific_Biosciences_Template_Preparation_and_Sequencing.pdf; etc.) can be used in conjunction with the methods and compositions provided herein. Suitable universal primers for nucleotide sequences suitable for use with the libraries of the present invention can be readily prepared using standard automated nucleic acid synthesis instruments and reagents commonly used in the art. Two different universal primers (or even mixtures) of a single type or separate types (e.g., a pair of universal amplification primers suitable for amplifying a primary library) can be used to generate the library of the present invention. The universal primers optionally include a tag (barcode) sequence, wherein the tag (barcode) sequence does not hybridize with an adapter sequence or a target nucleic acid sequence. The barcode sequence incorporated into the amplicon in the second universal amplification can, for example, be used to effectively identify the sample source, thereby generating a barcoded library. Therefore, the provided composition comprises a highly multiplexed barcoded target library. The provided composition also comprises a highly multiplexed barcoded labeled target library.

[0111] In some embodiments, the amplicon library includes a unique tag sequence located between the 5' first universal handle sequence and the 3' target-specific sequence, and wherein the unique tag sequence does not exhibit significant complementarity and / or hybridization with any portion of the unique tag sequence and / or the target nucleic acid sequence. 4 -10 9 Thus, in certain embodiments, each of the plurality of amplicons in the library comprises 10 4 -10 9 In some embodiments, each amplicon in the plurality of amplicons in the library comprises at least 1 different unique tag sequence and up to 10 5In certain embodiments, each target-specific amplicon in the library includes at least two and up to 10 9 In some embodiments, each of the plurality of amplicons in the library comprises a different combination of different tag sequences, each of which has two different unique tag sequences. In some embodiments, each of the plurality of amplicons in the library comprises a tag sequence, which comprises 4096 different tag sequences. In certain embodiments, each target-specific amplicon in the library comprises up to 16,777,216 different combinations of different tag sequences, each of which has two different unique tag sequences.

[0112] In some embodiments, a separate amplicon in a plurality of amplicons in a library comprises a unique tag sequence (e.g., contained in a tag adapter sequence) comprising different random tag sequences alternating with fixed tag sequences. In some embodiments, at least one unique tag sequence comprises at least one random sequence and at least one fixed sequence, or comprises a random sequence flanked by a fixed sequence on both sides, or comprises a fixed sequence flanked by a random sequence on both sides. In some embodiments, the unique tag sequence comprises a fixed sequence, and its length is 2-2000 nucleotides or base pairs. In some embodiments, the unique tag sequence comprises a random sequence, and its length is 2-2000 nucleotides or base pairs.

[0113] In some embodiments, the unique tag sequence comprises a sequence having at least one random sequence interspersed with a fixed sequence. In some embodiments, the individual tag sequences in the plurality of unique tags have the structure (N) n (X) x (M) m (Y) y, wherein "N" represents a random tag sequence generated from A, G, C, T, U or I, and wherein "N" is 2-10, indicating the nucleotide length of the "N" random tag sequence; wherein "X" represents a fixed tag sequence, and wherein "X" is 2-10, indicating the nucleotide length of the "X" random tag sequence; wherein "M" represents a random tag sequence generated from A, G, C, T, U or I, wherein the random tag sequence "M" is different or the same as the random tag sequence "N", and wherein "M" is 2-10, indicating the nucleotide length of the "M" random tag sequence; and wherein "Y" represents a fixed tag sequence, wherein the fixed tag sequence of "Y" is the same or different from the fixed tag sequence of "X", and wherein "Y" is 2-10, indicating the nucleotide length of the "Y" random tag sequence. In some embodiments, the fixed tag sequence "X" is the same in multiple tags. In some embodiments, the fixed tag sequence "X" is different in multiple tags. In some embodiments, the fixed tag sequence "Y" is the same in multiple tags. In some embodiments, the fixed tag sequence "Y" is different in the multiple tags. In some embodiments, the fixed tag sequence "(X)" within the multiple amplicons is different in the multiple tags. x ” and “(Y) y ” is the sequence alignment anchor.

[0114] In some embodiments, the random sequence within the unique tag sequence is represented by "N", and the fixed sequence is represented by "X". Therefore, the unique tag sequence is represented by N1N2N3X1X2X3 or N1N2N3X1X2X3N4N5N6X4X5X6. Optionally, the unique tag sequence can have a random sequence, in which some or all of the nucleotide positions are randomly selected from the group consisting of: A, G, C, T, U and I. For example, the nucleotides at each position within the random sequence are independently selected from any one of A, G, C, T, U or I, or from a subset of these six different types of nucleotides. Optionally, the nucleotides at each position within the random sequence are independently selected from any one of A, G, C or T. In some embodiments, the first fixed tag sequence "X1X2X3" is the same or different sequence in multiple tags. In some embodiments, the second fixed tag sequence "X4X5X6" is the same or different sequence in multiple tags. In some embodiments, the first fixed tag sequence "X1X2X3" and the second fixed tag sequence "X4X5X6" within the plurality of amplicons are sequence alignment anchors.

[0115] In some embodiments, the unique tag sequence comprises the sequence 5'-NNNACTNNNTGA-3', where "N" represents a position within the random sequence randomly generated from A, G, C, or T, calculated as 4 6 (or 4^6) is about 4096 possible different random labels, and the possible different combinations of two unique labels are 412 (or 4^12) is about 16.78 million. In some embodiments, 5'- NNN ACT NNN The underlined portion in TGA-3' is the sequence alignment anchor.

[0116] In some embodiments, a fixed sequence within a unique tag sequence is a sequence alignment anchor that can be used to generate error-corrected sequencing data. In some embodiments, a fixed sequence within a unique tag sequence is a sequence alignment anchor that can be used to generate error-corrected sequencing read families.

[0117] Kits, Systems

[0118] Also provided herein is a kit for preparing a library of a target nucleic acid using the method of the first or second aspect of the present invention. Embodiments of the kit include a supply source of at least one pair of target-specific adapters as defined herein that can produce a first amplified product, and optionally at least one pair of universal amplification primers that can anneal to the universal handle of the adapter and initiate the synthesis of an amplified product, which will include a target sequence of interest that is joined to a universal sequence. Adapters and / or primers can be provided in a ready-to-use kit, or more preferably in the form of a concentrate that needs to be diluted before use, or even in a lyophilized or dried form that needs to be restored before use. In certain embodiments, the kit further includes a supply source of a suitable diluent for diluting or restoring components. Optionally, the kit further includes a supply source of reagents, buffers, enzymes, dNTPs, etc. for amplification, digestion, repair, and / or purification when producing a library as provided herein. Non-limiting examples of such reagents are described in the materials and methods section of the accompanying examples. Other components optionally supplied in the kit include components suitable for purifying a library prepared using the provided method. In some embodiments, a kit for generating a target-specific library is provided, comprising a plurality of target-specific adapters having a 5' universal handle sequence, a 3' target-specific sequence and a cleavable group; a DNA polymerase; an adapter; dATP; dCTP; dGTP; dTTP and a digestion reagent. In some embodiments, the kit further comprises one or more antibodies, a repair reagent, a universal primer optionally comprising a nucleic acid barcode, a purification solution or column.

[0119] The specific features of the adapters included in the kit are described herein in relation to other aspects of the invention. The structure and properties of universal amplification primers are well known to those skilled in the art and can be implemented to be used in conjunction with the provided methods and compositions to adapt to specific analysis platforms (e.g., universal P1 primers and A primers as described herein have been described in the art and used for sequencing on an ion torrent sequencing platform). Similarly, additional and other universal adapter / primer sequences described and known in the art (e.g., Enomina universal adapter / primer sequences, Pacific Bioscience universal adapter / primer sequences, etc.) can be used in conjunction with the methods and compositions provided herein. Suitable primers for nucleotide sequences suitable for use with the adapters included in the kit can be easily prepared using standard automatic nucleic acid synthesis instruments and reagents commonly used in the art. The kit can include a supply source of a single type of universal primer or two different universal primers (or even a mixture) of a single type (e.g., a pair of amplification primers suitable for amplifying a template modified by an adapter in a first amplification). The kit may include at least one pair of adapters for the first amplification of the sample of interest according to the method of the present invention, plus at least two different amplification primers, which optionally carry different tag (barcode) sequences, wherein the tag (barcode) sequence does not hybridize with the adapter. The kit can be used to amplify at least two different samples, wherein each sample is amplified according to the method of the present invention respectively and the second amplification includes using a single universal primer with a barcode, and then merging the prepared sample libraries after library preparation. In some embodiments, the kit includes different universal primer pairs for the second amplification step described herein. In this case, the "universal" primer pair can have a substantially identical nucleotide sequence, but differs in some other feature or modification.

[0120] Also provide system, for example, for practicing the method provided herein and / or the system comprising the composition provided herein.In some embodiments, system contributes to the method carried out in automatic mode.In certain embodiments, system contributes to high throughput mode.In certain embodiments, system comprises for example fluid handling element, fluid containing element, for realizing and maintaining the heat source and / or radiator of required reaction temperature, and / or can move the component of system from one position to another position as needed (for example porous plate handling element).

[0121] sample

[0122] As defined herein, "sample" and its derivatives are used in their broadest sense and include any sample, culture and / or analog suspected of containing target nucleic acid. In some embodiments, the sample includes DNA, RNA, TNA, chimeric nucleic acid, hybrid nucleic acid, multiple forms of nucleic acid or any combination of two or more of the above. In some embodiments, the sample suitable for use with the method of the present invention includes any biological, clinical, surgical, agricultural, atmospheric or aquatic sample containing one or more target nucleic acids of interest. In some embodiments, the sample includes nucleic acid molecules obtained from animals (such as human or mammalian sources). In another embodiment, the sample includes nucleic acid molecules obtained from non-mammalian sources (such as plants, bacteria, viruses or fungi). In some embodiments, the source of nucleic acid molecules can be archived or extinct samples or species. In some embodiments, the sample includes, for example, isolated nucleic acid samples prepared from sources such as genomic DNA, RNA TNA, or prepared samples such as fresh frozen or formalin-fixed paraffin embedded (FFPE) nucleic acid samples. It is also envisioned that the sample from a single individual; a collection of nucleic acid samples from genetically related members; multiple nucleic acid samples from non-genetically related members; multiple nucleic acid samples (matches) from a single individual, such as tumor samples and normal tissue samples; or genetic material from a single source, which contains two different forms of genetic material, such as maternal and fetal DNA obtained from a maternal individual, or there is contaminating bacterial DNA in a sample containing plant or animal DNA. In some embodiments, the source of nucleic acid material comprises nucleic acid obtained from a newborn (e.g., a blood sample for newborn screening). In some embodiments, the method provided includes the amplification of multiple target-specific sequences from a single nucleic acid sample. In some embodiments, the method provided includes the target-specific amplification of two or more target sequences from two or more nucleic acid samples or species. In certain embodiments, the method provided includes the amplification of highly multiplexed target nucleic acid sequences from a single sample. In a specific embodiment, the method provided includes the amplification of highly multiplexed target nucleic acid sequences from more than one sample (each from the same source organism).

[0123] In some embodiments, the sample includes a mixture of target nucleic acids and non-target nucleic acids. In certain embodiments, the sample includes multiple initial polynucleotides, which include a mixture of one or more target nucleic acids and may include one or more non-target nucleic acids. In some embodiments, the sample including multiple polynucleotides includes a portion or aliquot of the original sample; in some embodiments, the sample includes multiple polynucleotides, which is a complete original sample. In some embodiments, the sample includes multiple initial polynucleotides isolated from the same source or the same individual at different time points.

[0124] In some embodiments, the nucleic acid sample comprises a cell-free nucleic acid from a biological fluid, a nucleic acid from a tissue, a nucleic acid from a biopsy tissue, a nucleic acid from a needle biopsy, a nucleic acid from a single cell, or a nucleic acid from two or more cells. In certain embodiments, a single reaction mixture contains 1ng-100ng of multiple initial polynucleotides. In some embodiments, multiple initial polynucleotides include formalin-fixed paraffin-embedded (FFPE) samples; genomic DNA; RNA; TNA; cell-free DNA or RNA or TNA; circulating tumor DNA or RNA or TNA; fresh frozen samples, or a mixture of two or more of the foregoing; and in some embodiments, multiple initial polynucleotides include nucleic acid reference standards. In some embodiments, the sample comprises nucleic acid molecules obtained from biopsies, tumors, scrapings, swabs, blood, mucus, urine, plasma, semen, hair, laser capture microdissection, surgical resection, and other clinical or laboratory-obtained samples. In some embodiments, the sample is an epidemiological, agricultural, forensic, or pathogenic sample. In certain embodiments, the sample comprises a reference. In some embodiments, the sample is a normal tissue or a well-documented tumor sample. In certain embodiments, the reference is a standard nucleic acid sequence (eg, Hg19).

[0125] Target nucleic acid sequence analysis

[0126] The methods and compositions provided by the present invention are particularly suitable for amplification, optionally labeling and preparing target sequences for subsequent analysis. Therefore, in some embodiments, the methods provided herein include analyzing the resulting library preparations. For example, the method includes analyzing the polynucleotide sequence of the target nucleic acid and, when appropriate, analyzing any tag sequence added to the target nucleic acid. In some embodiments in which multiple target nucleic acid regions are amplified, the methods provided include determining the polynucleotide sequence of multiple target nucleic acids. The methods provided further optionally include using a second tag sequence (e.g., a barcode sequence) to identify the source of the target sequence (or provide other information about the sample source). In certain embodiments, the use of the prepared library composition is provided, which is used to analyze the sequence of a nucleic acid library.

[0127] In a particular embodiment, the use of the prepared labeled library composition is provided, which is used to further analyze the sequence of the target nucleic acid library. In some embodiments, the sequence determination includes determining the abundance of at least one target sequence in the sample. In some embodiments, determining the sequence of the nucleic acid library includes determining the alleles that occur at low frequencies in the sample. In certain embodiments, determining the sequence of the nucleic acid library includes determining whether there are mutant target nucleic acids in multiple polynucleotides. In some embodiments, determining whether there are mutant target nucleic acids includes detecting the abundance level of at least one mutant target nucleic acid in multiple polynucleotides. For example, such determinations include detecting that at least one mutant target nucleic acid accounts for 0.05% to 1% of the original multiple polynucleotides in the sample, detecting at least one mutant target nucleic acid accounts for about 1% to about 5% of the polynucleotides in the sample, and / or detecting at least 85% 100% of the target nucleic acids in the sample. In some embodiments, determining whether there are mutant target nucleic acids includes detecting and identifying copy number changes and / or genetic fusion sequences in the sample.

[0128] In some embodiments, nucleic acid sequencing of amplified target sequences generated by means of the teachings of the present disclosure comprises resequencing or targeted resequencing. In some embodiments, nucleic acid sequencing further comprises comparing the nucleic acid sequencing results of the amplified target sequences against a reference nucleic acid sequence. In some embodiments, nucleic acid sequencing of target library sequences further comprises determining the presence or absence of mutations in the nucleic acid sequence. In some embodiments, nucleic acid sequencing comprises identifying genetic markers associated with a disease (e.g., cancer and / or a genetic disease).

[0129] In some embodiments, the prepared library of the target sequence of the disclosed method is used in various downstream analyses or analytical methods with or without further purification or manipulation. In some embodiments, the analysis comprises sequencing by traditional sequencing reactions, high-throughput next generation sequencing, targeted multiple array sequence detection, or any combination of two or more of the foregoing. In certain embodiments, the analysis is performed by high-throughput next generation sequencing. In a specific embodiment, sequencing is performed in a bidirectional manner, thereby generating sequence reads in the forward and reverse strands of any given amplicon.

[0130] In some embodiments, the library prepared according to the method provided herein is then further operated for other analysis. For example, the prepared library sequence is used in downstream enrichment techniques known in the art, such as bridge amplification or emPCR to produce a template library, which is then used in next generation sequencing. In some embodiments, the target nucleic acid library is used in enrichment applications and sequencing applications. For example, the sequence determination of the target nucleic acid library provided is achieved using any suitable DNA sequencing platform. In some embodiments, the library sequence of the disclosed method or the template library prepared subsequently is used for single nucleotide polymorphism (SNP) analysis, genotyping or epigenetic analysis, copy number change analysis, gene expression analysis, gene mutation analysis (including but not limited to detection, prognosis and / or diagnosis), detection and analysis of rare or low frequency allele mutations, nucleic acid sequencing (including but not limited to resequencing, targeted resequencing) and synthetic assembly analysis. In one embodiment, the prepared library sequence is used to detect mutations with an allele occurrence rate of less than 5%. In some embodiments, the methods disclosed herein are used to detect mutations with an allele occurrence rate of less than 4%, 3%, 2%, or about 1% in a nucleic acid population. In another embodiment, the library prepared as described herein is sequenced to detect and / or identify germline or somatic mutations from a nucleic acid molecule population. In certain embodiments, sequencing adapters are joined to the ends of the prepared libraries to generate multiple libraries suitable for nucleic acid sequencing.

[0131] In some embodiments, a method for preparing a target-specific amplicon library is provided for use in a variety of downstream processes or analytical methods, such as nucleic acid sequencing or clonal amplification. In some embodiments, a library is amplified using bridge amplification or emPCR to generate multiple clonal templates suitable for nucleic acid sequencing. For example, a secondary and / or tertiary amplification process is optionally performed after target-specific amplification, including (but not limited to) a library amplification step and / or a clonal amplification step. "Clonal amplification" refers to the production of multiple copies of a single molecule. Clonal amplification is performed using various methods known in the art. For example, emulsion PCR is a method and involves separating individual DNA molecules and beads coated with primers in an aqueous bubble within an oil phase. Then, the polymerase chain reaction (PCR) coats each bead with a cloned copy of the separated library molecule, and then fixes the beads for subsequent sequencing. Emulsion PCR (also known as "polony sequencing", commercialized by Agencourt, and recently acquired by Applied Biosystems) is used in the methods published by Marguilis et al. and Shendure and Porreca et al. Margulies et al., (2005) Nature 437:376-380; Shendure et al., Science 309(5741):1728-1732. Another method for clonal amplification is "bridge PCR", in which fragments are amplified when primers are attached to a solid surface. These methods, as well as other clonal amplification methods, produce many physically separated locations, each of which contains multiple copies of a single molecule polynucleotide fragment. Therefore, in some embodiments, one or more target-specific amplicons are amplified using, for example, bridge amplification or emPCR to produce multiple clonal templates suitable for nucleic acid sequencing.

[0132] In some embodiments, at least one library sequence to be cloned and amplified is connected to a load or particle. The load can comprise any suitable material and have any suitable shape, including, for example, flat, spherical or granular. In some embodiments, the support is a scaffold polymer particle as described in U.S. Publication Application No. 20100304982, which is incorporated herein by reference in its entirety. In certain embodiments, the method includes depositing an enrichment of at least a portion of the library sequence on a load (e.g., a sequencing load), wherein the load comprises an array of sequencing reaction sites. In some embodiments, the enrichment of the library sequence is connected to a sequencing reaction site on a load, wherein the load comprises 10 2 Up to 10 10 An array of sequencing reaction sites.

[0133] Sequencing means determining the information related to the nucleic acid sequence and can include identifying or determining part and all of the sequence information of the nucleic acid. Sequence information can be determined at varying degrees of statistical reliability or confidence. In some embodiments, sequence analysis comprises high-throughput, low-depth detection, such as by qPCR, rtPCR and / or array hybridization detection methods known in the art. In some embodiments, sequencing analysis comprises the determination of depth sequence evaluation, such as by Sanger sequencing (Sanger sequencing) or other high-throughput next-generation sequencing methods. Next-generation sequencing means using a method for determining many (usually thousands to billions) nucleic acid sequences in essentially large-scale parallel mode (e.g., wherein many sequences are read in a parallel mode, for example) or alternatively using an ultra-high-throughput continuous method that can be parallelized itself to carry out sequence determination. Therefore, in certain embodiments, the method of the present invention comprises sequencing analysis, which includes parallel sequencing of scale. Such methods include, but are not limited to, pyrosequencing (e.g., methods commercialized by 454 Life Sciences, Inc. of Branford, Conn.); sequencing by ligation (e.g., as commercialized by SOLiD of Carlsbad, Calif.); TM .Technology Life Technology Company) SOLiD TM .technology, Life Technologies, Inc. commercialized); sequencing by synthesis using modified nucleotides (such as TTruSeq by Illumina, Inc., San Diego, Calif. TM and HiSeq TM and MiSeq TM and / or NovaSeq TM Technology Commercialization; HeliScope by Helicos Biosciences Corporation, Cambridge, Mass. TM and PacBio of Pacific Biosciences of California, Inc. of Menlo Park, Calif. or RS systems, using ion detection techniques (e.g., IonTorrent TM technology, Ion Torrent in Carlsbad, California TMsequencing by DNA nanoballs (Complete Genomics, Inc., Mountain View, Calif.); nanopore-based sequencing technologies (e.g., developed by Oxford Nanopore Technologies, LTD, Oxford, UK), and similar highly parallel sequencing methods.

[0134] For example, in certain embodiments, the yield of libraries generated using the teachings of the present disclosure is sufficient for use in a variety of downstream applications, including the use of Ion Torrent TM PGM systems (e.g., PCR-mediated nucleic acid fragment libraries added to IonSphere TM Particles) (Life Technologies, Product No. 4467389) or Ion Torrent Proton TM Ion Xpress TM Template Kit. For example, instructions for preparing a template library from an amplicon library can be found in the IonXpress Template Kit User Guide (Life Technologies, Product No. 4465884), which is hereby incorporated by reference in its entirety. TM Instructions for nucleic acid sequencing on the Chip are described in the Ion Sequencing User Guide (Product No. 4467391), which is hereby incorporated by reference in its entirety.

[0135] The starting point of the sequencing reaction can be provided by annealing the sequencing primer to the product of the solid phase amplification reaction. In this regard, one or two adapters added during the formation of the template library can include a nucleotide sequence that enables the sequencing primer to anneal to the amplified product derived from the solid phase amplification of the complete genome or template library. Depending on the implementation of the embodiment of the present invention, the label sequence and / or the target nucleic acid sequence can be determined in a single read from a single sequencing primer or in multiple reads from two different sequencing primers. In the case of two reads from two sequencing primers, "label reads" and "target sequence reads" are performed in either order, and a suitable denaturation step is performed to remove the annealed primers after completing the first sequencing read.

[0136] In some embodiments, the sequencer is coupled to a server that applies parameters or software to determine the sequence of the amplified target nucleic acid molecule. In certain embodiments, the sequencer is coupled to a server that applies parameters or software to determine whether there are mutant alleles that occur at low frequencies in the sample.

[0137] Example

[0138] Example 1: Materials and Methods

[0139] Reverse transcription (RT) reaction method (21uL reaction) can be performed in samples where RNA and DNA are analyzed, such as FFPE RNA and cfTNA:

[0140] 1. Thaw 5x URT buffer at room temperature for at least 5 minutes. (Note: Check the tube for white precipitate. Vortex as needed to mix)

[0141]

[0142] 2. In a MicroAmp EnduraPlate 96-well plate, set up the RT reaction by adding the following components.

[0143] (5ng-15ng RNA or DNA / / 5ng-40ng cfTNA)

[0144]

[0145] 3. Mix the entire contents by vortexing or pipetting. Spin down briefly.

[0146] 4. Add 20 μl of Parol 40C oil on top of each reaction mixture.

[0147] 5. Load the plate into a thermal cycler (e.g., SimpliAmp Thermocycler) and run the following program:

[0148]

[0149]

[0150] Low cycle labeling PCR (38uL reaction volume + 20uL oil):

[0151] Assemble the tagmentation PCR reaction in the wells of a 96-well PCR plate:

[0152] FFPE DNA samples only

[0153] 1. Assemble the reaction by adding the following components to a MicroAmp EnduraPlate 96-well plate:

[0154] a. Prepare UDG mixture: 1ul + 5ul 5x URT buffer

[0155] b. Add 6ul of diluted UDG to 15ul of FFPE DNA sample.

[0156] c. Mix by vortexing. Spin down briefly to collect the reaction at the bottom of the well.

[0157] d. Add 20 μL of Parol 40C oil on top of each sample.

[0158] e. Carry out the following reaction:

[0159]

[0160] 2. Prepare the amplification master mix:

[0161]

[0162] 3. Add 17 μL of PCR master mix to 21 μL of UDG-treated FFPE DNA sample.

[0163] Set the pipette to 20 μL volume. Mix the reaction below the oil by pipetting up and down 20 times to ensure the reaction is well mixed without disturbing the oil phase. Briefly spin the plate down.

[0164] FFPE RNA and cfTNA samples only

[0165] 1. Add the components directly to the RT reaction in the RT step above:

[0166]

[0167] 2. Set the pipette to 20 μL volume. Mix the reaction below the oil by pipetting up and down 20 times to ensure the reaction is well mixed without disturbing the oil phase. Spin the plate down briefly.

[0168] 3. Perform a 3-cycle labeling PCR on the SimpliAmp using the following cycling conditions:

[0169] For FFPE DNA and RNA libraries:

[0170]

[0171] For cfTNA libraries,

[0172]

[0173] Digest-Fill-Ligation (45.6 μL reaction volume + 20 μL oil):

[0174] 1. Add 7.6 μL SUPA to the above PCR reaction wells. Add SUPA directly to the sample below the oil layer.

[0175] 2. Set the pipette to 25 μL. Mix the reaction below the oil layer by pipetting up and down 20 times. Spin the plate down briefly.

[0176] 3. Load the plate into the thermal cycler and run the following program:

[0177] stage temperature time Phase 1 30℃ 15 minutes Phase 2 50℃ 15 minutes Phase 3 55℃ 15 minutes Phase 4 25℃ 10 minutes Phase 5 98℃ 2 minutes Keep 4℃ ∞

[0178] Library amplification (~51 μL reaction volume + 20 μL oil)

[0179] 1. Carefully transfer 30 μL of the digestion-fill-ligation reaction above to the AmpliSeq HD Dual Barcode. Mix thoroughly by pipetting up and down 20 times. Transfer all reactions back to the original wells under the oil layer.

[0180] 2. Set the pipette to 30 μL. Mix the entire reaction under the oil by pipetting up and down 20 times. Spin the plate down briefly.

[0181] 3. Load the plate into the thermal cycler and run the following program:

[0182]

[0183] 2 rounds of AmpureXP library purification

[0184] According to the manufacturer's instructions, use 36.8ul The resulting repair samples were subjected to two rounds of purification using ELISA beads (Beckman Coulter, Inc.). Briefly:

[0185] Transfer the 46 µL of library reaction below the oil layer to a new clean well on the PCR plate.

[0186] 36.8 μl Agencourt TM AMPure TM XP reagent was added to each sample and mixed by pipetting, followed by incubation at room temperature for 5 minutes.

[0187] The plate was placed on a magnet until the solution in the wells became clear.

[0188] Carefully remove the supernatant; then remove the remaining supernatant.

[0189] Add 150uL 80% ethanol to 10mM Tris-HCl pH 8. Do not disturb the bead pellet.

[0190] Pipette the plate on the magnet 3 times with 5 seconds between each wash; remove the supernatant; repeat the wash step again. Remove any residual buffer from the wells using a pipette.

[0191] The wells were dried at room temperature for 5 minutes.

[0192] 30 uL of Low TE buffer was added to the wells and pipetted to resuspend the beads.

[0193] The solution was incubated at room temperature for 5 minutes and the plate was placed on a magnet until a clear solution was obtained.

[0194] Transfer 30uL of the eluate to a clean well on the plate.

[0195] Add 30 μL (1× volume) of Ampure XP beads to the wells; pipette in the wells to mix.

[0196] Repeat the above steps and elute after the second purification using 40uL of low TE buffer.

[0197] Transfer 40uL of library to a new clean well.

[0198] Library normalization via independent equalizer

[0199] First, Ion Library Equalizer TM Warm all reagents in the kit to room temperature. Vortex and centrifuge all reagents. Wash the Equalizer TM Beads (If you have done this before, skip to Add Equalizer TM beads and washed).

[0200] 1. For every 4 reactions, add 12 μL of beads to a clean 1.5-mL tube and 24 μL / reaction Equalizer TM Wash buffer.

[0201] 2. Place the test tube in the magnetic rack for 3 minutes or until the solution is completely clear.

[0202] 3. Carefully remove and discard the supernatant without disturbing the pellet.

[0203] 4. Remove from magnet, each reaction Equalizer TM Add 24 μL of wash buffer and resuspend.

[0204] Amplification of the library

[0205] 5. Remove the plate with purified libraries from the magnet and add 10 μL 5X DV-Amp mix and 2 μL Equalizer TM Primer (pink cap in the equalizer kit). Total volume = 52 μL

[0206] 6. Mix.

[0207] 7. Gently add 20 μL of Parol 40C oil on top of the sample.

[0208] 8. Run the following program on the thermal cycler:

[0209] 98C for 2 minutes

[0210] 9-cycle amplification of FFPE DNA / RNA or 6-cycle amplification of cfDNA:

[0211] 98C for 15 seconds

[0212] 64C for 1 minute

[0213] So

[0214] Keep at 4C for an unlimited time

[0215] 9. (Optional) After thermal cycling, centrifuge the plate to collect any droplets.

[0216] Equalizer TM Capture is added to the amplified library

[0217] 10. Add 10 μL of Equalizer Capture to each library amplification reaction below the oil layer.

[0218] 11. Mix up and down 10x.

[0219] 12. Incubate at room temperature for 5 minutes.

[0220] Adding an Equalizer TM Beads and wash

[0221] 13. Transfer 60 μL of amplified library sample below the oil layer to the well with washed beads.

[0222] 14. Mix thoroughly.

[0223] 15. Incubate at room temperature for 5 minutes.

[0224] 16. Place the plate on the magnet and incubate for 2 minutes or until the solution is clear.

[0225] 17. Remove the supernatant.

[0226] 18. Equalizer 150 μL TM Wash buffer was added to each reaction.

[0227] 19. With the plate still in the magnet, remove and discard the supernatant.

[0228] 20. Repeat the bead wash to elute the balanced library.

[0229] Elution of balanced library

[0230] 21. Remove the plate from the magnet and place 100 μL of Equalizer TM Elution buffer was added to each pellet.

[0231] 22. Pipette the mixture 5x with 50ul volume.

[0232] 23. Elute the library by incubating at 32°C for 5 minutes on a thermal cycler.

[0233] 24. Immediately remove the plate and place it on the magnet. Once the solution is clear, move it to a new well.

[0234] 25. Perform qPCR and adjust pool @ 100 pM for templating and sequencing.

[0235] Example 2: Compositions and Methods

[0236] The first step of the provided method comprises several rounds of amplification, for example three to six rounds of amplification, and in some instances, three rounds of amplification using forward and reverse adapters for each gene-specific target sequence. Each adapter contains a 5' universal sequence and a 3' gene-specific target sequence. In some embodiments, the adapter optionally includes a unique tag sequence located between the 5' universal and 3' gene-specific target sequences.

[0237] In certain embodiments where unique tag sequences are utilized, each gene-specific target adapter pair comprises a plurality of different unique tag sequences in each adapter. For example, each gene-specific target adapter comprises up to 4096 TAGs. Thus, each target-specific adapter pair comprises at least four and up to 16,777,216 possible combinations.

[0238] Each provided adapter includes a cleavable uracil in place of thymine at a specific position in the forward and reverse adapter sequences. The position of uracil (Us) is consistent for all forward and reverse adapters with unique tag sequences, wherein uracil (Us) is present at the 5' end and 3' end of the unique tag sequence flanking the sequence; and Us is present in each gene-specific target sequence region, although the position of each gene-specific target sequence will inevitably change. Design uracil flanking each unique tag sequence (UT) and in the gene-specific sequence region and calculate the Tm of such sequences to promote fragment dissociation at a temperature below the melting temperature of the universal handle sequence, which is designed to maintain hybridization at the selected temperature. There may be changes in the U in the flanking sequence of the UT region, however, the design keeps the melting temperature below the melting temperature of the universal handle sequence on each forward and reverse adapter. Exemplary adapter sequence structures include:

[0239] Forward Adaptor :

[0240] ------A handle-----------*UT*--------Gene specificity--

[0241] TCTGTACGGTGACAAGGCG- U -NNNACTNNNTGA- U -XXXXXXXXXXXXXXXX SEQ ID NO:1564

[0242] Reverse adapter

[0243] TGACAAGGCGTAGTCACGG- U -NNNACTNNNTGA- U -XXXXXXXXXXXXXXXX SEQ ID NO:1565

[0244] -----B handle-------------UT--------------Gene specificity-------

[0245] Wherein, each N is a base selected from A, C, G or T and the constant segment of the UT region is used as an anchor sequence to ensure correct identification of the variable (N) portion. The constant and variable regions of the UT can be significantly modified (e.g., alternative constant sequences, >3N / segment), as long as the Tm of the UT region remains lower than the Tm of the universal handle region. Importantly, there is no cleavable uracil in each forward (e.g., TCTGTACGGTGACAAGGCG (SEQ ID NO: 1566) and reverse (e.g., TGACAAGGCGTAGTCACGG (SEQ ID NO: 1567) universal handle sequence) universal handle sequence. In embodiments of the present invention, the universal sequence has been designed to adapt to subsequent amplification and sequencing sequence addition on the ION Torrent platform, however, those skilled in the art will understand that such universal sequences may be suitable for use with other universal sequences that are more suitable for alternative sequencing platforms (e.g., ILLUMINA sequencing system, QIAGEN sequencing system, PACBIO sequencing system, BGI sequencing system or others).

[0246] Methods for using the provided compositions include preparing libraries by AmpliSeq HD technology and slight variations thereof and using reagents and kits available from Thermo Fisher Scientific. SuperFiU DNA includes modifications in the uracil binding pocket (e.g., AA 36) and the B-family polymerase catalytic domain (e.g., AA 762). SuperFiU is described in U.S. Patent Publication No. US2021 / 0147817 filed on June 26, 2017, which is incorporated herein by reference. The ability of polymerases to utilize uracil and / or any alternative cleavable residues (e.g., inosine, etc.) contained in the adapter sequence may be limited. In certain embodiments, it may be advisable to use a mixture of polymerases to reduce enzyme-specific PCR errors.

[0247] The second step of the method involves partial digestion of the resulting amplicon and any unused uracil-containing adapters. For example, when uracil is incorporated as a cleavable site, digestion and repair comprises enzymatic cleavage of uridine monophosphate from the resulting primers, primer dimers, and amplicons, and melting of DNA fragments, followed by repair of gapped amplicons by polymerase filling and joining. This step reduces and may eliminate primer-dimer products in multiplex PCR. In some cases, digestion and repair are performed in a single step. In some cases, it may be necessary to briefly separate the digestion and repair steps. For example, a heat-labile polymerase inhibitor can be used in conjunction with the method so that digestion is performed at a relatively low temperature (25°C-40°C), followed by initiating repair by raising the temperature enough to destroy polymerase-inhibitor interactions (e.g., polymerase-Ab), but not high enough to melt the universal handle sequence.

[0248] Uracil-DNA glycosylase (UDG) can be used to remove uracil, leaving an abasic site that can be acted upon by several enzymes or enzyme combinations, including (but not limited to): APE 1-apurinic / apyrimidinic endonuclease; FPG-formamidopyrimidinic [fapy]-DNA glycosylase; Nth-endonuclease IIIEndo VIII-endonuclease VIII; PNK-polynucleotide kinase; Taq-Thermus aquaticus DNA polymerase; DNA pol I-DNA polymerase I; Polβ-human DNA polymerase β. In a specific embodiment, the method uses the human apurinic / apyrimidinic endonuclease, APE1. APE1 activity leaves behind a 3'-OH and a 5' deoxyribose-phosphate (5'-dRP). Removal of the 5'-dRP can be achieved by a variety of enzymes, including recJ, polymerase β, Taq, DNA polI, or any DNA polymerase with 5'-3' exonuclease activity. Removal of the 5'-dRP by any of these enzymes can produce a 5'-phosphate end that can be joined. In another embodiment, UDG activity removes uracil and leaves an abasic site, which is removed by FPG, leaving 3' and 5'-phosphates. Next, the 3'-phosphate is removed by T4 PNK, leaving a polymerase-extendable 3'-OH. Next, the 5'-deoxyribose phosphate can be removed by polymerase beta, fpg, Nth, Endo VIII, Taq, DNA pol I, or any other DNA polymerase with 5'-3' exonuclease activity. In a specific embodiment, Taq DNA polymerase is utilized.

[0249] The repair filling process can be accomplished by almost any polymerase, which may be the amplification polymerase used for amplification in step 1 or by any polymerase added in step 2, including (but not limited to): Phusion DNA polymerase; Phusion U DNA polymerase; SuperFi DNA polymerase; SuperFi U DNA polymerase; TAQ; PolβT4 DNA polymerase; and T7 DNA polymerase. The ligation repair of the amplicon can be performed by a variety of ligases, including (but not limited to): T4 DNA ligase; T7 DNA ligase; Taq DNA ligase In a specific embodiment of the method, Taq DNA polymerase is used and ligation repair is achieved by T7 DNA ligase.

[0250] The final step of library preparation involves amplifying the repaired amplicons by standard PCR protocols using universal primers that contain sequences complementary to universal handle sequences on the 5' and 3' ends of the prepared amplicons. For example, the A-universal primer and the P1 universal primer (each part of the Ion Express Adaptor Kit (Thermo Fisher Scientific, Inc.)) can optionally contain a sample-specific barcode. The final library amplification step can be performed by a number of polymerases, including but not limited to; Phusion DNA polymerase; Phusion U DNA polymerase; SuperFi DNA polymerase; SuperFi U DNA polymerase; Taq DNA polymerase; Veraseq ultra DNA polymerase.

[0251] Example 3: Measurement content and method

[0252] Using primers to target sequences specific for the targets in Table 1, the adapters each included 4096 unique tag sequences for each gene-specific target sequence, resulting in an estimated 16,777,216 different unique tag combinations for each gene-specific target sequence pair.

[0253] The library preparation is performed according to the method described above. The library prepared for templated preparation is prepared, sequenced and analyzed. Sequencing can be performed by a variety of known methods, including (but not limited to) synthetic sequencing, ligation sequencing and / or hybridization sequencing. The Ion Torrent platform (Thermo Fisher Scientific) has been used to sequence in the examples herein, however, the library can be prepared and adapted for analysis, such as sequencing, using any other platform, such as Illumina, Qiagen, PacBio, etc. Multiple metrics can be used to analyze the results to evaluate performance, for example:

[0254] ○ Number of Families (Captured Input DNA, ng). The number of families is a measure of the number of families mapped to a single target. In this case, each unique molecular signature is a family.

[0255] ○ Uniformity is a measure of the percentage of target bases covered by at least 0.2x the average read depth. Using this metric ensures that the technology does not selectively underamplify certain targets.

[0256] o Positive / Negative: When control samples with known mutations are used for analysis (eg Acrometrix Oncology Hotspot Control DNA, Thermo Fisher Scientific), the number of true positives can be tracked.

[0257] ■ True Positives: The number of true positive notifications regarding the number of mutations that were present and correctly identified.

[0258] ■ False Positives (FP): Number of false positive calls regarding the number of mutations that are present (hotspots and full targets) but known not to be present in the sample.

[0259] ■ False Negatives (FN) (if acrometrix puncture was used): the number of false negative calls regarding the number of mutations that were present but not identified.

[0260] ○ On-target / off-target is the percentage of mapped reads that are aligned / misaligned in the target region. Use this metric to ensure that the technology is primarily amplifying the targets that the panel was designed for.

[0261] ○ Track low quality to ensure the data is worth analyzing. This metric is an overall system metric and is not directly related to this technology.

[0262] Table 1: Accurate determination of gene content by variant class

[0263]

[0264]

[0265] Clinical evidence is defined as the number of instances where a gene / variant combination appears in drug labels, guidelines, and / or clinical trials. Tables 2 and 3 depict the top genes / variants and indications associated with the provided assays, as supported by clinical evidence.

[0266]

[0267] Assays available on drug labeling and / or guidelines (NCCN and ESMO) covering up to 29 gene and variant combinations

[0268] Table 4: Cancer indications ranked by clinical evidence

[0269]

[0270]

[0271] Example 4: Results

[0272] Primers were designed using the composition design methods provided herein, and those targeting oncology genes of the panel target genes as described in Table 1 above were used, wherein the library amplification step utilized two primer pairs (placing two universal sequences on each end of the amplicon, e.g., one A-universal handle and one P1-universal handle on each end) to achieve bidirectional sequencing as described herein. The prepared libraries were sequenced using the Ion Gene Studio templating / and sequencing kit and instrument (Thermo Fisher Scientific) and / or the fully integrated library preparation, templating and sequencing system, Genexus (Thermo Fisher Scientific). The performance of the instant panel indicated that the technology was able to appropriately detect targeted mutations, copy number variations, and fusions as expected.

[0273] SNV and CNV detection in multiple cancer types from FFPE

[0274]

[0275]

[0276] Fusion detection in multiple cancer types from FFPE

[0277]

[0278] Detection of variants in clinical plasma samples with known variants

[0279]

[0280]

[0281]

[0282] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art will appreciate that many variations, changes and replacements may be employed in practicing the present invention. It is contemplated that the following claims define the scope of the present invention and thus cover methods and structures within the scope of these claims and their equivalents.

[0283] Table A. Primer sequences for current oncology assays, FWD pool and REV pool

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348] Each L is independently A, C, G or T.

[0349] In view of the fact that the principles of the present disclosure can be applied to many possible embodiments, it should be recognized that the illustrated embodiments are only preferred embodiments and should not be considered to limit the scope of the present invention. Specifically, the scope of the present invention is defined by the appended claims. The inventor therefore claims that the present invention fully belongs to the scope and spirit of the claims.

Claims

1. A composition for performing a single-flow multiplex assay for executable oncology biomarkers in a sample, the composition consisting of multiple sets of primer pair reagents, the multiple sets of primer pair reagents targeting multiple target sequences to detect low-level targets in the sample, wherein the target sequences are selected for target genes selected from the group consisting of the following functions: DNA hotspot mutation genes, copy number variation (CNV) genes, intergenic fusion genes, and intragenic fusion genes selected from the genes in Table 1.

2. The composition of claim 1, wherein one or more executable oncology biomarkers in the sample determine changes in oncology activity in the sample that are indicative of a potential diagnosis, prognosis, candidate treatment options, and / or adverse events.

3. A composition according to claim 1 or claim 2, wherein the target gene consists of the genes in Table 1.

4. The composition of any one of claims 1 to 3, wherein the plurality of target sequences comprises amplicon sequences detected by the primers of Table A.

5. The composition of any one of claims 1 to 4, wherein the plurality of target sequences comprises each of the amplicon sequences detected by the primers of Table A.

6. The composition according to any one of claims 1 to 5, wherein the plurality of primer reagents are selected from the primers of Table A.

7. The composition of any one of claims 1 to 6, wherein the plurality of primer reagents consists of each of the primers of Table A.

8. A multiplex assay comprising the composition according to any one of claims 1 to 7.

9. A test kit comprising the composition according to any one of claims 1 to 7 or the multiplex assay according to claim 8.

10. A method for determining the presence of one or more actionable oncology biomarkers in a biological sample, the method comprising: Multiplex amplification of a plurality of target sequences from a biological sample, wherein amplification comprises contacting at least a portion of the sample with a composition according to any one of claims 1 to 7 and a polymerase under amplification conditions, thereby generating amplified target sequences; Each target sequence in the plurality of target sequences is detected; wherein the detection of one or more executable oncology biomarkers compared to a control sample determines a change in oncology activity in the sample that is indicative of a potential diagnosis, prognosis, candidate treatment regimen, and / or adverse event.

11. The method according to claim 10, wherein the target sequence is selected for a target gene selected from the group consisting of the following functions: a DNA hotspot mutation gene, a copy number variation (CNV) gene, an intergenic fusion gene, and an intragenic fusion gene selected from the genes in Table 1.

12. The method according to claim 10 or claim 11, wherein the target gene consists of the genes in Table 1.

13. The method of any one of claims 10 to 12, wherein the plurality of target sequences comprises the amplicon sequences detected by the primers of Table A.

14. The method of any one of claims 10 to 13, wherein the plurality of target sequences comprises each of the amplicon sequences detected by the primers of Table A.

15. The method according to any one of claims 10 to 14, wherein the plurality of primer reagents are selected from the primers of Table A.

16. The method according to any one of claims 10 to 15, wherein the plurality of primer reagents consists of each of the primers of Table A.

17. The method according to any one of claims 10 to 16, wherein the biological sample comprises a tumor and / or tissue surrounding the tumor.

18. The method according to any one of claims 10 to 17, wherein the biological sample and the control sample are from the same individual.

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