Compositions and methods for tumor assays

Abstract

Methods and compositions are provided for preparing a library of target nucleic acid sequences that are useful for evaluating genetic mutations for tumor biomarker profiling of a sample. In particular, target-specific primer panels are provided that allow selective amplification of tumor biomarker target sequences in a sample. In one aspect, the present invention relates to target-specific primers that are useful for selective amplification of one or more target sequences associated with tumor 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 can be used to detect the presence of genetic variants of one or more target sequences associated with tumors.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority under and the benefit of U.S. Provisional Application No. 63 / 342,867, filed May 17, 2022, under 35 U.S.C. §119(e), which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application incorporates by reference the electronic sequence listing material filed concurrently herewith. The nucleic acid 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, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but it is understood that the complementary strand is included by any reference to the displayed strand. The electronic sequence listing material was submitted as an extensible markup language (.xml) file entitled "TP109605WO1_ST26", created on May 8, 2023, which has a file size of approximately 8,088,321 bytes, and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to compositions of libraries of target nucleic acids and methods for preparing same and uses thereof. [Background technology]

[0004] Advances in cancer therapy have begun to provide promising results across oncology. Targeted therapies, immune checkpoint inhibitors, cancer vaccines, and T-cell therapy have shown sustainable results in more responsive populations than traditional chemotherapy. However, effective identification of responsive candidates and / or monitoring response has proven challenging. There is a pressing need to better understand the tumor microenvironment, tumor evolution, and biomarkers of drug response. Higher throughput, systematic, and standardized assay solutions that can efficiently and effectively detect multiple relevant biomarkers in a variety of sample types are desirable. Summary of the Invention

[0005] In one aspect of the present invention, compositions are provided for single-stream multiplex determination of actionable tumor biomarkers in samples. In some embodiments, the compositions consist of multiple primer reagents directed to multiple target sequences to rapidly and effectively detect low levels of targets in samples. The compositions provided target tumor gene sequences, where multiple gene sequences are selected from among DNA hotspot mutation genes, copy number variation (CNV) genes, intergenic fusion genes, and intragenic fusion genes. In certain embodiments, the target genes are selected from the genes in Table 1. In certain embodiments, the target genes consist of the genes in Table 1. The compositions provided maximize the detection of key biomarkers, such as EGFR, ALK, BRAF, ROS1, HER2, MET, NTRK, and RET, in one day from various samples (e.g., FFPE tissue, plasma) in an integrated and automated workflow.

[0006] In some embodiments, a plurality of actionable target genes in a sample is determined to be indicative of a potential diagnosis, prognosis, potential treatment regimen, and / or change in tumor activity in the sample. In certain embodiments, a composition provided comprises a plurality of primer reagents selected from Table A. In some embodiments, a multiplex assay is provided comprising the composition of the invention. In some embodiments, a test kit is provided comprising the composition of the invention.

[0007] In another aspect of the invention, a method is provided for determining actionable tumor biomarkers in a biological sample. Such a method comprises performing a multiplex amplification of a plurality of target sequences from a biological sample comprising the target sequences. The amplification comprises contacting at least a portion of a sample comprising the plurality of target sequences of interest with a plurality of target-specific primers in the presence of a polymerase under amplification conditions to generate a plurality of amplified target sequences. The method further comprises detecting the presence of each of the plurality of target tumor sequences, wherein detection of one or more actionable tumor biomarkers compared to a control sample determines a change in tumor activity in the sample indicative of a potential diagnosis, prognosis, potential treatment regimen, and / or adverse event. The methods described herein utilize the compositions of the invention provided herein. In some embodiments, the target genes are 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 genes are selected from the genes in Table 1. In certain embodiments, the target genes consist of the genes in Table 1.

[0008] Still further, the use of the provided compositions and kits comprising the provided compositions for the analysis of sequences of nucleic acid libraries is an additional aspect of the present invention. In some embodiments, the analysis of sequences of the resulting libraries allows for the detection of low frequency alleles, improved detection of gene fusions and novel fusions, and / or detection of genetic mutations in a sample of interest and / or in a plurality of samples of interest. In certain embodiments, manual, partially automated, and fully automated implementations of the use of the provided compositions and methods are contemplated. In certain embodiments, the use of the provided compositions is implemented in a fully integrated library preparation, templating, and sequencing system for the genetic analysis of samples. In certain embodiments, the use of the provided compositions and methods of the present invention benefits research and clinical applications, including first-line testing of tissue and / or plasma specimens, and continuous monitoring of specimens for the detection of biomarker relapse and / or resistance.

[0009] 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.

[0010] An efficient method for the generation of targeted libraries containing actionable tumor biomarkers from complex samples is desirable for various nucleic acid analyses. The present invention provides, inter alia, a method for preparing libraries of targeted nucleic acid sequences, allowing for the rapid generation of highly multiplexed targeted libraries containing unique tag sequences, and the resulting library compositions are useful for various applications, including sequencing applications. The compositions provided are designed for the detection of mutations, copy number variations (CNVs), and gene fusions in tissue and plasma derived samples. The compositions provided include targeted primer panels and reagents for use with high throughput samples to provide a next generation workflow for genetic analysis. In certain embodiments, the use is implemented in a fully integrated sample-to-analysis system. The novel features of the present invention are set forth in detail in the appended claims, and a complete understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description describing exemplary embodiments in which the principles of the present invention are utilized. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and Internet web pages, are expressly incorporated by reference in their entirety for any purpose. Where the definition of a term in an incorporated reference differs from the definition provided in the present teachings, the definition provided in the present teachings shall prevail. It will be understood that "about" is implied before temperatures, concentrations, times, etc. discussed in the present teachings, such that only minor and insignificant deviations are within the scope of the present teachings herein. In this application, the use of the singular includes the plural unless specifically stated otherwise. It is noted that as used herein, the singular forms "a," "an," and "the," as well as any singular use of a word, include plural referents unless clearly and unambiguously limited to one referent. Similarly, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" are not intended to be limiting. It should be understood that both general descriptions are exemplary and explanatory only and are not limiting of the present invention.

[0012] Unless otherwise defined, scientific and technical terms used in connection with the invention described herein shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, the terminology utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization used herein are well known and commonly used in the art. The practice of the subject matter may employ conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant techniques), cell biology, and biochemistry that are within the skill of the art, unless otherwise indicated. Such conventional techniques include, but are not limited to, preparation of synthetic polynucleotides, polymerization techniques, chemical and physical analysis of polymer particles, preparation of nucleic acid libraries, nucleic acid sequencing and analysis, and the like. Specific illustrations of suitable techniques may be used by reference to the examples provided herein. Other equivalent conventional procedures may also be used. Such conventional techniques and explanations can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Hermanson, Bioconjugate Techniques, Second Edition (Academic Press, 2008), Merkus, Particle Size Measurements (Springer, 2009), Rubinstein and Colby, Polymer Physics (Oxford University Press, 2003), etc. As utilized in accordance with the embodiments provided herein, the following terms shall be understood to have the following meanings, unless otherwise indicated.

[0013] As used herein, "amplify", "amplifying" or "amplification reaction" and their derivatives generally refer to an act or process in which at least a portion of a nucleic acid molecule (called a template nucleic acid molecule) is replicated or copied to at least one additional nucleic acid molecule. The additional nucleic acid molecule optionally comprises a sequence that is substantially identical or substantially complementary to at least some portion of the template nucleic acid molecule. The template target nucleic acid molecule may be single-stranded or double-stranded. The additional resulting replicated nucleic acid molecule may be independently single-stranded or double-stranded. In some embodiments, the amplification comprises a template-dependent in vitro enzyme-catalyzed reaction for the generation of at least one copy of at least some portion of the target nucleic acid molecule or the generation of at least one copy of a target nucleic acid sequence that is complementary to at least some portion of the target nucleic acid molecule. The amplification optionally comprises linear or exponential replication of the nucleic acid molecule. In some embodiments, such amplification is performed using isothermal conditions, while in other embodiments, such amplification may comprise thermal cycling. In some embodiments, the amplification is a multiplex amplification that comprises simultaneous amplification of multiple target sequences in a single amplification reaction. At least some of the target sequences can be located on the same nucleic acid molecule or on different target nucleic acid molecules included in a single amplification reaction. In some embodiments, "amplification" includes amplification of at least some portion of DNA-based nucleic acid and / or RNA-based nucleic acid, whether alone or in combination. The amplification reaction can include single-stranded or double-stranded nucleic acid substrates and can further include any amplification process known to those of skill in the art. In some embodiments, the amplification reaction includes polymerase chain reaction (PCR). In some embodiments, the amplification reaction includes isothermal amplification.

[0014] As used herein, "amplification conditions" and derivatives (e.g., conditions for amplification) 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, thermal cycling conditions, or a combination of isothermal 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 a nucleic acid, such as one or more target sequences, or an amplified target sequence linked to one or more adapters, e.g., an adapter-linked amplified target sequence. Generally, amplification conditions include an amplification or nucleic acid synthesis catalyst (e.g., a polymerase), a primer having a degree of complementarity to the nucleic acid to be amplified, and a nucleotide (e.g., deoxyribonucleoside triphosphate, dNTP) that facilitates extension of the primer when hybridized to the nucleic acid. Amplification conditions may require hybridization or annealing of a primer to the nucleic acid, extension of the primer, and a denaturation step in which the extended primer is separated from the nucleic acid sequence being amplified. Typically, but not necessarily, amplification conditions may include thermal cycling. In some embodiments, amplification conditions include multiple cycles in which the steps of annealing, extension, and separation are repeated. Typically, amplification conditions include a denaturation step in which the denaturation step is repeated, such as in the case of a nucleic acid sequence that is amplified by ... ++ or Mn ++ Cations such as MgCl 2 etc.), and optionally may also include various adjusters of ionic strength.

[0015] As used herein, "target sequence", "target nucleic acid sequence", or "target sequence of interest" and derivatives 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 is present in double-stranded form and includes at least a portion of the specific nucleotide sequence to be amplified or synthesized, or its complement, prior to the addition of target-specific primers or additional adapters. The target sequence can include a nucleic acid to which a primer useful in an amplification or synthesis reaction can hybridize prior to extension by a polymerase. In some embodiments, the term refers to a nucleic acid sequence in which the sequence identity, ordering, or location of nucleotides are determined by one or more of the methods of the present disclosure.

[0016] As used herein, the term "portion" and variations thereof, when used in reference to a given nucleic acid molecule, e.g., a primer or a 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.

[0017] As used herein, "contacting" and its derivatives, when used in reference to two or more components, generally refers to any process in which the approach, proximity, mixing, or intermingling of the referenced components is promoted or accomplished without necessarily requiring physical contact of such components, including mixing solutions containing any one or more of the referenced components with one another. The referenced components may be contacted in any particular order or combination, and the particular order of listing of the components is not limiting. For example, "contacting A with B and C" encompasses embodiments in which A is contacted first with B and then with C, as well as embodiments in which C is contacted with A and then with B, as well as embodiments in which a mixture of A and C is contacted with B, and the like. Moreover, such contacting does not necessarily require that the end result of the contacting process is a mixture containing all of the referenced components, so long as all of the referenced components are present at the same time or are included in the same mixture or solution at the same time at some point during the contacting process. For example, "contacting A with B and C" can 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 can then be removed, leaving only B. When one or more of the contacting reference components comprises a plurality (e.g., "contacting a target sequence with a plurality of target-specific primers and a polymerase"), each member of the plurality can be viewed as an individual component of the contacting process, such that the contacting can include contacting any one or more members of the plurality with any other member of the plurality, and / or with any other reference component, in any order or combination (e.g., some but not all of the plurality of target-specific primers can be contacted with the target sequence, then with the polymerase, and then with other members of the plurality of target-specific primers).

[0018] As used herein, the term "primer" and its derivatives generally refer to any polynucleotide that can hybridize to a target sequence of interest. In some embodiments, a primer can also serve to initiate nucleic acid synthesis. Typically, a primer serves as a substrate to which nucleotides can be polymerized by a polymerase; however, in some embodiments, a primer can be incorporated into a synthetic nucleic acid strand to provide a site to which another primer can hybridize to initiate synthesis of a new strand that is complementary to the synthetic nucleic acid molecule. A primer can be composed of any combination of nucleotides or analogs thereof, which can be optionally linked to form a linear polymer of any suitable length. In some embodiments, a primer is a single-stranded oligonucleotide or polynucleotide. (For purposes of this disclosure, the terms "polynucleotide" and "oligonucleotide" are used interchangeably herein and do not necessarily indicate a difference in length between the two). In some embodiments, a primer is double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to initiate the synthesis of an extension product. The length of the primer will depend on many factors, including temperature, source of primer, and use of the method. In some embodiments, the primer acts as an initiation point for amplification or synthesis when exposed to amplification or synthesis conditions, which may occur in a template-dependent manner, optionally resulting in the formation of a primer extension product that is complementary to at least a portion of the target sequence. Exemplary amplification or synthesis conditions may include contacting the primer with a polynucleotide template (e.g., a template that includes a target sequence), nucleotides, and an inducing agent such as a polymerase at a suitable temperature and pH to induce polymerization of nucleotides to the end of the target-specific primer. If double-stranded, the primer may be optionally treated to separate its strands before being used to prepare primer extension products.In some embodiments, the primer is an oligodeoxyribonucleotide or oligoribonucleotide. In some embodiments, the primer can comprise one or more nucleotide analogs. The exact length and / or composition, including sequence, of the target-specific primer can affect many properties, including melting temperature (Tm), GC content, formation of secondary structures, repetitive nucleotide motifs, expected length of primer extension products, degree of coverage across the nucleic acid molecule of interest, number of primers present in a single amplification or synthesis reaction, presence of nucleotide analogs or modified nucleotides in the primer, and the like. In some embodiments, a primer can pair with a compatible primer in an 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 that is substantially complementary to at least a portion of a strand of the nucleic acid molecule, and the reverse primer of the primer pair comprises a sequence that is substantially identical to at least a portion of the strand. In some embodiments, the forward primer and the reverse primer can hybridize to opposite strands of a nucleic acid duplex. Optionally, a forward primer primes synthesis of a first nucleic acid strand and a reverse primer primes synthesis of a second nucleic acid strand, and the first and second strands can be substantially complementary to each other or hybridize to form a double-stranded nucleic acid molecule. In some embodiments, one end of the amplification or synthesis product is defined by the forward primer and the other end of the amplification or synthesis product is defined by the reverse primer. In some embodiments, when amplification or synthesis of a long primer extension product is required, such as amplification of an exon, coding region, or gene, several primer pairs can be created that span more than the desired length to allow sufficient amplification of the region. In some embodiments, the primers can include one or more cleavable groups. In some embodiments, the primer length ranges from about 10 to about 60 nucleotides, from about 12 to about 50 nucleotides, and from about 15 to about 40 nucleotides in length.Typically, when a primer is exposed to amplification conditions in the presence of dNTPs and polymerase, it can hybridize to the corresponding target sequence and undergo primer extension.In some cases, the specific nucleotide sequence or part of the primer is known at the start of the amplification reaction or can be determined by one or more of the methods disclosed herein.In some embodiments, the primer comprises one or more cleavable groups at one or more positions within the primer.

[0019] As used herein, "target-specific primer" and its derivatives generally refer to a single- or double-stranded polynucleotide, typically an oligonucleotide, that comprises at least one sequence that is at least 50% complementary, 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 to at least a portion of a nucleic acid molecule that comprises a target sequence. In such cases, the target-specific primer and the target sequence are described as "corresponding" to each other. In some embodiments, a target-specific primer can hybridize to at least a portion of its corresponding target sequence (or to the complement of the target sequence), and such hybridization can optionally be performed under standard hybridization conditions or stringent hybridization conditions. In some embodiments, a target-specific primer cannot hybridize to the target sequence or its complement, but can hybridize to a portion of a nucleic acid strand that comprises the target sequence or its complement. In some embodiments, the target-specific primer comprises at least one sequence that is at least 75% complementary, 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 to at least a portion of the target sequence itself, and in other embodiments, the target-specific primer comprises at least one sequence that is at least 75% complementary, 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 to at least a portion of a nucleic acid molecule other than the target sequence. In some embodiments, the target-specific primer is substantially non-complementary to other target sequences present in the sample, and optionally the target-specific primer is substantially non-complementary to other nucleic acid molecules present in the sample.In some embodiments, nucleic acid molecules present in a sample that do not contain or correspond to a target sequence (or the complement of a target sequence) are referred to as "non-specific" sequences or "non-specific nucleic acids." In some embodiments, a target-specific primer is designed to contain a nucleotide sequence that is substantially complementary to at least a portion of its corresponding target sequence. In some embodiments, a target-specific primer is at least 95% complementary, or at least 99% complementary, or identical over its entire length to at least a portion of a nucleic acid molecule that contains its corresponding target sequence. In some embodiments, a target-specific primer can be at least 90%, at least 95% complementary, at least 98% complementary, or at least 99% complementary, or identical over its entire length to at least a portion of its corresponding target sequence. In some embodiments, a forward target-specific primer and a reverse target-specific primer define a target-specific primer pair that can be used to amplify a target sequence via template-dependent primer extension. Typically, each primer of a target-specific primer pair comprises at least one sequence that is substantially complementary to at least a portion of a nucleic acid molecule that comprises a corresponding target sequence, but is less than 50% complementary to at least one other target sequence in the sample. In some embodiments, the amplification can be performed using multiple target-specific primer pairs in a single amplification reaction, each primer pair comprising a forward target-specific primer and a reverse target-specific primer, each comprising at least one sequence that is substantially complementary or substantially identical to a corresponding target sequence in the sample, and each primer pair having a different corresponding target sequence. In some embodiments, the target-specific primer can be substantially non-complementary at its 3' end or its 5' end to any other target-specific primer present in the amplification reaction. In some embodiments, the target-specific primer can comprise minimal cross-hybridization to other target-specific primers in the amplification reaction. In some embodiments, the target-specific primer comprises minimal cross-hybridization to non-specific sequences in the amplification reaction mixture.In some embodiments, the target-specific primer comprises minimal self-complementarity. In some embodiments, the target-specific primer comprises one or more cleavable groups located near or around the central nucleotide of the target-specific primer. In some embodiments, one or more target-specific primers comprise only non-cleavable nucleotides at the 5' end of the target-specific primer. In some embodiments, the target-specific primer comprises minimal nucleotide sequence overlap at the 3' 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.

[0020] As used herein, the term "adapter" refers to a nucleic acid molecule that can be used to manipulate a polynucleotide of interest. In some embodiments, the adapter is used in the amplification of one or more target nucleic acids. In some embodiments, the adapter is used in a reaction for sequencing. In some embodiments, the adapter has one or more ends that lack a 5' phosphate residue. In some embodiments, the adapter comprises, consists of, or consists essentially of at least one priming site. Such a priming site containing an adapter can be referred to as a "primer" adapter. In some embodiments, the adapter priming site can be useful in a PCR process. In some embodiments, the adapter comprises a nucleic acid sequence that is substantially complementary to the 3' or 5' end of at least one target sequence in the sample, referred to herein as a gene-specific target sequence, target-specific sequence, or target-specific primer. In some embodiments, the adapter comprises a nucleic acid sequence that is substantially non-complementary to the 3' or 5' end of any target sequence present in the sample. In some embodiments, the adapter comprises a single-stranded or double-stranded linear oligonucleotide that is not substantially 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 of the sample. In some embodiments, suitable adaptor lengths range from about 10-75 nucleotides, about 12-50 nucleotides, and about 15-40 nucleotides. In general, the adaptor can comprise any combination of nucleotides and / or nucleic acids. In some aspects, the adaptor comprises one or more cleavable groups at one or more locations. In some embodiments, the adaptor comprises a sequence that is substantially identical or substantially complementary to at least a portion of a primer, e.g., a universal primer. In some embodiments, the adaptor comprises a tag sequence that aids in cataloguing, identification, or sequencing.In some embodiments, the adaptor acts as a substrate for amplification of the target sequence in the presence of a polymerase and dNTPs, particularly under suitable temperature and pH conditions.

[0021] As used herein, "polymerase" and its derivatives generally refer to any enzyme capable of catalyzing the polymerization of nucleotides (including analogs thereof) into a nucleic acid chain. Typically, but not necessarily, such nucleotide polymerization may occur in a template-dependent manner. Such polymerases may include, without limitation, naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fused, or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives, or fragments thereof that retain the ability to catalyze such polymerization. Optionally, the polymerase may be a mutant polymerase that includes one or more mutations involving the substitution of one or more amino acids with other amino acids, the insertion or deletion of one or more amino acids from the polymerase, or the combination of two or more portions of a polymerase. Typically, a polymerase includes one or more active sites at which catalysis of nucleotide binding and / or nucleotide polymerization may occur. Some exemplary polymerases include, without limitation, DNA polymerases and RNA polymerases. As used herein, the term "polymerase" and variations thereof also refer to a fusion protein comprising at least two moieties linked together, a first moiety comprising a peptide capable of catalyzing the polymerization of nucleotides into a nucleic acid strand and linked to a second moiety comprising a second polypeptide. In some embodiments, the second polypeptide can comprise a reporter enzyme or a processivity enhancing domain. Optionally, the polymerase can have 5' exonuclease activity or terminal transferase activity. In some embodiments, the polymerase can be optionally reactivated, for example, by the use of heat, chemicals, or by re-adding a new amount of the polymerase to the reaction mixture. In some embodiments, the polymerase can comprise a hot start polymerase and / or an aptamer-based polymerase, which can be optionally reactivated.

[0022] As used herein, the terms "identity" and "identical" and variations thereof, when used in reference to two or more nucleic acid or polypeptide sequences, refer to the sequence similarity of two or more sequences (e.g., nucleotide or polypeptide sequences). In the context of two or more homologous sequences, the identity or homology percentage of a sequence or subsequence thereof refers to the percentage of all monomeric units (e.g., nucleotides or amino acids) that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity). The identity percentage can extend beyond a specified region when compared and aligned for maximum correspondence over a comparison window, or using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection. Sequences are said to be "substantially identical" if there is at least 85% identity at the amino acid or nucleotide level. Preferably, identity exists over a region that is at least about 25, 50, or 100 residues in length, or over the entire length of at least one of the comparison sequences. Exemplary algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res. 25:3389-3402 (1977). Other methods include algorithms such as those of Smith & Waterman, Adv. Appl. Math. 2:482 (1981) and Needleman & Wunsch, J. Mol. Biol. 48:443 (1970). 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.

[0023] The terms "complementary" and "complement" as used herein 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 can undergo cumulative base pairing at two or more respective corresponding positions in an antiparallel orientation, as in a hybridized duplex. Such base pairing can proceed according to any set of established rules, for example, according to the Watson-Crick base pairing rules, or according to some other base pairing paradigm. Optionally, there can be "complete" or "total" complementarity between a first nucleic acid sequence and a second nucleic acid sequence, where each nucleotide in the first nucleic acid sequence can undergo stabilized base pairing interactions 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 one nucleic acid sequence are complementary to the residues in the other nucleic acid sequence. In some embodiments, at least 50% but less than 100% of the residues of one nucleic acid sequence are complementary to the residues in the other nucleic acid sequence. In some embodiments, at least 70%, 80%, 90%, 95%, or 98% but less than 100% of the residues of one nucleic acid sequence are complementary to the residues in the other nucleic acid sequence. A sequence is said to be "substantially complementary" if at least 85% of the residues of one nucleic acid sequence are complementary to the residues in the other nucleic acid sequence. In some embodiments, two complementary or substantially complementary sequences can hybridize to 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 the other nucleic acid sequence. A sequence is said to be "substantially non-complementary" if less than 15% of the residues of one nucleic acid sequence are complementary to the residues in the other nucleic acid sequence. In some embodiments, two non-complementary or substantially non-complementary sequences cannot hybridize to each other under standard or stringent hybridization conditions. A "mismatch" exists at any position where the two opposing nucleotides are not complementary. Complementary nucleotides include those nucleotides that are efficiently incorporated by DNA polymerases opposite each other during DNA replication under physiological conditions.In typical embodiments, complementary nucleotides can form base pairs with each other, such as AT / U and GC base pairs formed through specific Watson-Crick hydrogen bonds between nucleotides and / or nucleobases of polynucleotides in antiparallel positions, or base pairs formed through some other type of base pairing paradigm. Complementarity of other artificial base pairs can be based on other types of hydrogen bonds and / or hydrophobicity of the bases and / or shape complementarity between the bases.

[0024] As used herein, "amplified target sequence" and its derivatives generally refer to a nucleic acid sequence generated by amplifying / amplifying a target sequence using target-specific primers and the methods provided herein. An amplified target sequence can be either of the same sense (i.e., the positive strand generated during the second or subsequent even round of amplification) or antisense (i.e., the negative strand generated during the first or subsequent odd round of amplification) with respect to the target sequence. For purposes of this disclosure, an amplified target sequence is typically less than 50% complementary to any portion of another amplified target sequence in the reaction.

[0025] As used herein, the terms "ligate", "linking" and derivatives generally refer to the act or process of covalently linking two or more molecules together, e.g., covalently linking two or more nucleic acid molecules to one another. In some embodiments, linking comprises joining nicks between adjacent nucleotides of a nucleic acid. In some embodiments, linking comprises forming a covalent bond between an end of a first nucleic acid molecule and an end of a second nucleic acid molecule. In some embodiments, e.g., in embodiments where the nucleic acid molecules being linked comprise conventional nucleotide residues, linking comprises forming a covalent bond between the 5' phosphate group of one nucleic acid and the 3' hydroxyl group of the second nucleic acid, thereby forming a linked nucleic acid molecule. In some embodiments, any means for joining nicks or joining the 5' phosphate to the 3' hydroxyl between adjacent nucleotides can be used. In an exemplary embodiment, an enzyme such as a ligase can be used.

[0026] As used herein, "ligase" and its derivatives generally refer to any agent capable of catalyzing the ligation of two substrate molecules. In some embodiments, a ligase comprises an enzyme capable of catalyzing the joining of nicks between adjacent nucleotides of a nucleic acid. In some embodiments, a ligase comprises an enzyme capable of catalyzing the formation of a covalent bond between the 5' phosphate of one nucleic acid molecule and the 3' hydroxyl of another nucleic acid molecule, thereby forming a ligated 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.

[0027] As defined herein, a "cleavable group" generally refers to any moiety that can be cleaved under appropriate conditions once incorporated into a nucleic acid. For example, a cleavable group can be incorporated into a target-specific primer, an amplified sequence, an adapter, or a nucleic acid molecule of a sample. In an exemplary embodiment, a target-specific primer can include a cleavable group that is incorporated into an amplification product and then cleaved after amplification, thereby removing a portion or all of the target-specific primer from the amplification product. A cleavable group can be cleaved or otherwise removed from a target-specific primer, an amplified sequence, an adapter, or a nucleic acid molecule of a sample by any acceptable means. For example, a cleavable group can be removed from a target-specific primer, an amplified sequence, an adapter, or a nucleic acid molecule of a sample by enzymes, heat, photo-oxidation, or chemical treatment. In one aspect, a cleavable group can include a non-naturally occurring nucleobase. For example, an oligodeoxyribonucleotide can include one or more RNA nucleobases, such as uracil, which can be removed by uracil glycosylase. In some embodiments, the cleavable group can comprise 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, xanthosine, inosine, dihydrouridine, or 5-methylcytidine). The modified nucleobases or nucleotides can be removed from the nucleic acid by enzymatic, chemical, or thermal means. In one embodiment, the cleavable group can comprise a moiety that can be removed from the primer upon exposure to ultraviolet light (i.e., bromodeoxyuridine) after amplification (or synthesis). In another embodiment, the cleavable group can comprise a methylated cytosine. Typically, the methylated cytosine can be cleaved from the primer upon, for example, sodium bisulfite treatment after induction of amplification (or synthesis). In some embodiments, the cleavable moiety can comprise a restriction site.For example, the primer or target sequence can include nucleic acid sequences specific for one or more restriction enzymes, and following amplification (or synthesis), the primer or target sequence can be treated with one or more restriction enzymes to remove the cleavable groups. Typically, one or more cleavable groups can be included at one or more locations with the target-specific primers, amplified sequences, adapters, or nucleic acid molecules of the sample.

[0028] As used herein, "digestion," "digestion step," and derivatives thereof generally refer to any process in which cleavable groups are cleaved or otherwise removed from target-specific primers, amplified sequences, adapters, or nucleic acid molecules of a sample. In some embodiments, the digestion step comprises a chemical, thermal, photo-oxidative, or digestive process.

[0029] As used herein, the term "hybridization" is consistent with its use in the art and generally refers to the process by which two nucleic acid molecules undergo base pairing interactions. Two nucleic acid molecules are said to be hybridized when any portion of one nucleic acid molecule is base paired with any portion of the other nucleic acid molecule, and it is not necessary that the two nucleic acid molecules are hybridized over their entire length, and in some embodiments, at least one of the nucleic acid molecules may contain portions that are not hybridized to the other nucleic acid molecule. The phrase "hybridizing under stringent conditions" and its variants generally refers to conditions under which hybridization of a target-specific primer to a target sequence occurs in the presence of high hybridization temperatures and low ionic strength. As used herein, the phrase "standard hybridization conditions" and its variants generally refers to conditions under which hybridization of a primer to an oligonucleotide (i.e., a target sequence) occurs in the presence of low hybridization temperatures and high ionic strength. In one exemplary embodiment, standard hybridization conditions include an aqueous environment containing about 100 mM magnesium sulfate, about 500 mM Tris sulfate at pH 8.9, and about 200 mM ammonium sulfate at about 50-55° C., or equivalents.

[0030] As used herein, the term "end" and variations thereof, when used in reference to a nucleic acid molecule, e.g., a target sequence or an amplified target sequence, can include the terminal 30 nucleotides, the terminal 20, and even more typically the terminal 15 nucleotides of a nucleic acid molecule. A linear nucleic acid molecule consisting of a series of linked contiguous nucleotides typically includes at least two ends. In some embodiments, one end of a nucleic acid molecule can include a 3' hydroxyl group or its equivalent and can be referred to as the "3' end" and its derivatives. Optionally, the 3' end includes a 3' hydroxyl group that is not linked to the 5' phosphate group of a mononucleotide pentose ring. Typically, the 3' end includes one or more 5' linked nucleotides located adjacent to a nucleotide that includes an unlinked 3' hydroxyl group, typically the 30 nucleotides located adjacent to the 3' hydroxyl, typically the terminal 20, and even more typically the terminal 15 nucleotides. Generally, the one or more linked nucleotides can be expressed as a percentage of the nucleotides present in the oligonucleotide, or can be provided as a number of linked nucleotides adjacent to an unlinked 3' hydroxyl. For example, the 3' end can comprise less than 50% of the nucleotide length of the oligonucleotide. In some embodiments, the 3' end does not comprise any unbound 3' hydroxyl group, but can comprise any moiety that can serve as a site for attachment of a nucleotide by primer extension and / or nucleotide polymerization. In some embodiments, the term "3' end", for example, when referring to a target-specific primer, can comprise the terminal 10 nucleotides, the terminal 5 nucleotides, the terminal 4, 3, 2, or fewer nucleotides at the 3' end. In some embodiments, the term "3' end", when referring to a target-specific primer, can comprise nucleotides located 10 nucleotides or less from the 3' terminus. As used herein, the "5' end" and derivatives thereof generally refer to the end of a nucleic acid molecule, e.g., a target sequence or an amplified target sequence, including a free 5' phosphate group or its equivalent.In some embodiments, the 5' end includes a 5' phosphate group that is not attached to the 3' hydroxyl of an adjacent mononucleotide pentose ring. Typically, the 5' end includes one or more linked nucleotides located adjacent to the 5' phosphate, typically 30 nucleotides located adjacent to the nucleotide that includes the 5' phosphate group, typically the terminal 20, and even more typically the terminal 15 nucleotides. Generally, the one or more linked nucleotides can be expressed as a percentage of the nucleotides present in the oligonucleotide, or can be provided as a number of linked nucleotides adjacent to the 5' phosphate. For example, the 5' end can be less than 50% of the nucleotide length of the oligonucleotide. In another exemplary embodiment, the 5' end can include about 15 nucleotides adjacent to the nucleotide that includes the terminal 5' phosphate. In some embodiments, the 5' end does not include any unlinked 5' phosphate group, but can include a 3' hydroxyl group, or any moiety that can function as a site of attachment to the 3' end of another nucleic acid molecule. In some embodiments, the term "5' end" can include, for example, the terminal 10 nucleotides, the terminal 5 nucleotides, the terminal 4, 3, 2, or fewer nucleotides at the 5' end when referring to a target-specific primer. In some embodiments, the term "5' end" when referring to a target-specific primer can include nucleotides located 10 or less from the 5' terminus. In some embodiments, the 5' end of a target-specific primer can include only non-cleavable nucleotides, e.g., nucleotides that do not contain one or more cleavable groups as disclosed herein, or cleavable nucleotides as readily determined by one of skill in the art. The "first end" and "second end" of a polynucleotide refer to the 5' end or 3' end of the polynucleotide. Either the first end or the second end of a polynucleotide can be the 5' end or the 3' end of the polynucleotide, and the terms "first" and "second" are not meant to indicate that the ends are specifically the 5' end or the 3' end.

[0031] As used herein, "tag", "barcode", "unique tag" or "tag sequence" and derivatives thereof generally refer to a unique short (6-14 nucleotide) nucleic acid sequence within an adapter or primer that can function as a "key" to distinguish or separate multiple amplified target sequences in a sample. For purposes of this disclosure, the barcode or unique tag sequence is incorporated into the nucleotide sequence of the adapter or primer. As used herein, "barcode sequence" refers to a nucleic acid fixed sequence that is sufficient to allow identification of a sample or source of a nucleic acid sequence of interest. The barcode sequence can be, but need not be, a subsection of the original nucleic acid sequence on which the identification is based. In some embodiments, the barcode is 5-20 nucleic acids long. In some embodiments, the barcode comprises analog nucleotides, e.g., L-DNA, LNA, PNA, etc. As used herein, "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 together with a second unique tag sequence, is sufficient to allow identification of a single target nucleic acid molecule in a sample. A unique tag sequence can, but need not, comprise a subsection of the original target nucleic acid sequence. In some embodiments, the unique tag sequence is between 2 and 50 nucleotides or base pairs, or between 2 and 25 nucleotides or base pairs, or between 2 and 10 nucleotides or base pairs in length. A unique tag sequence can comprise at least one random sequence interspersed with fixed sequences.

[0032] As used herein, "comparable maximum minimum melting temperature" 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 the cleavable group. The hybridization temperatures of each nucleic acid fragment generated by the adapter or target-specific primer are compared to determine the maximum minimum temperature required to prevent hybridization of the nucleic acid sequence from the target-specific primer or adapter or a fragment or portion thereof to the respective target sequence. Once the maximum hybridization temperature is known, it is possible to manipulate the adapter or target-specific primer, for example by shifting the position of one or more cleavable groups along the length of the primer, to achieve a comparable maximum minimum melting temperature for each nucleic acid fragment, thereby optimizing the digestion and repair steps of the library preparation.

[0033] 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 excludes removing the reaction mixture from a first container to a second container to complete the series of steps. Generally, an addition-only process excludes manipulation of the reaction mixture outside of the container containing the reaction mixture. Typically, an addition-only process is amenable to automation and high throughput.

[0034] As used herein, "polymerization conditions" and its derivatives generally refer to conditions suitable for nucleotide polymerization. In typical embodiments, such nucleotide polymerization is catalyzed by a polymerase. In some embodiments, the polymerization conditions include conditions for primer extension, optionally in a template-dependent manner, resulting in the production of a synthesized nucleic acid sequence. In some embodiments, the polymerization conditions include a polymerase chain reaction (PCR). Typically, the polymerization conditions are sufficient to synthesize nucleic acids and include the use of a reaction mixture that includes a polymerase and nucleotides. The polymerization conditions can include conditions for annealing of a target-specific primer to a target sequence and extension of the primer in a template-dependent manner in the presence of a polymerase. In some embodiments, the polymerization conditions can be performed using thermal cycling. In addition, the polymerization conditions can include multiple cycles in which the steps of annealing, extension, and separation of the two nucleic acid strands are repeated. Typically, the polymerization conditions include the use of a reaction mixture that includes a polymerase and nucleotides. 2 Generally, polymerization of one or more nucleotides to form a nucleic acid chain involves the nucleotides being linked together via phosphodiester bonds, although alternative linkages may be possible in the context of certain nucleotide analogs.

[0035] As used herein, the term "nucleic acid" refers to natural nucleic acids, artificial nucleic acids, their analogs, or combinations thereof, including polynucleotides and oligonucleotides. As used herein, the terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to single- and double-stranded polymers of nucleotides, including, but not limited to, 2'-deoxyribonucleotides (nucleic acid) and ribonucleotides (RNA) linked by internucleotide phosphodiester bonds, e.g., 3'-5' and 2'-5', inverted bonds, e.g., 3'-3' and 5'-5', branched structures, or analog nucleic acids. Polynucleotides are defined as any of the following: + , N.H. 4 + , Trialkylammonium, Mg 2+ , Na +and associated counterions such as . Oligonucleotides can be composed of only deoxyribonucleotides, only ribonucleotides, or chimeric mixtures thereof. Oligonucleotides can be composed of nucleobase and sugar analogs. Polynucleotides typically range in size from a few monomeric units, e.g., 5-40, when they are more commonly and frequently referred to in the art as oligonucleotides, to several thousand monomeric nucleotide units, when they are more commonly and frequently referred to in the art as polynucleotides; however, for purposes of this disclosure, both oligonucleotides and polynucleotides can be of any suitable length. Unless otherwise indicated, whenever an oligonucleotide sequence is represented, it will be understood that the nucleotides are in 5'-3' order from left to right, with "A" indicating deoxyadenosine, "C" indicating deoxycytidine, "G" indicating deoxyguanosine, "T" indicating thymidine, and "U" indicating deoxyuridine. As discussed herein and known in the art, mononucleotides are typically reacted to form oligonucleotides via the attachment of the 5' phosphate or equivalent group of one nucleotide to the 3' hydroxyl or equivalent group of an adjacent nucleotide, optionally via a phosphodiester or other suitable linkage, and therefore oligonucleotides and polynucleotides are said to have a "5' end" and a "3' end."

[0036] As used herein, the term "polymerase chain reaction" ("PCR") refers to the method of K.B. Mullis, U.S. Patent Nos. 4,683,195 and 4,683,202, incorporated herein by reference, which describes a method for increasing the concentration of a segment of a polynucleotide of interest in a mixture of genomic DNA 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 cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double-stranded polynucleotide of interest. To carry out the amplification, the mixture is denatured and the primers are then annealed to their complementary sequences within the polynucleotide of the target molecule. Following annealing, the primers are extended with a polymerase to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing, and extension constitute one "cycle" and there can be many "cycles") to obtain a high concentration of the amplified segment of the desired polynucleotide of interest. The length of the amplified segment (amplicon) of the desired polynucleotide of interest is determined by the relative positions of the primers with respect to each other, and therefore this length is a controllable parameter. By repeating the process, the method is called "polymerase chain reaction" (hereinafter "PCR"). The desired amplified segment of the polynucleotide of interest is said to be "PCR amplified" because it becomes the predominant nucleic acid sequence (in terms of concentration) in the mixture. As defined herein, a target nucleic acid molecule in a sample containing multiple target nucleic acid molecules is amplified via PCR. In a modification of the method discussed above, the target nucleic acid molecule can be PCR amplified using multiple different primer pairs, in some cases more than one primer pair per target nucleic acid molecule of interest, thereby forming a multiplex PCR reaction. Using multiplex PCR, it is possible to simultaneously amplify multiple nucleic acid molecules of interest from a sample to form an amplified target sequence.Additionally, the amplified target sequences can be analyzed by a number of different methodologies (e.g., quantification by bioanalyzer or qPCR, hybridization with a labeled probe, incorporation of a biotinylated primer followed by avidin-enzyme conjugate detection, and detection of the amplified target sequences). 32 It is also possible to detect the presence of a nucleic acid sequence by incorporation of P-labeled deoxynucleotide triphosphates (e.g., dCTP or dATP). Any oligonucleotide sequence can be amplified with an appropriate set of primers, allowing the 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 created by the multiplex PCR process disclosed herein are themselves efficient substrates for subsequent PCR amplification or a variety of downstream assays or manipulations.

[0037] 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, the multiplex amplification is performed such that some or all of the target sequences are amplified in a single reaction vessel. The "plexie" or "plex" of a given multiplex amplification generally refers to the number of different target-specific sequences amplified during that single multiplex amplification. In some embodiments, the plexie can be about 12-plex, 24-plex, 48-plex, 96-plex, 192-plex, 384-plex, 768-plex, 1536-plex, 3072-plex, 6144-plex, or more.

[0038] composition A single-stream multiplex next-generation sequencing workflow has been developed to determine tumor biomarkers of actionable tumors in a sample to determine the tumor status in the sample. The tumor high-precision assay composition and method of the present invention provide a specific and robust solution for biomarker screening to understand the mechanisms involved in tumor immune response. Thus, a composition for multiplex library preparation is provided, which is used in combination with manual or automated next-generation sequencing technologies and workflow solutions (e.g., Ion Torrent™ NGS workflow) to evaluate low-level biomarker targets in various sample types to assess tumor status.

[0039] Thus, compositions are provided for single-stream multiplex determination of actionable tumor biomarkers in a sample. In some embodiments, the compositions consist of multiple sets of primer pair reagents directed to multiple target sequences to detect low-level targets in a sample, and the target genes are selected from tumor response genes consisting 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 genes are selected from tumor genes consisting of one or more functions in Table 1. In some embodiments, the target genes are selected from one or more actionable target genes in the sample that determine changes in tumor activity in the sample that indicate potential diagnosis, prognosis, potential treatment regimes, and / or likelihood of adverse events. Overall, the various functions of genes that make up the provided multiplex panel of the present invention provide a comprehensive concept that recommends an actionable approach to cancer therapy.

[0040] In certain embodiments, the target tumor sequence is directed to a sequence that has a mutation associated with cancer. In some embodiments, the target sequence or amplified target sequence is directed to a cancer-associated cancer, such as 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), gynecological (e.g., uterine, ovarian, cervical, endometrial, fallopian), colorectal cancer, gallbladder cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, bladder cancer, prostate cancer, testicular cancer, urothelial cancer, liver cancer, or other cancers. The present invention is directed to sequences having mutations associated with one or more solid tumor cancers selected from the group consisting of: pancreatic cancer (e.g., hepatocellular, HCC), lung cancer (e.g., non-small cell lung, small cell lung), kidney (renal cell) cancer, pancreatic cancer (e.g., adenocarcinoma, ductal), thyroid cancer, cholangiocarcinoma, pituitary tumor, Wilms' tumor, Kaposi's sarcoma, hairy cell carcinoma, osteosarcoma, thymic carcinoma, skin cancer, melanoma, cardiac cancer, oral and laryngeal cancer, neuroblastoma, mesothelioma, and other solid tumors (thymic, bone, soft tissue, oral SCC, myelofibrosis, synovial sarcoma). In one embodiment, the mutations may include substitutions, insertions, inversions, point mutations, deletions, mismatches, and translocations. 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 the group consisting of 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 cancer-associated mutant biomarker is located in at least one of the genes provided in Table 1.

[0041] In some embodiments, the one or more mutant tumor sequences are located in at least one of the genes selected from Table 1. In some embodiments, the one or more mutant sequences exhibit cancer activity.

[0042] In some embodiments, the one or more mutant sequences indicate the likelihood of a patient responding to a therapeutic agent. In some embodiments, the one or more mutant tumor biomarker sequences indicate the likelihood of a patient not responding to a therapeutic agent. In certain embodiments, the relevant therapeutic agent may be a tumor therapy, including, but not limited to, kinase inhibitors, cell signaling inhibitors, checkpoint blockade, T cell therapy, and therapeutic vaccines.

[0043] 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 cancer, such as 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), gynecological (e.g., uterine, ovarian, cervical, endometrial, fallopian), colorectal cancer, gallbladder cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, bladder cancer, prostate cancer, testicular cancer, urothelial cancer, The present invention is directed to mutations associated with one or more solid tumor cancers selected from the group consisting of: liver cancer (e.g., hepatocellular, HCC), lung cancer (e.g., non-small cell lung, small cell lung), kidney (renal cell) cancer, pancreatic cancer (e.g., adenocarcinoma, ductal), thyroid cancer, cholangiocarcinoma, pituitary tumor, Wilms' tumor, Kaposi's sarcoma, hairy cell carcinoma, osteosarcoma, thymic carcinoma, skin cancer, melanoma, cardiac cancer, oral and laryngeal cancer, neuroblastoma, mesothelioma, and other solid tumors (thymus, bone, soft tissue, oral SCC, myelofibrosis, synovial sarcoma). In one embodiment, the mutations may include substitutions, insertions, inversions, point mutations, deletions, mismatches, and translocations. In one embodiment, the mutations may include copy number variations. In one embodiment, the mutations may include germline or somatic mutations. 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 the group consisting of 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.

[0044] In one embodiment, the cancer associated mutation is located in at least one of the genes provided in Table 1. In some embodiments, the mutant target sequence is directed to 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 the amplicon sequence of each of the genes provided in Table 1.

[0045] In some embodiments, the composition comprises any one or more of the tumor target specific primer pairs provided in Table A. In some embodiments, the composition comprises all of the tumor target specific primer pairs provided in Table A. In some embodiments, any one or more of the tumor target specific primer pairs provided in Table A can be used to amplify a target sequence present in a sample by the methods described herein, as disclosed.

[0046] In some embodiments, the tumor target specific primers from Table A comprise 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 comprise 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 cancer-associated target specific primers is at least 90% identical to at least one nucleic acid sequence generated using a target specific primer selected from SEQ ID NOs: 1-1559. In some embodiments, at least one of the tumor-associated target specific primers is complementary over its entire length to at least one target sequence in the sample. 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 individual exons that have a mutation associated with cancer. In one embodiment, the amplified target sequence is directed to an individual exon that has a mutation associated with cancer.

[0047] method The provided method of the present invention includes an efficient procedure that allows for the rapid preparation of highly multiplexed libraries suitable for downstream analysis.The method optionally allows for the incorporation of one or more unique tag sequences.Certain methods include streamlined, add-only procedures that facilitate very rapid library generation.

[0048] Provided herein is a method for determining tumor activity in a sample. In some embodiments, the method includes multiplex amplification of multiple tumor sequences from a biological sample, the amplification includes contacting at least a portion of the sample with a set of multiple primer pair reagents directed to the multiple target sequences and a polymerase under amplification conditions, thereby generating amplified target expression sequences. The method further includes detecting the presence of mutations in one or more target sequences in the sample, and mutations in one or more tumor markers compared to a control determine a change in tumor activity in the sample. In some embodiments, the tumor sequences of the method are selected from tumor response genes consisting 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 genes are selected from tumor genes consisting of one or more functions in Table 1. In some embodiments, the target genes are selected from one or more actionable target genes in the sample that determine a change in tumor activity in the sample that is indicative of a potential diagnosis, prognosis, potential treatment regimen, and / or potential adverse event. Overall, the various functions of the genes constituting the provided multiplex panel of the present invention provide a comprehensive concept that suggests an actionable approach to cancer therapy.

[0049] In certain embodiments, the target tumor 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 cancer-associated cancer, such as 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), gynecological (e.g., uterine, ovarian, cervical, endometrial, fallopian), colorectal cancer, gallbladder cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, bladder cancer, prostate cancer, testicular cancer, urothelial cancer, liver cancer, or other cancers. The present invention is directed to sequences having mutations associated with one or more solid tumor cancers selected from the group consisting of: pancreatic cancer (e.g., hepatocellular, HCC), lung cancer (e.g., non-small cell lung, small cell lung), kidney (renal cell) cancer, pancreatic cancer (e.g., adenocarcinoma, ductal), thyroid cancer, cholangiocarcinoma, pituitary tumor, Wilms' tumor, Kaposi's sarcoma, hairy cell carcinoma, osteosarcoma, thymic carcinoma, skin cancer, melanoma, cardiac cancer, oral and laryngeal cancer, neuroblastoma, mesothelioma, and other solid tumors (thymic, bone, soft tissue, oral SCC, myelofibrosis, synovial sarcoma). In one embodiment, the mutations may include substitutions, insertions, inversions, point mutations, deletions, mismatches, and translocations. 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 the group consisting of 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 cancer-associated mutant biomarker is located in at least one of the genes provided in Table 1.

[0050] In some embodiments, the one or more mutant tumor sequences of the method are located in at least one of the genes selected from Table 1. In some embodiments, the one or more mutant sequences exhibit cancer activity.

[0051] In some embodiments, the one or more mutant sequences of the method indicate the likelihood of the patient responding to a therapeutic agent. In some embodiments, the one or more mutant tumor biomarker sequences indicate the likelihood of the patient not responding to a therapeutic agent. In certain embodiments, the relevant therapeutic agent may be a tumor therapy, including, but not limited to, a kinase inhibitor, a cell signaling inhibitor, a checkpoint blockade, a T cell therapy, and a therapeutic vaccine.

[0052] 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 the method is directed to a mutation associated with cancer. 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 the method is directed to a mutation associated with cancer. The present invention is directed to mutations associated with one or more solid tumor cancers selected from the group consisting of cancer, liver cancer (e.g., hepatocellular, HCC), lung cancer (e.g., non-small cell lung, small cell lung), kidney (renal cell) cancer, pancreatic cancer (e.g., adenocarcinoma, ductal), thyroid cancer, cholangiocarcinoma, pituitary tumor, Wilms' tumor, Kaposi's sarcoma, hairy cell carcinoma, osteosarcoma, thymic carcinoma, skin cancer, melanoma, cardiac cancer, oral and laryngeal cancer, neuroblastoma, mesothelioma, and other solid tumors (thymus, bone, soft tissue, oral SCC, myelofibrosis, synovial sarcoma). In one embodiment, the mutations may include substitutions, insertions, inversions, point mutations, deletions, mismatches, and translocations. In one embodiment, the mutations may include copy number variations. In one embodiment, the mutations may include germline or somatic mutations. 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 the group consisting of 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.

[0053] In one embodiment, the cancer associated mutation is located in at least one of the genes provided in Table 1. In some embodiments, the mutant target sequence is directed to 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 the amplicon sequence of each of the genes provided in Table 1.

[0054] In some embodiments, the method comprises the use of any one or more of the tumor target specific primer pairs provided in Table A. In some embodiments, the method comprises the use of all of the tumor target specific primer pairs provided in Table A. In some embodiments, the use of any one or more of the tumor target specific primer pairs provided in Table A can be used to amplify a target sequence present in a sample by the methods described herein, as disclosed.

[0055] In some embodiments, the method includes the use of tumor target specific primers from Table A, which 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 method includes detection of an 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, the method includes the use of at least one of the cancer-associated target specific primers that is at least 90% identical to at least one nucleic acid sequence generated using a target specific primer selected from SEQ ID NOs: 1-1559. In some embodiments, at least one of the tumor-associated target specific primers is complementary over its entire length to at least one target sequence in the sample. 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 a terminal 3' nucleotide. In one embodiment, the amplified target sequences are directed to one or more individual exons that have a mutation associated with cancer. In one embodiment, the amplified target sequences of the present method are directed to individual exons that have a mutation associated with cancer.

[0056] In some embodiments, the method includes the detection and, optionally, identification of clinically actionable markers. As defined herein, the term "clinically actionable marker" includes clinically actionable mutations and / or clinically actionable expression patterns that are known or may be associated by one of skill in the art with a prognosis of the treatment of cancer. In one embodiment, the prognosis of the treatment of cancer includes the identification of mutations and / or expression patterns associated with responsiveness or non-responsiveness of the cancer to a drug, drug combination, or treatment regimen. In one embodiment, the method includes the amplification of multiple target sequences from a population of nucleic acid molecules that are associated with or correlated with the onset, progression, or remission 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 genes provided in Table 1. In some embodiments, the amplified target sequence may include any one or more amplified target sequences generated using 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 of a gene from Table 1.

[0057] 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 adaptors capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid(s) undergo a first amplification, and digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and prepare a partially digested target amplicon, thereby generating a gapped double-stranded amplicon. The method further includes repairing the partially digested target amplicon, and then amplifying the repaired target amplicon in a second amplification using a universal primer, thereby generating a library of target nucleic acid sequences. Each of the plurality of adaptors used in the methods herein includes a universal handle sequence, a target nucleic acid sequence, a cleavable portion, and optionally one or more tag sequences. At least two and up to 100,000 target-specific adaptor pairs are included in the methods provided, and the target nucleic acid sequence of each adaptor includes at least one cleavable portion, and the universal handle sequence does not include a cleavable portion. In some embodiments where any tag sequence is included in at least one adaptor, the cleavable moiety is included in the adaptor sequence adjacent to either end of the tag sequence.

[0058] In one aspect of the present invention, a method for preparing a tagged library of target nucleic acid sequences is provided. In some embodiments, the method includes contacting a nucleic acid sample with a plurality of adaptors capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid(s) undergo a first amplification, and digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and prepare a partially digested target amplicon, thereby generating a gapped double-stranded amplicon. The method further includes repairing the partially digested target amplicon, and then amplifying the repaired target amplicon in a second amplification using a universal primer, thereby generating a library of target nucleic acid sequences. Each of the plurality of adaptors used in the methods herein includes a universal handle sequence, a target nucleic acid sequence, a cleavable portion, and one or more tag sequences. At least two and up to 100,000 target-specific adaptor pairs are included in the provided methods, where 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 portion is included adjacent to either end of the tag sequence.

[0059] In certain embodiments, the comparable maximum-minimum melting temperature of each universal sequence is higher than the comparable maximum-minimum melting temperature of each target nucleic acid sequence and each tag sequence present in the adaptor.

[0060] In some embodiments, each of the adaptors comprises a unique tag sequence, as further described herein, and further comprises a cleavable group adjacent to either end of the tag sequence in each adaptor, respectively. In some embodiments in which unique tag sequences are used, each target-specific amplicon sequence generated comprises at least one distinct sequence and up to 10 distinct sequences. 7 In certain embodiments, each target-specific pair of adapters of the plurality comprises up to 16,777,216 different adapter combinations comprising different tag sequences.

[0061] In some embodiments, the method comprises contacting the plurality of gapped polynucleotide products with a digestion reagent and a repair reagent simultaneously, hi some embodiments, the method comprises contacting the plurality of gapped polynucleotide products with a digestion reagent and then a repair reagent sequentially.

[0062] Digestion reagents useful in the methods provided herein include any reagent capable of cleaving a cleavable site present in an adaptor, and in some embodiments include, but are not limited to, one or a combination of uracil DNA glycosylase (UDG), apurinic endonuclease (e.g., APE1), RecJf, formamidopyrimidine [fapy]-DNA glycosylase ([fapy]-DNA glycosylase, fpg), Nth endonuclease III, endonuclease VIII, polynucleotide kinase (PNK), Taq DNA polymerase, DNA polymerase I, and / or human DNA polymerase beta.

[0063] Repair reagents useful in the methods provided herein include any reagent capable of repairing gapped amplicons, and in some embodiments include, but are not limited to, any one or combination of Phusion DNA polymerase, Phusion U DNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, human DNA polymerase beta, T4 DNA polymerase and / or T7 DNA polymerase, SuperFiU DNA polymerase, E. coli DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq DNA ligase, and / or 9°N DNA ligase.

[0064] Thus, in certain embodiments, the digestion and repair reagents include one or a combination of 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 beta, as well as any one or a combination of Phusion DNA polymerase, Phusion U DNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, human DNA polymerase beta, T4 DNA polymerase and / or T7 DNA polymerase, SuperFiU DNA polymerase, E. 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 combination of 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 combination of 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.

[0065] In some embodiments, the method includes digestion and repair steps that are performed in a single step, while in other embodiments, the method includes digestion and repair steps that are performed at different temperatures and separated in time.

[0066] In some embodiments, the methods of the invention are performed and one or more of the method steps are performed in a manual mode. In certain embodiments, the methods of the invention are performed and each of the method steps are performed manually. In some embodiments, the methods of the invention are performed and one or more of the method steps are performed in an automated mode. In certain embodiments, the methods of the invention are performed and each of the method steps are automated. In some embodiments, the methods of the invention are performed and one or more of the method steps are performed in a combination of manual and automated modes.

[0067] In some embodiments, the method of the present invention includes 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, 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.

[0068] In some embodiments, the purification step comprises performing a solid-phase attachment reaction, a solid-phase immobilization reaction, or gel electrophoresis. In certain embodiments, the purification step comprises a separation performed using Solid Phase Reversible Immobilization (SPRI) beads. In certain embodiments, the purification step comprises a separation performed using SPRI beads, where the SPRI beads comprise paramagnetic beads.

[0069] In some embodiments, the method includes contacting a nucleic acid sample with a plurality of adaptors capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid(s) undergo a first amplification, digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and prepare a partially digested target amplicon, thereby generating a gapped double-stranded amplicon. The method further includes repairing the partially digested target amplicon, then purifying the repaired amplicon, then amplifying the repaired target amplicon in a second amplification using a universal primer, thereby generating a library of target nucleic acid sequences, and then purifying the resulting library. Each of the plurality of adaptors used in the methods herein comprises a universal handle sequence, a target nucleic acid sequence, a cleavable portion, and optionally one or more tag sequences. At least two and up to 100,000 target-specific adaptor pairs are included in the methods provided, and the target nucleic acid sequence of each adaptor comprises at least one cleavable portion, and the universal handle sequence does not comprise a cleavable portion. In some embodiments where any tag sequence is included in at least one adaptor, the cleavable moiety is included in the adaptor sequence adjacent to either end of the tag sequence.

[0070] In some embodiments, the method includes contacting a nucleic acid sample with a plurality of adaptors capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid(s) undergo a first amplification, digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and prepare a partially digested target amplicon, thereby generating a gapped double-stranded amplicon. The method further includes repairing the partially digested target amplicon and purifying the repaired amplicon, then amplifying the repaired target amplicon in a second amplification using a universal primer, thereby generating a library of target nucleic acid sequences, and then purifying the resulting library. Each of the plurality of adaptors used in the methods herein includes a universal handle sequence, a target nucleic acid sequence, a cleavable portion, and one or more tag sequences. At least two and up to 100,000 target-specific adaptor pairs are included in the provided methods, where 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 portion is included adjacent to either end of the tag sequence.

[0071] In some embodiments, the method includes contacting a nucleic acid sample with a plurality of adaptors capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid(s) undergo a first amplification, digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and prepare a partially digested target amplicon, thereby generating a gapped double-stranded amplicon. The method further includes repairing the partially digested target amplicon, then purifying the repaired amplicon, then amplifying the repaired target amplicon in a second amplification using a universal primer, thereby generating a library of target nucleic acid sequences, and then purifying the resulting library. Each of the plurality of adaptors used in the methods herein comprises a universal handle sequence, a target nucleic acid sequence, a cleavable portion, and optionally one or more tag sequences. At least two and up to 100,000 target-specific adaptor pairs are included in the methods provided, and the target nucleic acid sequence of each adaptor comprises at least one cleavable portion, and the universal handle sequence does not comprise a cleavable portion. In some embodiments where any tag sequence is included in at least one adaptor, the cleavable moiety is included in the adaptor sequence adjacent to either end of the tag sequence.In some embodiments, the digestion and repair reagents include one or a combination of 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 beta, as well as any one or a combination of Phusion DNA polymerase, Phusion U DNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, human DNA polymerase beta, T4 DNA polymerase and / or T7 DNA polymerase, SuperFiU DNA polymerase, E. 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 combination of 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 combination of 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.

[0072] In some embodiments, the method includes contacting a nucleic acid sample with a plurality of adaptors capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid(s) undergo a first amplification, digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and prepare a partially digested target amplicon, thereby generating a gapped double-stranded amplicon. The method further includes repairing the partially digested target amplicon and purifying the repaired amplicon, then amplifying the repaired target amplicon in a second amplification using a universal primer, thereby generating a library of target nucleic acid sequences, and then purifying the resulting library. Each of the plurality of adaptors used in the methods herein includes a universal handle sequence, a target nucleic acid sequence, a cleavable portion, and one or more tag sequences. At least two and up to 100,000 target-specific adaptor pairs are included in the provided methods, where 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 portion is included adjacent to either end of the tag sequence.In some embodiments, the digestion and repair reagents include one or a combination of 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 beta, as well as any one or a combination of Phusion DNA polymerase, Phusion U DNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, human DNA polymerase beta, T4 DNA polymerase, and / or T7 DNA polymerase, SuperFiU DNA polymerase, E. 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 combination of 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 combination of 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.

[0073] In certain embodiments, the methods of the invention are carried out in a single, additive workflow reaction, allowing for the rapid generation of highly multiplexed targeted libraries. For example, in one embodiment, a method for preparing a library of target nucleic acid sequences includes contacting a nucleic acid sample with a plurality of adaptors capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid(s) undergo a first amplification, digesting the resulting first amplification product to reduce or eliminate the resulting primer dimers and prepare a partially digested target amplicon, thereby generating a gapped double-stranded amplicon. The method further includes repairing the partially digested target amplicon, and then amplifying the repaired target amplicon in a second amplification using a universal primer, thereby generating a library of target nucleic acid sequences, and purifying the resulting library. In certain embodiments, the purification includes a single or repeated separation step that is carried out following the generation of the library after the second amplification, and other method steps are carried out in a single reaction vessel without the necessary transfer of any portion (aliquot) of the product generated in the step to another reaction vessel. Each of the plurality of adaptors used in the methods herein comprises a universal handle sequence, a target nucleic acid sequence, a cleavable portion, and optionally one or more tag sequences. At least two and up to 100,000 target-specific adaptor pairs are included in the methods provided, where the target nucleic acid sequence of each adaptor comprises at least one cleavable portion, and the universal handle sequence does not comprise a cleavable portion. In some embodiments where any tag sequence is included in at least one adaptor, the cleavable portion is included in the adaptor sequence adjacent to either end of the tag sequence.

[0074] In another embodiment, a method for preparing a tagged library of target nucleic acid sequences is provided, comprising contacting a nucleic acid sample with a further plurality of adaptors capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid(s) undergo a first amplification, digesting the resulting first amplification products to reduce or eliminate resulting primer dimers and prepare partially digested target amplicons, thereby generating gapped double-stranded amplicons. The method further comprises repairing the partially digested target amplicons, and then amplifying the repaired target amplicons in a second amplification using a universal primer, thereby generating a library of target nucleic acid sequences, and purifying the resulting library. In certain embodiments, the purification comprises a single or repeated separation step, and other method steps are optionally performed in a single reaction vessel without the necessary transfer of any portion of the products generated in the steps to another reaction vessel. Each of the plurality of adaptors used in the methods herein comprises a universal handle sequence, a target nucleic acid sequence, a cleavable portion, and one or more tag sequences. At least two and up to 100,000 target-specific adaptor pairs are included in the provided methods, where 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 portion is included adjacent to either end of the tag sequence.

[0075] In one embodiment, a method for preparing a library of target nucleic acid sequences includes contacting a nucleic acid sample with a plurality of adaptors capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid(s) undergo a first amplification, digesting the resulting first amplification products to reduce or eliminate resulting primer dimers and prepare partially digested target amplicons, thereby generating gapped double-stranded amplicons. The method further includes repairing the partially digested target amplicons, and then amplifying the repaired target amplicons in a second amplification using a universal primer, thereby generating a library of target nucleic acid sequences, and purifying the resulting library.

[0076] In some embodiments, the digestion reagent comprises any one or any combination of uracil DNA glycosylase (UDG), apurinic endonuclease (e.g., 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 beta. In certain embodiments, the digestion reagent comprises any one or any combination of uracil DNA glycosylase (UDG), apurinic endonuclease (e.g., 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 beta, wherein the digestion reagent lacks formamidopyrimidine [fapy]-DNA glycosylase (fpg).

[0077] In some embodiments, the digestion reagent comprises a single-stranded DNA exonuclease that degrades in the 5' to 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 RecJf exonuclease. In certain embodiments, the digestion reagent comprises APE1 and RecJf, and the cleavage reagent comprises an apurinic / apyrimidinic endonuclease. In certain embodiments, the digestion reagent comprises AP endonuclease (APE1).

[0078] In some embodiments, the repair reagent comprises at least one DNA polymerase and the gap-filling reagent comprises any one or any combination of Phusion DNA polymerase, Phusion U DNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, human DNA polymerase beta, T4 DNA polymerase, and / or T7 DNA polymerase, and / or SuperFi U DNA polymerase. In some embodiments, the repair reagent further comprises a plurality of nucleotides.

[0079] In some embodiments, the repair reagent comprises an ATP-dependent or ATP-independent ligase, the repair reagent comprises any one or any combination of E. coli DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq DNA ligase, 9°N DNA ligase.

[0080] In certain embodiments, the digestion and repair reagents include one or a combination of 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 beta, as well as any one or a combination of Phusion DNA polymerase, Phusion U DNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, human DNA polymerase beta, T4 DNA polymerase and / or T7 DNA polymerase, SuperFiU DNA polymerase, E. 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 reagent comprises any one or combination of 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 purification comprises a single or repeated separation step following the generation of the second post-amplification library, and the method steps are performed in a single reaction vessel up to the first purification without the necessary transfer of any portion of the products generated in the steps to another reaction vessel. Each of the multiple adaptors used in the methods herein comprises a universal handle sequence, a target nucleic acid sequence, a cleavable portion, and optionally one or more tag sequences. At least two and up to 100,000 target-specific adaptor pairs are included in the methods provided, and the target nucleic acid sequence of each adaptor comprises at least one cleavable portion, and the universal handle sequence does not comprise a cleavable portion. In some embodiments, where any tag sequence is included in at least one adaptor, the cleavable portion is included in the adaptor sequence adjacent to either end of the tag sequence.

[0081] In another embodiment, a method for preparing a tagged library of target nucleic acid sequences is provided, comprising contacting a nucleic acid sample with a further plurality of adaptors capable of amplifying one or more target nucleic acid sequences in the sample under conditions in which the target nucleic acid(s) undergo a first amplification, digesting the resulting first amplification products to reduce or eliminate resulting primer dimers and prepare partially digested target amplicons, thereby generating gapped double-stranded amplicons. The method further comprises repairing the partially digested target amplicons, and then amplifying the repaired target amplicons in a second amplification using a universal primer, thereby generating a library of target nucleic acid sequences, and purifying the resulting library. In certain embodiments, the digestion and repair reagents include one or a combination of 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 beta, as well as any one or a combination of Phusion DNA polymerase, Phusion U DNA polymerase, SuperFi DNA polymerase, Taq DNA polymerase, human DNA polymerase beta, T4 DNA polymerase and / or T7 DNA polymerase, SuperFiU DNA polymerase, E. 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 reagent comprises any one or combination of uracil DNA glycosylase (UDG), apurinic endonuclease (e.g., APE1), Taq DNA polymerase, Phusion U DNA polymerase, SuperFiU DNA polymerase, and T7 DNA ligase.In certain embodiments, purification includes a single or repeated separation step following the generation of the second post-amplification library, while other method steps are performed in a single reaction vessel without the necessary transfer of any portion (aliquot) of the product generated in the step to another reaction vessel. Each of the multiple adaptors used in the methods herein includes a universal handle sequence, a target nucleic acid sequence, a cleavable portion, and one or more tag sequences. At least two and up to 100,000 target-specific adaptor pairs are included in the methods provided, where the target nucleic acid sequence of each adaptor includes at least one cleavable portion, the universal handle sequence does not include a cleavable portion, and the cleavable portion is included adjacent to either end of the tag sequence.

[0082] In some embodiments, the adaptor-dimer by-products resulting from the first amplification of the method steps are substantially removed from the resulting library. In certain embodiments, the enriched population of amplified target nucleic acids contains a reduced amount of adaptor-dimer by-products. In certain embodiments, the adaptor-dimer by-products are eliminated.

[0083] 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 2-3 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.

[0084] composition Additional aspects of the invention include compositions comprising a plurality of nucleic acid adaptors, as well as library compositions prepared according to the methods of the invention. The compositions provided are useful in conjunction with the methods described herein, as well as for additional analyses and applications known in the art.

[0085] Thus, a composition is provided that includes a plurality of nucleic acid adaptors, each of the plurality of adaptors includes a 5' universal handle sequence, optionally one or more tag sequences, and a 3' target nucleic acid sequence, each adaptor includes a cleavable portion, the target nucleic acid sequence of the adaptor includes at least one cleavable portion, and if a tag sequence is present, the cleavable portion is included adjacent to either end of the tag sequence, and the universal handle sequence does not include a cleavable portion. At least 2 and up to 100,000 target-specific adaptor pairs are included in the provided composition. The provided composition allows for the rapid generation of highly multiplexed targeting libraries.

[0086] In some embodiments, the compositions provided include a plurality of nucleic acid adaptors, each of the plurality of adaptors includes a 5' universal handle sequence, one or more tag sequences, and a 3' target nucleic acid sequence, each adaptor includes a cleavable portion, the target nucleic acid sequence of the adaptor includes at least one cleavable portion, the cleavable portion is included on either end adjacent to the tag sequence, and the universal handle sequence does not include a cleavable portion. At least two and up to 100,000 target-specific adaptor pairs are included in the compositions provided. The compositions provided allow for the rapid generation of highly multiplexed tagged targeting libraries.

[0087] The primer / adapter composition may be single-stranded or double-stranded. In some embodiments, the adapter composition comprises single-stranded adapters. In some embodiments, the adapter composition comprises double-stranded adapters. In some embodiments, the adapter composition comprises a mixture of single-stranded and double-stranded adapters.

[0088] In some embodiments, the composition comprises a plurality of adaptors capable of amplifying one or more target nucleic acid sequences, comprising a multiplex of adaptor pairs capable of amplifying at least two different target nucleic acid sequences, wherein the target specific primer sequences are 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, 10000, 11000, or 12000 or more target specific adaptor pairs. In some embodiments, the target specific adaptor pair comprises about 15 nucleotides to about 40 nucleotides in length, and at least one nucleotide is replaced with a cleavable group. In some embodiments, the cleavable group is a uridine nucleotide. In some embodiments, the target specific adaptor pair is designed for amplification of an exon, gene, exome, or region of a genome associated with a clinical or pathological condition, for example, amplification of one or more sites containing one or more mutations (e.g., driver mutations) associated with cancer, e.g., lung cancer, colon cancer, breast cancer, etc., or amplification of mutations associated with a genetic disease, e.g., cystic fibrosis, muscular dystrophy, etc. In some embodiments, the target specific adaptor pair when hybridized to a target sequence and amplified as provided herein generates a library of adaptor ligated amplified target sequences that are about 100 to about 600 base pairs in length. In some embodiments, the adaptor ligated amplified target sequences are not over-represented in the library by more than 30% compared to the remainder of the other adaptor ligated amplified target sequences in the library. In some embodiments, the adaptor-ligated amplified target sequence library is substantially homogeneous with respect to the GC content, amplified target sequence length, or melting temperature (Tm) of each target sequence.

[0089] In some embodiments, the target-specific primer sequence of the adaptor pair in the composition of the invention is a target-specific sequence capable of amplifying a specific region of a nucleic acid molecule. In some embodiments, the target-specific adaptor is capable of amplifying genomic DNA or cDNA. In some embodiments, the target-specific adaptor is capable of amplifying mammalian nucleic acid, such as, but not limited to, human DNA or RNA, mouse DNA or RNA, bovine DNA or RNA, canine DNA or RNA, equine DNA or RNA, or any other mammal of interest. In other embodiments, the target-specific adaptor comprises a sequence directed to amplify a plant nucleic acid of interest. In other embodiments, the target-specific adaptor comprises a sequence directed to amplify 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.

[0090] As described herein, each of the plurality of adapters comprises a 5' universal handle sequence. In some embodiments, the universal handle sequence comprises 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 provided adapter does not show significant complementarity and / or hybridization to any portion of the unique tag sequence and / or target nucleic acid sequence of interest. In some embodiments, the first universal handle sequence comprises 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 comprises 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 and second universal handle sequences correspond to forward and reverse universal handle sequences, and in certain embodiments, the same first and second universal handle sequences are included for each of the multiple target-specific adapter pairs. Such forward and reverse universal handle sequences are targeted with the universal primer to perform a second amplification of the repaired amplicons in the generation of a library according to the method of the present invention. 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), and the first and second 5' universal handle sequences do not show significant hybridization to any portion of the target nucleic acid sequence of interest.

[0091] 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 methods and compositions provided to suit specific analytical platforms. The universal handle sequences of the adapters provided herein are appropriately adapted 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 are used for sequencing on Ion Torrent sequencing platforms (Ion Xpress™ adapters, 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, for example, at 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, for example, at 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 of appropriate nucleotide sequences for use with the adapters of the present invention are readily prepared using standard automated nucleic acid synthesis equipment and reagents in routine use in the art. Included for use in the methods of the invention are one single type of universal primer or two different distinct types (or even mixtures) of universal primers, e.g., a pair of universal amplification primers suitable for amplifying the repaired amplicon in a second amplification.The universal primer optionally contains a different tag (barcode) sequence, which does not hybridize to the adapter. The barcode sequence incorporated into the amplicon in the second universal amplification can be utilized, for example, for effective identification of the sample source.

[0092] 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, wherein the unique tag sequence does not exhibit significant complementarity and / or hybridization to any portion of the unique tag sequence and / or target nucleic acid sequence of interest. In some embodiments, the plurality of primer adapter pairs comprises 10 4 ~10 9 Thus, in certain embodiments, each target-specific adapter pair generated has a combination of 10 4 ~10 9 In some embodiments, the plurality of primer adapters comprises at least one different unique tag sequence and up to 10 5 In some embodiments, the plurality of primer adapters comprises at least one different unique tag sequence and up to 10 unique tag sequences. 5 In certain embodiments, each target-specific amplicon generated comprises at least two and up to ten target-specific adapters each comprising a different tag sequence, each having two different unique tag sequences. 9 In some embodiments, the plurality of primer adapters comprises each target-specific adapter comprising 4096 different tag sequences. In certain embodiments, each target-specific amplicon generated comprises up to 16,777,216 different adapter combinations comprising different tag sequences, each having two different unique tag sequences.

[0093] In some embodiments, each primer adaptor in the plurality of adaptors comprises a unique tag sequence (e.g., contained in a tag adaptor) that comprises 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 on both sides by a fixed sequence, or comprises a fixed sequence flanked on both sides by a random sequence. In some embodiments, the unique tag sequence comprises a fixed sequence that is between 2 and 2000 nucleotides or base pairs in length. In some embodiments, the unique tag sequence comprises a random sequence that is between 2 and 2000 nucleotides or base pairs in length.

[0094] In some embodiments, the unique tag sequence comprises a sequence having at least one random sequence interspersed with fixed sequences. In some embodiments, each tag sequence in the plurality of unique tags has the structure (N) n (X) x (M) m (Y) ywherein "N" represents a random tag sequence generated from A, G, C, T, U, or I, "n" is 2-10 and represents the nucleotide length of the "N" random tag sequence, "X" represents a fixed tag sequence, "x" is 2-10 and represents the nucleotide length of the "X" random tag sequence, "M" represents a random tag sequence generated from A, G, C, T, U, or I, the random tag sequence "M" is different or the same as the random tag sequence "N", "m" is 2-10 and represents the nucleotide length of the "M" random tag sequence, "Y" represents a fixed tag sequence, the fixed tag sequence of "Y" is the same or different from the fixed tag sequence of "X", and "y" is 2-10 and represents 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 the multiple tags. In some embodiments, the fixed tag sequence "Y" is different in the multiple tags. In some embodiments, the fixed tag sequence "(X)" in the multiple adaptors is different in the multiple tags. x " and "(Y) y " is a sequence alignment anchor.

[0095] In some embodiments, the random sequence in the unique tag sequence is represented by "N" and the fixed sequence is represented by "X." Thus, the unique tag sequence is represented by N 1 N 2 N 3 X 1 X 2 X 3 Or N 1 N 2 N 3 X 1 X 2 X 3 N 4 N 5 N 6 X 4 X 5 X 6Optionally, the unique tag sequence can have a random sequence where 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 nucleotide at each position in the random sequence is independently selected from any one of A, G, C, T, U, or I, or selected from a subset of these six different types of nucleotides. Optionally, the nucleotide at each position in the random sequence is independently selected from any one of A, G, C, or T. In some embodiments, the first fixed tag sequence "X 1 X 2 X 3 " are identical or different sequences within multiple tags. In some embodiments, the second fixed tag sequence "X 4 X 5 X 6 " is the same or different in the multiple tags. In some embodiments, the first fixed tag sequence "X 1 X 2 X 3 " and a second fixed tag sequence "X 4 X 5 X 6 " is a sequence alignment anchor.

[0096] 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, and the number of distinct random tags that can result is 4. 6 (or 4^6), which is approximately 4096, so the number of possible different combinations of two unique tags is 4 12 (or 4^12), which is approximately 16.78 million. NNN ACT NNN The underlined portion of TGA-3' is the sequence alignment anchor.

[0097] In some embodiments, the fixed sequences within the unique tag sequence are sequence alignment anchors that can be used to generate error-corrected sequencing data, in some embodiments, the fixed sequences within the unique tag sequence are sequence alignment anchors that can be used to generate a family of error-corrected sequencing reads.

[0098] The adaptors provided herein include at least one cleavable moiety. In some embodiments, the cleavable moiety is in the 3' target specific sequence. In some embodiments, the cleavable moiety is at or near the junction between the 5' first universal handle sequence and the 3' target specific sequence. In some embodiments, the cleavable moiety is at or near the junction between the 5' first universal handle sequence and the unique tag sequence, as well as at or near the junction between the unique tag sequence and the 3' target specific sequence. The cleavable moiety can be present in a modified nucleotide, nucleoside, or nucleobase. In some embodiments, the cleavable moiety can include a nucleobase that does not naturally occur in the target sequence of interest.

[0099] In some embodiments, at least one cleavable moiety in the plurality of adaptors is a uracil base, a uridine, or a deoxyuridine nucleotide. In some embodiments, the cleavable moiety is within the 3' target specific sequence and the junction between the 5' universal handle sequence and the unique tag sequence and / or the 3' target specific sequence, and at least one cleavable moiety in the plurality of adaptors is cleavable with uracil DNA glycosylase (UDG). In some embodiments, the cleavable moiety is cleaved to provide a susceptible abasic site, and at least one enzyme capable of reacting with the abasic site generates a gap comprising an extendable 3' end. In certain embodiments, the resulting gap comprises a 5'-deoxyribose phosphate group. In certain embodiments, the resulting gap comprises an extendable 3' end and a 5' linkable phosphate group.

[0100] In another embodiment, inosine can be incorporated into DNA-based nucleic acids as a cleavable group. In one 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 to create an abasic site.

[0101] When a cleavable moiety is present, the location of at least one cleavable moiety in the adapter does not significantly change the melting temperature (Tm) of any given double-stranded adapter in the plurality of double-stranded adapters. The melting temperatures (Tm) of any two given double-stranded adapters from the plurality of double-stranded adapters are substantially the same, and the melting temperatures (Tm) of any two given double-stranded adapters do not differ from each other by more than 10°C. However, within each of the plurality of adapters, the melting temperatures of sequence regions are different, for example, the comparable maximum-minimum melting temperature of the universal handle sequence is higher than the comparable maximum-minimum melting temperature of any unique tag sequence and / or target-specific sequence of any of the adapters. This localized difference in the comparable maximum-minimum melting temperature can be adjusted to optimize the digestion and repair of amplicons, and ultimately improved effectiveness of the methods provided herein.

[0102] Further provided are compositions comprising a nucleic acid library generated by the method of the present invention. Thus, compositions comprising a plurality of amplified target nucleic acid amplicons are provided, each of the plurality of amplicons comprising a 5' universal handle sequence, optionally a first unique tag sequence, an intermediate target nucleic acid sequence, optionally a second unique tag sequence, and a 3' universal handle sequence. The provided compositions comprise at least 2 and up to 100,000 target-specific amplicons. The provided compositions comprise highly multiplexed targeted libraries. In some embodiments, the provided compositions comprise a plurality of nucleic acid amplicons, each of the plurality of amplicons comprising 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. The provided compositions comprise at least 2 and up to 100,000 target-specific tagged amplicons. The provided compositions comprise highly multiplexed tagged targeted libraries.

[0103] 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, 10000, 11000, or 12000 or more target specific amplicons. In some embodiments, the target specific amplicon comprises one or more exons, genes, exomes, or regions of the genome associated with a clinical or pathological condition, e.g., an amplicon that includes one or more sites that contain one or more mutations (e.g., driver mutations) associated with cancer, e.g., lung cancer, colon cancer, breast cancer, etc., or an amplicon that includes a mutation associated with a genetic disease, e.g., cystic fibrosis, muscular dystrophy, etc. In some embodiments, the target specific amplicon comprises a library of adaptor-ligated amplicon target sequences that are about 100 to about 750 base pairs in length.

[0104] As described herein, each of the plurality of amplicons comprises a 5' universal handle sequence. In some embodiments, the universal handle sequence comprises 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 provided adapter does not exhibit significant complementarity and / or hybridization to any portion of the unique tag sequence of interest and / or the target nucleic acid sequence. In some embodiments, the first universal handle sequence comprises 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 comprises 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 and second universal handle sequences correspond to forward and reverse universal handle sequences, and in certain embodiments, the same first and second universal handle sequences are included for each of the plurality of target-specific amplicons. Such forward and reverse universal handle sequences are targeted with the universal primers to perform a second amplification of the preliminary library composition in the generation of the amplification products obtained according to the methods of the present invention. 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), and the first and second 5' universal handle sequences do not show significant hybridization to any portion of the target nucleic acid sequence of interest.

[0105] 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 methods and compositions provided to suit specific analytical platforms. The universal handle sequences of the adapters and amplicons provided herein are appropriately adapted 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 are used for sequencing on Ion Torrent sequencing platforms (Ion Xpress™ adapters, Thermo Fisher Scientific). Similarly, additional and other universal adaptor / primer sequences described and known in the art (e.g., Illumina universal adaptor / primer sequences can be found, for example, at support.illumina.com / content / dam / illumina-support / documents / documentation / chemistry_documentation / experiment-design / illumina-adapter-sequences_1000000002694-01.pdf; PacBio universal adaptor / primer sequences can be found, for example, at 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 of appropriate nucleotide sequences for use with the libraries of the present invention are readily prepared using standard automated nucleic acid synthesis equipment and reagents in routine use in the art. One single type or two different distinct types (or even mixtures) of universal primers may be used in generating the libraries of the invention, for example a pair of universal amplification primers suitable for amplifying a preliminary library.The universal primer optionally comprises a tag (barcode) sequence, and the tag (barcode) sequence does not hybridize to the adapter sequence or the target nucleic acid sequence. The barcode sequence incorporated into the amplicon in the second universal amplification can be utilized, for example, for effective identification of the sample source, thereby generating a barcoded library. The compositions provided thus comprise highly multiplexed barcoded targeting libraries. The compositions provided also comprise highly multiplexed barcoded tagged targeting libraries.

[0106] In some embodiments, the amplicon library comprises a unique tag sequence located between the 5' first universal handle sequence and the 3' target specific sequence, wherein the unique tag sequence does not exhibit significant complementarity and / or hybridization to any portion of the unique tag sequence and / or the target nucleic acid sequence. In some embodiments, the plurality of amplicons comprises 10 4 ~10 9 Thus, in certain embodiments, each of the multiple amplicons in the library has a combination of 10 different tag sequences. 4 ~10 9 In some embodiments, each of the multiple amplicons in the library comprises at least one different unique tag sequence and up to 10 5 In certain embodiments, each target-specific amplicon in the library comprises at least 2 and up to 10 distinct tag sequences, each having two distinct unique tag sequences. 9 In some embodiments, each of the plurality of amplicons in the library comprises a tag sequence that 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 having two different unique tag sequences.

[0107] In some embodiments, each amplicon in a plurality of amplicons of a library comprises a unique tag sequence (e.g., comprised in a tag adapter sequence) that comprises 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 on both sides by a fixed sequence, or comprises a fixed sequence flanked on both sides by a random sequence. In some embodiments, the unique tag sequence comprises a fixed sequence that is between 2 and 2000 nucleotides or base pairs in length. In some embodiments, the unique tag sequence comprises a random sequence that is between 2 and 2000 nucleotides or base pairs in length.

[0108] In some embodiments, the unique tag sequence comprises a sequence having at least one random sequence interspersed with fixed sequences. In some embodiments, each tag sequence in the plurality of unique tags has the structure (N) n (X) x (M) m (Y) ywherein "N" represents a random tag sequence generated from A, G, C, T, U, or I, "n" is 2-10 and represents the nucleotide length of the "N" random tag sequence, "X" represents a fixed tag sequence, "x" is 2-10 and represents the nucleotide length of the "X" random tag sequence, "M" represents a random tag sequence generated from A, G, C, T, U, or I, the random tag sequence "M" is different or the same as the random tag sequence "N", "m" is 2-10 and represents the nucleotide length of the "M" random tag sequence, "Y" represents a fixed tag sequence, the fixed tag sequence of "Y" is the same or different from the fixed tag sequence of "X", and "y" is 2-10 and represents 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 the plurality of tags. In some embodiments, the fixed tag sequence "Y" is different in the plurality of tags. In some embodiments, the fixed tag sequence "(X)" in the plurality of amplicons is different in the plurality of tags. x " and "(Y) y " is a sequence alignment anchor.

[0109] In some embodiments, the random sequence in the unique tag sequence is represented by "N" and the fixed sequence is represented by "X." Thus, the unique tag sequence is represented by N 1 N 2 N 3 X 1 X 2 X 3 Or N 1 N 2 N 3 X 1 X 2 X 3 N 4 N 5 N 6 X 4 X 5 X 6Optionally, the unique tag sequence can have a random sequence where 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 nucleotide at each position in the random sequence is independently selected from any one of A, G, C, T, U, or I, or selected from a subset of these six different types of nucleotides. Optionally, the nucleotide at each position in the random sequence is independently selected from any one of A, G, C, or T. In some embodiments, the first fixed tag sequence "X 1 X 2 X 3 " are identical or different sequences within multiple tags. In some embodiments, the second fixed tag sequence "X 4 X 5 X 6 " is the same or different in the plurality of tags. In some embodiments, the first fixed tag sequence "X 1 X 2 X 3 " and a second fixed tag sequence "X 4 X 5 X 6 " is a sequence alignment anchor.

[0110] 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, and the number of distinct random tags that can result is 4. 6 (or 4^6), which is approximately 4096, so the number of possible different combinations of two unique tags is 4 12 (or 4^12), which is approximately 16.78 million. In some embodiments, the underlined portion of 5'-NNNACTNNNTGA-3' is a sequence alignment anchor.

[0111] In some embodiments, the fixed sequences within the unique tag sequence are sequence alignment anchors that can be used to generate error-corrected sequencing data, in some embodiments, the fixed sequences within the unique tag sequence are sequence alignment anchors that can be used to generate a family of error-corrected sequencing reads.

[0112] Kits, Systems Further provided herein is a kit for use in preparing a library of target nucleic acids using the method of the first or second aspect of the invention. An embodiment of the kit comprises a supply of at least one pair of target-specific adaptors as defined herein capable of generating a first amplification product, and optionally a supply of at least one universal pair of amplification primers capable of annealing to the universal handles of the adaptors and initiating the synthesis of an amplification product, the amplification product comprising the target sequence of interest linked to the universal sequence. The adaptors and / or primers may be provided in a ready-to-use kit, or more preferably as a concentrate that requires dilution before use, or in a lyophilized or dried form that requires further reconstitution before use. In certain embodiments, the kit further comprises a supply of a suitable diluent for dilution or reconstitution of the components. Optionally, the kit further comprises a supply of reagents, buffers, enzymes, dNTPs, etc. for use in carrying out amplification, digestion, repair, and / or purification in the generation of the libraries provided herein. Non-limiting examples of such reagents are as described in the Materials and Methods section of the accompanying examples. Further components optionally provided in the kit include components suitable for purification of libraries prepared using the provided methods. In some embodiments, a kit is provided for generating a target-specific library that includes a 5' universal handle sequence, a 3' target-specific sequence, and a plurality of target-specific adaptors having a cleavable group, a DNA polymerase, adaptors, dATP, dCTP, dGTP, dTTP, and a digestion reagent. In some embodiments, the kit further includes one or more antibodies, a repair reagent, a universal primer optionally including a nucleic acid barcode, a purification solution, or a column.

[0113] The specific features of the adapters for inclusion in the kit are as described elsewhere herein in relation to other aspects of the invention. The structure and properties of universal amplification primers are well known to those of skill in the art and can be implemented for use with the methods and compositions provided to suit a particular analytical platform (e.g., as described herein, universal P1 and A primers have been described in the art and utilized for sequencing on the Ion Torrent sequencing platform). Similarly, additional and other universal adapter / primer sequences described and known in the art (e.g., Illumina universal adapter / primer sequences, PacBio universal adapter / primer sequences, etc.) can be used with the methods and compositions provided herein. Suitable primers of appropriate nucleotide sequences for use with the adapters included in the kit are readily prepared using standard automated nucleic acid synthesis equipment and reagents in routine use in the art. The kit can include a supply of one single type of universal primer or separate types (or even mixtures) of two different universal primers, e.g., a pair of amplification primers suitable for amplifying the adapter-modified template in the first amplification. The kit may include at least two different amplification primers, optionally with different tag (barcode) sequences, for the first amplification of the sample of interest according to the method of the present invention, in addition to at least one pair of adapters, where the tag (barcode) sequences do not hybridize to the adapter. The kit may be used to amplify at least two different samples, each sample being amplified separately according to the method of the present invention, and the second amplification using a single universal primer with a barcode, and then pooling the prepared sample libraries after library preparation. In some embodiments, the kit includes different universal primer pairs for use in the second amplification step described herein. In this context, a "universal" primer pair has substantially identical nucleotide sequences, but may differ with respect to some other feature or modification.

[0114] Further provided are systems, e.g., systems used to practice the methods provided herein and / or including the compositions provided herein. In some embodiments, the systems facilitate methods performed in an automated mode. In certain embodiments, the systems facilitate high throughput modes. In certain embodiments, the systems include, for example, fluid handling elements, fluid containing elements, heat sources and / or heat sinks to achieve and maintain desired reaction temperatures, and / or robotic elements (e.g., multi-well plate handling elements) that can move components of the system from location to location as needed.

[0115] sample As defined herein, "sample" and its derivatives are used in its broadest sense and include any specimen, culture, and / or analog suspected of containing a target nucleic acid. In some embodiments, a 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 foregoing. In some embodiments, samples useful in connection with the methods of the invention include any biological, clinical, surgical, agricultural, air, or water-based specimen containing one or more target nucleic acids of interest. In some embodiments, a sample includes nucleic acid molecules obtained from an animal, such as a human or mammalian source. In another embodiment, a sample includes nucleic acid molecules obtained from a non-mammalian source, such as a plant, bacteria, virus, or fungus. In some embodiments, the source of the nucleic acid molecule may be an archived or extinct sample or species. In some embodiments, a sample includes an isolated nucleic acid sample prepared from a source, such as, for example, genomic DNA, RNA TNA, or a prepared sample, such as, for example, a fresh frozen or formalin-fixed paraffin-embedded (FFPE) nucleic acid specimen. It is also contemplated that the sample may be from a single individual, a collection of nucleic acid samples from genetically related members, multiple nucleic acid samples from genetically unrelated members, multiple nucleic acid samples from a single individual (matched), such as a tumor sample and a normal tissue sample, or genetic material from a single source containing two different forms of genetic material, such as maternal and fetal DNA obtained from a maternal subject, or the presence of 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 methods provided include amplification of multiple target specific sequences from a single nucleic acid sample. In some embodiments, the methods provided include target specific amplification of two or more target sequences from two or more nucleic acid samples or species. In certain embodiments, the methods provided include highly multiplexed amplification of target nucleic acid sequences from a single sample. In certain embodiments, the methods provided include highly multiplexed amplification of target nucleic acid sequences from multiple samples, each from the same source organism.

[0116] In some embodiments, the sample comprises a mixture of target and non-target nucleic acids. In certain embodiments, the sample comprises a plurality of initial polynucleotides, which may comprise a mixture of one or more target nucleic acids and one or more non-target nucleic acids. In some embodiments, the sample comprising a plurality of polynucleotides comprises a portion or aliquot of the original sample, and in some embodiments, the sample comprises a plurality of polynucleotides that is the entire original sample. In some embodiments, the sample comprises a plurality of initial polynucleotides isolated from the same source or the same subject at different time points.

[0117] In some embodiments, the nucleic acid sample comprises cell-free nucleic acid from bodily fluids, nucleic acid from tissue, nucleic acid from biopsy tissue, nucleic acid from needle biopsy, nucleic acid from a single cell, or nucleic acid from two or more cells. In certain embodiments, the single reaction mixture contains 1-100 ng of a plurality of initial polynucleotides. In some embodiments, the plurality of initial polynucleotides comprises a formalin-fixed paraffin-embedded (FFPE) sample, genomic DNA, RNA, TNA, cell-free DNA or RNA or TNA, circulating tumor DNA or RNA or TNA, fresh frozen sample, or a mixture of two or more of the above, and in some embodiments, the plurality of initial polynucleotides comprises a nucleic acid reference standard. In some embodiments, the sample comprises nucleic acid molecules obtained from biopsies, tumors, scrapings, swabs, blood, mucus, urine, plasma, semen, hair, laser capture microdissections, surgical resections, 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 (e.g., Hg19).

[0118] Targeted nucleic acid sequence analysis The methods and compositions of the present invention provided are particularly suitable for amplifying and optionally tagging and preparing target sequences for subsequent analysis. Thus, in some embodiments, the methods provided herein include analyzing the resulting library preparation. For example, the methods include analyzing the polynucleotide sequence of the target nucleic acid, and, if applicable, 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 the multiple target nucleic acids. The methods provided optionally further 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 for analyzing the sequences of a nucleic acid library.

[0119] In certain embodiments, the use of the prepared tagged library composition is provided for further analyzing the sequence of the target nucleic acid library. In some embodiments, the determination of the sequence comprises determining the abundance of at least one of the target sequences in the sample. In some embodiments, the determination of low frequency alleles in the sample is included in the determination of the sequence of the nucleic acid library. In certain embodiments, the determination of the presence of mutant target nucleic acids in the plurality of polynucleotides is included in the determination of the sequence of the nucleic acid library. In some embodiments, the determination of the presence of mutant target nucleic acids comprises detecting the abundance level of at least one mutant target nucleic acid in the plurality of polynucleotides. For example, such determination comprises detecting that at least one mutant target nucleic acid is present at 0.05% to 1% of the original plurality of polynucleotides in the sample, detecting that at least one mutant target nucleic acid is present at about 1% to about 5% of the polynucleotides in the sample, and / or detecting at least 85% to 100% of the target nucleic acids in the sample. In some embodiments, the determination of the presence of mutant target nucleic acids comprises detecting and identifying copy number variations and / or gene fusion sequences in the sample.

[0120] In some embodiments, nucleic acid sequencing of the amplified target sequences generated by the teachings of the present disclosure comprises de novo sequencing or targeted resequencing. In some embodiments, nucleic acid sequencing further comprises comparing the results of nucleic acid sequencing of the amplified target sequences to a reference nucleic acid sequence. In some embodiments, nucleic acid sequencing of the target library sequences further comprises determining the presence or absence of a mutation in the nucleic acid sequence. In some embodiments, nucleic acid sequencing comprises identifying genetic markers associated with disease (e.g., cancer and / or genetic disease).

[0121] In some embodiments, the prepared library of target sequences of the disclosed method is used in various downstream analyses or assays, with or without further purification or manipulation. In some embodiments, the analysis includes sequencing by conventional sequencing reaction, high-throughput next-generation sequencing, targeted multiplex 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 certain embodiments, the sequencing is performed in a bidirectional manner, thereby generating sequence reads on both the forward and reverse strands for any given amplicon.

[0122] In some embodiments, the library prepared according to the methods provided herein is then further manipulated for additional analysis. For example, the prepared library sequences are used in downstream enrichment techniques known in the art, such as bridge amplification or emPCR, to generate a template library that is then used in next-generation sequencing. In some embodiments, the target nucleic acid library is used in enrichment and sequencing applications. For example, sequencing of the provided target nucleic acid library is accomplished using any suitable DNA sequencing platform. In some embodiments, the library sequences of the disclosed methods or the template library prepared subsequently are used for nucleic acid sequencing, including single nucleotide polymorphism (SNP) analysis, genotyping or epigenetic analysis, copy number variation analysis, gene expression analysis, analysis of genetic variations, including but not limited to detection, prognosis and / or diagnosis, detection, and analysis of rare or low frequency allelic variations, including but not limited to de novo sequencing, targeted resequencing, and synthetic assembly analysis. In one embodiment, the prepared library sequences are used to detect variations at an allele frequency of less than 5%. In some embodiments, the methods disclosed herein are used to detect mutations in a population of nucleic acids with an allele frequency of less than 4%, 3%, 2%, or about 1%. In another embodiment, a library prepared as described herein is sequenced to detect and / or identify germline or somatic mutations from a population of nucleic acid molecules. In certain embodiments, sequencing adaptors are ligated to the ends of the prepared library to generate a plurality of libraries suitable for nucleic acid sequencing.

[0123] In some embodiments, methods for preparing target-specific amplicon libraries are provided for use in various downstream processes or assays, such as nucleic acid sequencing or clonal amplification. In some embodiments, the library is amplified using bridge amplification or emPCR to generate multiple clonal templates suitable for nucleic acid sequencing. For example, optionally, the target-specific amplification is followed by a secondary and / or tertiary amplification process, including, but not limited to, a library amplification step and / or a clonal amplification step. "Clonal amplification" refers to the generation of multiple copies of individual molecules. Various methods known in the art are used for clonal amplification. For example, emulsion PCR is one method, which involves isolating individual DNA molecules with primer-coated beads in water bubbles in an oil phase. Polymerase chain reaction (PCR) then coats each bead with a clonal copy of the isolated library molecule, and these beads are subsequently immobilized for later sequencing. Emulsion PCR is used in methods published by Margulies et al. and Shendure and Porreca et al. (also known as "polony sequencing" and commercialized by Agencourt and recently acquired by Applied Biosystems). Margulies et al. (2005) Nature 437:376-380, Shendure et al., Science 309(5741):1728-1732. Another method for clonal amplification is "bridge PCR", where fragments are amplified with primers attached to a solid surface. These methods, like other methods of clonal amplification, generate multiple physically isolated loci, each containing multiple copies derived from a single molecule polynucleotide fragment. Thus, in some embodiments, one or more target-specific amplicons are amplified using bridge amplification or emPCR to generate multiple clonal templates suitable for, for example, nucleic acid sequencing.

[0124] In some embodiments, at least one of the library sequences to be clonally amplified is attached to a support or particle. The support can be composed of any suitable material and can have any suitable shape, including, for example, flat, spheroidal, or microparticle. In some embodiments, the support is a scaffold polymer particle as described in U.S. Patent Application Publication No. 2010 / 0304982, which is incorporated herein by reference in its entirety. In certain embodiments, the method includes depositing at least a portion of the enriched population of library sequences onto a support (e.g., a sequencing support), the support comprising a series of sequencing reaction sites. In some embodiments, the enriched population of library sequences is attached to a sequencing reaction site on the support, the support comprising a series of 10 2 ~10 10 The sequence comprises sequencing reaction sites.

[0125] Sequencing refers to the determination of information regarding the sequence of a nucleic acid, and may include the identification or determination of partial and complete sequence information of a nucleic acid. The sequence information may be determined with different degrees of statistical certainty or reliability. In some embodiments, the sequence analysis includes high-throughput, low-depth detection, such as by qPCR, rtPCR, and / or array hybridization detection methodologies known in the art. In some embodiments, the sequence analysis includes detailed sequence assessment determination, such as by Sanger sequencing or other high-throughput next-generation sequencing methods. Next-generation sequencing refers to sequencing using methods that determine large numbers (typically thousands to billions) of nucleic acid sequences in an essentially massively parallel manner, e.g., large numbers of sequences are read out, e.g., in parallel, or alternatively, using an ultra-high-throughput continuous process that may itself be parallelized. Thus, in certain embodiments, the methods of the invention include sequence analysis, including massively parallel sequencing.Such methods include pyrosequencing (commercially available, for example, by 454 Life Sciences, Inc., Branford, Conn.); sequencing by ligation (for example, SOLiD™ technology, Life Technologies, Inc., Carlsbad, Calif.); sequencing by synthesis using modified nucleotides (for example, TruSeq™, HiSeq™, and MiSeq™, and / or NovaSeq™ technologies by Illumina, Inc., San Diego, Calif.; HelisCOpe™ by Helicos Biosciences Corporation, Cambridge, Mass.; and PacBio Sequel® or RS systems by Pacific Biosciences of California, Inc., Menlo Park, Calif.), sequencing by ion detection technology (for example, Ion Torrent™ technology, Life Technologies, Carlsbad, Calif.); sequencing of DNA nanoballs (Complete Genomics, Inc., Mountain View, CA). View, Calif.); nanopore-based sequencing technologies (such as those developed by Oxford Nanopore Technologies, LTD, Oxford, UK), as well as similar highly parallelized sequencing methods.

[0126] For example, in certain embodiments, libraries generated by the teachings of the present disclosure are in sufficient yield to be used in a variety of downstream applications, including the Ion Torrent™ PGM system (e.g., PCR-mediated addition of nucleic acid fragment libraries onto Ion Sphere™ particles) (Life Technologies, part number 4467389) or the Ion Xpress™ template kit using the Ion Torrent Proton™ PGM system. For example, instructions for preparing a template library from an amplicon library can be found in the Ion Xpress template kit user guide (Life Technologies, part number 4465884), which is incorporated herein by reference in its entirety. Instructions for subsequent loading of the template library onto an Ion Torrent™ chip for nucleic acid sequencing are described in the Ion Sequencing User Guide (part number 4467391), which is incorporated herein by reference in its entirety.

[0127] The starting point of the sequencing reaction may be provided by annealing a sequencing primer to the product of the solid-phase amplification reaction. In this regard, one or both of the adapters added during the formation of the template library may contain a nucleotide sequence that allows annealing of the sequencing primer to the amplification product derived from the whole genome or solid-phase amplification of the template library. Depending on the implementation of the embodiment of the present invention, the tag sequence and / or the target nucleic acid sequence may 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, the "tag read" and the "target sequence read" may be performed in either order, with a suitable denaturation step to remove the annealed primers after the first sequencing read is completed.

[0128] 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, hi certain embodiments, the sequencer is coupled to a server that applies parameters or software to determine the presence of low frequency mutant alleles present in the sample.

[0129] Example Example 1 Compositions and Methods Reverse transcription (RT) reactions (21 uL reactions) can be performed on samples where RNA and DNA are to be analyzed, e.g., FFPE RNA and cfTNA. 1. Thaw 5x URT buffer at room temperature for at least 5 minutes. (Note: Check for white precipitate in the tube. Vortex to mix if necessary.)

[0130] [Table 1] 2. In a MicroAmp EnduraPlate 96-well plate, set up the RT reaction by adding the following components: (5-15ng RNA or DNA / / 5-40ng cfTNA)

[0131] [Table 2] 3. Mix the entire contents by vortexing or pipetting. Spin down briefly. 4. Add 20 μl of Parol 40C oil to the top of each reaction mix. 5. Load the plate into a thermocycler (e.g., SimpliAmp thermocycler) and run the following program:

[0132] [Table 3]

[0133] Low cycle tagmentation PCR (38uL reaction volume + 20uL oil): Assemble tagmentation PCR reactions in 96-well PCR plate wells.

[0134] FFPE DNA samples only 1. Assemble the reaction by adding the following components to a MicroAmp EnduraPlate 96-well plate: a. Prepare UDG mix: 1ul + 5ul of 5x URT buffer b. Add 6 ul of diluted UDG to 15 μl of FFPE DNA sample. c. Mix by vortexing. Spin briefly to collect reactants at the bottom of the wells. d. Add 20 μL of Parol 40C oil on top of each sample. e. Carry out the reaction as follows:

[0135] [Table 4] 2. Preparation of Amplification Master Mix:

[0136] [Table 5] 3. Add 17 μL of PCR master mix to 21 μL of UDG-treated FFPE DNA sample.

[0137] Set the pipette to a volume of 20 µL. Mix the reactions under the oil by pipetting up and down 20 times to thoroughly mix the reactions without disturbing the oil phase. Spin the plate down briefly.

[0138] FFPE RNA and cfTNA samples only 1. Add components from the RT step above directly to the RT reaction.

[0139] [Table 6] 2. Set the pipette to a volume of 20 μL. Mix the reactions under the oil by pipetting up and down 20 times to thoroughly mix the reactions without disturbing the oil phase. Spin the plate down briefly. 3. Run 3 cycles of tagmentation PCR on a SimpliAmp using the following cycling conditions:

[0140] For FFPE DNA and RNA libraries:

[0141] [Table 7]

[0142] For cfTNA libraries,

[0143] [Table 8]

[0144] Digest-fill-ligation (45.6 μL reaction volume + 20 μL oil): 1. Add 7.6 μL of SUPA to each of the PCR reaction wells described above. Add SUPA directly to the sample below the oil layer. 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. 3. Load the plate into the thermocycler and run the following program:

[0145] [Table 9]

[0146] Library amplification (approximately 51 μL reaction volume + 20 μL oil) 1. Carefully transfer 30 μL of the digest-fill-ligate reaction described above into the AmpliSeq HD Dual Barcode. Mix well by pipetting up and down 20 times. Transfer all reactions back into the original wells under the oil layer. 2. Set the pipette to 30 μL. Mix the entire reaction under oil by pipetting up and down 20 times. Spin the plate down briefly. 3. Load the plate into the thermocycler and run the following program:

[0147] [Table 10]

[0148] 2-Round AmpureXP Library Purification The resulting repaired sample is purified using two rounds of 36.8 ul of Ampure® beads (Beckman Coulter, Inc.) according to the manufacturer's instructions. Briefly: Transfer 46 µL of the library reaction beneath the oil layer to a new, clean well of the PCR plate. Add 36.8 μl of Agencourt™ AMPure™ XP reagent to each sample, mix by pipetting, and incubate at room temperature for 5 minutes. The plate is placed on a magnet until the solution in the wells becomes clear. Carefully remove the supernatant, then remove the remaining supernatant. Add 150 uL of 80% ethanol in 10 mM Tris-HCl pH 8. Do not disturb the bead pellet. Toggle the plate on the magnet three times with 5 s intervals, remove the supernatant, and repeat the washing step one more time. Use a pipette to remove any remaining buffer in the wells. The wells are allowed to dry for 5 minutes at room temperature. Add 30 uL of low TE buffer to the wells and pipette up and down to resuspend the beads. Incubate the solution at room temperature for 5 minutes and then place the plate on a magnet to allow the solution to clear. Transfer 30 uL of the eluent to a clean well on the plate. Add 30 μL (1× volume) of Ampure XP beads to the above wells and mix by pipetting up and down in the wells. After the second purification, elute using 40 uL of low TE buffer and repeat the steps above. Transfer 40 uL of library to a new clean well.

[0149] Library normalization with individual equalizers First, warm all Ion Library Equalizer™ kit reagents to room temperature. Vortex and centrifuge all reagents. Wash Equalizer™ beads (skip to Adding and Washing Equalizer™ Beads if done previously). 1. For every 4 reactions, add 12 μL of beads to a clean 1.5 mL tube and 24 μL / reaction of Equalizer™ Wash Buffer. 2. Place the tube on the magnetic rack for 3 minutes or until the solution becomes completely clear. 3. Carefully remove and discard the supernatant without disturbing the pellet. 4. Remove from magnet and add 24 μL Equalizer™ Wash Buffer per reaction and resuspend. Amplify the library. 5. Remove the plate containing the purified library from the magnet and add 10 μL of 5×DV-Amp Mix and 2 μL of Equalizer™ primer (pink cap from Equalizer kit). Total volume = 52 μL 6. Mix. 7. Gently add 20 μL of Parol 40C oil on top of the sample. 8. Run the following program in the thermocycler: 98C for 2 minutes 9 cycles of amplification for FFPE DNA / RNA, 6 cycles of amplification for cfTNA: 98C for 15 seconds 64C for 1 minute Next Infinitely hold with 4C 9. (Optional) After thermal cycling, centrifuge the plate to collect droplets. Add Equalizer™ Capture to the amplified library 10. Add 10 μL of Equalizer Capture to each library amplification reaction below the oil layer. 11. Mix up and down 10 times. 12. Incubate at room temperature for 5 minutes. Add Equalizer™ beads and wash. 13. Transfer 60 μL of the amplified library sample beneath the oil layer to the wells containing the washed beads. 14. Mix thoroughly. 15. Incubate at room temperature for 5 minutes. 16. Place the plate in the magnet and then incubate for 2 minutes or until the solution is clear. 17. Remove the supernatant. 18. Add 150 μL of Equalizer™ Wash Buffer to each reaction. 19. With the plate still on the magnet, remove and discard the supernatant. 20. Repeat bead wash to elute equalized libraries. Elute the equalized libraries. 21. Remove plate from magnet and add 100 μL of Equalizer™ Elution Buffer to each pellet. 22. Mix by pipetting 5 times in a volume of 50ul. 23. Elute the library by incubating in a thermocycler at 32°C for 5 minutes. 24. Immediately remove, place plate in magnet and as soon as solution becomes clear transfer to new wells. 25. Perform qPCR and adjust pool to 100 pM for templating and sequencing.

[0150] Example 2 Compositions and Methods The first step of the provided methods includes several rounds of amplification, for example, 3-6 cycles of amplification, in a particular example, 3 cycles of amplification using a forward and reverse adapter 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.

[0151] In a specific embodiment where unique tag sequences are utilized, each gene-specific target adapter pair includes multiple different unique tag sequences in each adapter. For example, each gene-specific target adapter includes up to 4096 TAGs. Thus, each target-specific adapter pair includes at least 4 and up to 16,777,216 possible combinations.

[0152] Each of the provided adapters contains a cleavable uracil instead of a thymine at a specific position in the forward and reverse adapter sequences. The position of the uracil (U) is consistent for all forward and reverse adapters with a unique tag sequence, and when present, the uracil (U) is adjacent to the 5' and 3' ends of the unique tag sequence, and although the position of each gene-specific target sequence necessarily varies, the U is present in each of the gene-specific target sequence regions. The uracil adjacent to each unique tag sequence (UT) and in the gene-specific sequence region is designed, along with the sequence and the calculated Tm of such sequence, to promote fragment dissociation at a temperature lower than the melting temperature of the universal handle sequence that is designed to remain hybridized at the selected temperature. Although variation of the U in the adjacent sequence of the UT region is possible, the design maintains the melting temperature lower than that of the universal handle sequence for each of the forward and reverse adapters. Exemplary adapter sequence structures include the following:

[0153] Forward adapter:

[0154] [Table 11] where each N is a base selected from A, C, G, or T, and the constant section 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 altered (e.g., alternative constant sequences, >3N per section) as long as the Tm of the UT region remains below that of the universal handle region. Importantly, no cleavable uracils are present in each of the forward (e.g., TCTGTACGGTGACAAGGCG (SEQ ID NO: 1566) and reverse (e.g., TGACAAGGCGTAGTCACGG (SEQ ID NO: 1567)) universal handle sequences. In this example, the universal sequences are designed to accommodate subsequent amplification and addition of sequences on the ION Torrent platform, however, one of skill in the art will appreciate that such universal sequences can be adapted to use other universal sequences that may be more applicable to alternative sequencing platforms (e.g., ILLUMINA sequencing system, QIAGEN sequencing system, PACBIO sequencing system, BGI sequencing system, etc.).

[0155] Methods of using the provided compositions include preparation of libraries with AmpliSeq HD technology and minor variations thereof, as well as the use of reagents and kits available from Thermo Fisher Scientific. SuperFiU DNA contains modifications in the uracil binding pocket (e.g., AA 36) and the family B polymerase catalytic domain (e.g., AA 762). SuperFiU is described in U.S. Patent Application Publication No. 2021 / 0147817, filed June 26, 2017 (incorporated herein by reference). Polymerase enzymes may be limited in their ability to utilize uracil and / or any alternative cleavable residues (e.g., inosine, etc.) contained in the adapter sequence. In certain embodiments, it may be advantageous to use a mixture of polymerases to reduce enzyme-specific PCR errors.

[0156] The second step of the method involves partial digestion of the resulting amplicons and any unused uracil-containing adaptors. For example, if uracil is incorporated as a cleavable site, digestion and repair involves enzymatic cleavage of uridine monophosphate from the resulting primers, primer-dimers, and amplicons, melting the DNA fragments, and then repairing the gapped amplicons by polymerase fill-in and ligation. This step reduces and potentially eliminates primer-dimer products that occur in multiplex PCR. In some instances, digestion and repair are performed in a single step. In certain instances, it may be desirable to separate the digestion and repair steps in time. For example, a thermolabile polymerase inhibitor may be utilized with the method such that digestion occurs at a lower temperature (25-40°C) and repair is activated by increasing the temperature not high enough to melt the universal handle sequence, but high enough to disrupt the polymerase-inhibitor interaction (e.g., polymerase-Ab).

[0157] The uracil can be removed using a uracil-DNA glycosylase (UDG) enzyme, leaving an abasic site, which can be acted upon by several enzymes or combinations of enzymes, including, but not limited to: APE1-apurinic / apyrimidinic endonuclease, FPG-formamidopyrimidine [fapy]-DNA glycosylase, Nth endonuclease III, Endo VIII-endonuclease VIII, PNK-polynucleotide kinase, Taq-Thermus aquaticus DNA polymerase, DNA pol I-DNA polymerase I, Polβ-human DNA polymerase beta. In a particular implementation, the method uses a human apurinic / apyrimidinic endonuclease, APE1. The APE1 activity leaves a 3'-OH and a 5'-deoxyribose-phosphate (5'-dRP). Removal of the 5'-dRP can be accomplished by a number of enzymes, including recJ, polymerase beta, Taq, DNA pol I, or any DNA polymerase with 5'-3' exonuclease activity. Removal of the 5'-dRP by any of these enzymes creates a ligatable 5'-phosphate end. In another implementation, UDG activity removes the uracil, leaving an abasic site, which is removed by FPG, leaving a 3' and 5'-phosphate. The 3'-phosphate is then removed by T4 PNK, leaving a 3'-OH capable of polymerase extension. The 5'-deoxyribose phosphate can then 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 particular implementation, Taq DNA polymerase is utilized.

[0158] The repair fill-in process can be accomplished by almost any polymerase, possibly by the amplifying polymerase used for amplification in step 1, or 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, Pol beta, T4 DNA polymerase, and T7 DNA polymerase. Ligation repair of the amplicon can be performed by a number of ligases, including but not limited to: T4 DNA ligase, T7 DNA ligase, Taq DNA ligase. In a particular implementation of the method, Taq DNA polymerase is utilized and ligation repair is accomplished by T7 DNA ligase.

[0159] The final step of library preparation involves amplifying the repaired amplicons by standard PCR protocols using universal primers that contain sequences complementary to the 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 Adapter 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.

[0160] Example 3 Assay Content and Method Together with primers directed to the target sequences specific for the targets in Table 1, the adapters each contain 4096 unique tag sequences for each gene-specific target sequence, resulting in an estimate of 16,777,216 different unique tag combinations for each gene-specific target sequence pair.

[0161] The preparation of the library was performed according to the method described above. The prepared library is prepared for templating and sequencing and analyzed. Sequencing can be performed by various known methods, including but not limited to sequencing by synthesis, sequencing by ligation, and / or sequencing by hybridization. Sequencing was performed in the examples herein using the Ion Torrent platform (Thermo Fisher Scientific, Inc.), but the library can be prepared and adapted for analysis, e.g., sequencing, using any other platform, e.g., Illumina, Qiagen, PacBio, etc. The results can be analyzed using several metrics to evaluate performance, such as, for example, the following: Family count (ng of captured input DNA) The median family count is a measure of the number of families that map to individual targets. In this case, each unique molecular tag is a family. Uniformity is a measure of the percentage of target bases that are covered by at least 0.2x the average read depth. This metric is used to ensure that a technology does not selectively underamplify a particular target. Positive / Negative: If control samples with known mutations are utilized and analyzed (e.g., Acrometrix Oncology Hotspot Control DNA, Thermo Fisher Scientific, Inc.), the number of true positives can be tracked. True Positives: The number of true positives informs how many mutations were present and correctly identified. False Positives (FP): (hotspot and total target) The number of false positives informs the number of mutations known not to be present in the sample but determined to be present. False Negatives (FN) (if acrometrix spike-in is used) The number of false negatives tells us the number of mutations that were present but not identified. On / Off Target is the percentage of mapped reads that are aligned / not aligned across the target region. This metric is used to ensure that the technology primarily amplifies the targets for which the panel was designed. Poor quality is tracked to ensure that the data is worth analyzing. This metric is a general system metric and is not directly related to this technology.

[0162] [Table 12] (In Table 1, ALK, FGFR1, FGFR2, FGFR3, NTRK1, NTRK2, NTRK3, and RET are shown in bold.)

[0163] 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 show the top genes / variants and indications associated with the provided assays that are supported by clinical evidence.

[0164] [Table 13]

[0165] [Table 14]

[0166] The combinations of up to 29 genes and variants targeted by the provided assays are described in the drug label and / or guidelines (NCCN and ESMO).

[0167] [Table 15]

[0168] Results of Example 4 Primers were designed using the composition design approach provided herein, and the library amplification step utilized two primer pairs to enable bidirectional sequencing as described herein (with two universal sequences placed at each end of the amplicon, e.g., A-universal handle and P1-universal handle at each end), targeted to tumor genes using those of the panel target genes as described above in Table 1. The prepared libraries were sequenced using Ion Gene Studio template / and sequencing kits and instrumentation (Thermo Fisher Scientific, Inc.), and / or the fully integrated library preparation, template, and sequencing system Genexus (Thermo Fisher Scientific, Inc.). Performance of the instant panel indicates that the technology can adequately detect mutations, copy number variations, and fusions of interest.

[0169] [Table 16]

[0170] [Table 17]

[0171] [Table 18]

[0172] [Table 19]

[0173] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0174] [Table 20-1]

[0175] [Table 20-2]

[0176] [Table 20-3]

[0177] [Table 20-4]

[0178] [Table 20-5]

[0179] [Table 20-6]

[0180] [Table 20-7]

[0181] [Table 20-8]

[0182]

Table 20-9

[0183]

Table 20-10

[0184]

Table 20-11

[0185]

Table 20-12

[0186]

Table 20-13

[0187]

Table 20-14

[0188]

Table 20-15

[0189]

Table 20-16

[0190]

Table 20-17

[0191]

Table 20-18

[0192]

Table 20-19

[0193]

Table 20-20

[0194]

Table 20-21

[0195]

Table 20-22

[0196]

Table 20-23

[0197]

Table 20-24

[0198]

Table 20-25

[0199]

Table 20-26

[0200]

Table 20-27

[0201]

Table 20-28

[0202]

Table 20-29

[0203]

Table 20-30

[0204]

Table 20-31

[0205]

Table 20-32

[0206]

Table 20-33

[0207]

Table 20-34

[0208]

Table 20-35

[0209]

Table 20-36

[0210]

Table 20-37

[0211]

Table 20-38

[0212]

Table 20-39

[0213]

Table 20-40

[0214]

Table 20-41

[0215]

Table 20-42

[0216]

Table 20-43

[0217]

Table 20-44

[0218]

Table 20-45

[0219]

Table 20-46

[0220]

Table 20-47

[0221]

Table 20-48

[0222] In view of the numerous possible embodiments to which the principles of this disclosure may be applied, it should be recognized that the illustrated embodiments are merely examples and should not be construed as limiting the scope of the disclosure. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. A composition for single-stream multiplexing of actionable tumor biomarkers in a sample, wherein the composition comprises multiple sets of primer-pair reagents directed to multiple target sequences for detecting low-level targets in the sample, wherein the target sequences are selected for target genes selected from the group consisting of DNA hotspot mutation genes, copy number variation (CNV) genes, intergene fusion genes, and intragene fusion genes, selected from the genes in Table 1.

2. The composition according to claim 1, wherein one or more actionable tumor biomarkers in the sample determine changes in tumor activity in the sample that indicate potential diagnosis, prognosis, candidate treatment regimens, and / or adverse events.

3. The composition according to claim 1, wherein the target gene includes the genes listed in Table 1.

4. The composition according to claim 1, wherein the target gene consists of the genes listed in Table 1.

5. The composition according to claim 1, wherein the plurality of target sequences include amplicon sequences detected by primers from Table A.

6. The composition according to claim 1, wherein each of the plurality of target sequences includes an amplicon sequence detected by the primers from Table A.

7. The composition according to claim 1, wherein the plurality of primer reagents are selected from the primers in Table A.

8. The composition according to claim 1, wherein each of the plurality of primer reagents is included in Table A.

9. A test kit comprising the composition described in claim 1.

10. A method for determining the presence of one or more actionable tumor biomarkers in a biological sample, Multiple amplification of multiple target sequences from a biological sample, wherein amplification includes contacting at least a portion of the sample with the composition and polymerase described in claim 1 under amplification conditions, thereby generating amplified target sequences. A method comprising detecting each of the aforementioned plurality of target sequences, wherein the detection of one or more actionable tumor biomarkers compared to a control sample determines a change in tumor activity in the sample indicating 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 DNA hotspot mutation genes, copy number variation (CNV) genes, intergene fusion genes, and intragene fusion genes, selected from the genes in Table 1, with the following functions:

12. The method according to claim 10, wherein the target gene includes the genes listed in Table 1.

13. The method according to claim 10, wherein the target gene consists of the genes listed in Table 1.

14. The method according to claim 10, wherein the plurality of target sequences include amplicon sequences detected by primers from Table A.

15. The method according to claim 10, wherein the plurality of target sequences each include an amplicon sequence detected by a primer from Table A.

16. The method according to claim 10, wherein the plurality of primer reagents are selected from the primers in Table A.

17. The method according to claim 10, wherein the plurality of primer reagents each include one of the primers in Table A.

18. The method according to claim 10, wherein the biological sample and the control sample are from the same individual.

19. The method according to claim 10, wherein the control sample is a sample having a known mutation.

20. The method according to claim 10, wherein the sample is isolated from the same source or the same object at different points in time.