COMPOSITIONS AND METHODS FOR ASSESSING IMMUNE RESPONSE
The present invention provides methods and compositions that are useful for assessing gene expression for tumor immune response profile of a sample. In particular, a target-specific primer panel is provided that allows for selective amplification of immune response target sequences in a sample. In one aspect, the invention relates to target-specific primers useful for selective amplification of one or more target sequences associated with immune response. In some aspects, amplified target sequences obtained using the disclosed methods, and compositions can be used in various processes including nucleic acid sequencing and used to detect the presence of genetic variants and/or expression levels of one or more targeted sequences associated with immune response.
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This application claims priority to and the benefit of U.S. Provisional Application No. 62/398,756 filed Sep. 23, 2016 and U.S. Provisional Application No. 62/419,091 filed Nov. 8, 2016. The entire contents of each of the aforementioned applications are incorporated herein by reference.
SEQUENCE LISTINGThis application hereby incorporates by reference the material of the electronic Sequence Listing filed concurrently herewith. The material in the electronic Sequence Listing is submitted as a text (.txt) file entitled “LT01193_ST25.txt” created on Sep. 22, 2017, and is herein incorporated by reference in its entirety.
BACKGROUNDAdvances in cancer immunotherapies have started to provide promising results across oncology. Immune checkpoint inhibitors, cancer vaccines and T-cell therapies have shown sustainable results in responsive populations over conventional or targeted therapies. However, effective identification of responsive candidates and/or monitoring response has proven challenging. The need of a better understanding of the tumor microenvironment, tumor-lymphocyte interactions and drug response biomarkers is immediate. In particular, for example, while the presence of PD-L1 has been reported to be a promising marker to predict positive response to anti-PD-L1 therapy, current methods using immunohistochemistry to measure PD-L1 protein levels are inefficient and highly variable. Higher-throughput, systematic and standardized solutions are a more desirable alternative.
BRIEF SUMMARY OF THE INVENTIONIn one aspect of the invention compositions are provided for a single stream multiplex determination of an immune response in a sample. In some embodiments the composition consists of a plurality of primer pair reagents directed to a plurality of target sequences to measure the expression levels of the targets in the sample. Provided compositions target immune response genes and housekeeping gene sequences wherein the plurality of immune response target gene sequences are selected from targets among the following function: checkpoint pathways, T cell related signaling pathways, markers of tumor infiltrating lymphocytes (TILS), tumor markers, and housekeeping genes. In some embodiments the plurality of immune response target genes are selected from targets among immune checkpoint pathways and targets; T and B cell signaling genes, markers of lymphocyte subsets, interferon signaling genes, cytokine signaling genes; tumor markers, tumor antigens, and proliferation markers. In particular embodiments, provided compositions include a plurality of primer pair reagents selected from Table 2. In some embodiments a multiplex assay comprising compositions of the invention is provided. In some embodiments a test kit comprising compositions of the invention is provided.
In another aspect of the invention, methods are provided for determining immune response activity in a biological sample. Such methods comprise performing multiplex amplification of a plurality of target expression sequences from a biological sample containing target sequences. Amplification comprises contacting at least a portion of the sample comprising multiple target sequences of interest using a plurality of target-specific primer pairs in the presence of a polymerase under amplification conditions to produce a plurality of amplified target expression sequences. The methods further comprise detecting the level of expression of each of the plurality of target immune response sequences, wherein a change in the level of expression of one or more target immune response markers as compared with a control sample determines a change in immune response activity in the sample. The methods described herein utilize compositions of the invention provided herein.
The accompanying drawings, which are incorporated into and form a part of the specification, illustrate one or more exemplary embodiments and serve to explain the principles of various exemplary embodiments. The drawings are exemplary and explanatory only and are not to be construed as limiting or restrictive in any way.
The following description of various exemplary embodiments is exemplary and explanatory only and is not to be construed as limiting or restrictive in any way. Other embodiments, features, objects, and advantages of the present teachings will be apparent from the description and accompanying drawings, and from the claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which these inventions belong.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of features is not necessarily limited only to those features but may include other features not expressly listed or inherent to such process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive-or and not to an exclusive-or.
As used herein, “amplify”, “amplifying” or “amplification reaction” and their derivatives, refer to any action or process whereby at least a portion of a nucleic acid molecule (referred to as a template nucleic acid molecule) is replicated or copied into at least one additional nucleic acid molecule. The additional nucleic acid molecule optionally includes sequence that is substantially identical or substantially complementary to at least some portion of the template nucleic acid molecule. The template nucleic acid molecule can be single-stranded or double-stranded and the additional nucleic acid molecule can independently be single-stranded or double-stranded. In some embodiments, amplification includes a template-dependent in vitro enzyme-catalyzed reaction for the production of at least one copy of at least some portion of the nucleic acid molecule or the production of at least one copy of a nucleic acid sequence that is complementary to at least some portion of the nucleic acid molecule. Amplification optionally includes linear or exponential replication of a nucleic acid molecule. In some embodiments, such amplification is performed using isothermal conditions; in other embodiments, such amplification can include thermocycling. In some embodiments, the amplification is a multiplex amplification that includes the simultaneous amplification of a plurality of target sequences in a single amplification reaction. At least some of the target sequences can be situated on the same nucleic acid molecule or on different target nucleic acid molecules included in the single amplification reaction. In some embodiments, “amplification” includes amplification of at least some portion of DNA- and RNA-based nucleic acids alone, or in combination. The amplification reaction can include single or double-stranded nucleic acid substrates and can further including any of the amplification processes known to one of ordinary skill in the art. In some embodiments, the amplification reaction includes polymerase chain reaction (PCR).
As used herein, “amplification conditions” and its derivatives, refers to conditions suitable for amplifying one or more nucleic acid sequences. Such amplification can be linear or exponential. In some embodiments, the amplification conditions can include isothermal conditions or alternatively can include thermocyling conditions, or a combination of isothermal and thermocycling conditions. In some embodiments, the conditions suitable for amplifying one or more nucleic acid sequences includes polymerase chain reaction (PCR) conditions. Typically, the amplification conditions refer to a reaction mixture that is sufficient to amplify nucleic acids such as one or more target sequences, or to amplify an amplified target sequence ligated to one or more adapters, e.g., an adapter-ligated amplified target sequence. Amplification conditions include a catalyst for amplification or for nucleic acid synthesis, for example a polymerase; a primer that possesses some degree of complementarity to the nucleic acid to be amplified; and nucleotides, such as deoxyribonucleotide triphosphates (dNTPs) to promote extension of the primer once hybridized to the nucleic acid. The amplification conditions can require hybridization or annealing of a primer to a nucleic acid, extension of the primer and a denaturing step in which the extended primer is separated from the nucleic acid sequence undergoing amplification. Typically, but not necessarily, amplification conditions can include thermocycling; in some embodiments, amplification conditions include a plurality of cycles where the steps of annealing, extending and separating are repeated. Typically, the amplification conditions include cations such as Mg++ or Mn++ (e.g., MgCl2, etc) and can also include various modifiers of ionic strength.
As used herein, “target sequence” or “target sequence of interest” and its derivatives, refers to any single or double-stranded nucleic acid sequence that can be amplified or synthesized according to the 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 particular nucleotide sequence to be amplified or synthesized, or its complement, prior to the addition of target-specific primers or appended adapters. Target sequences can include the nucleic acids to which primers useful in the amplification or synthesis reaction can hybridize prior to extension by a polymerase. In some embodiments, the term refers to a nucleic acid sequence whose sequence identity, ordering or location of nucleotides is determined by one or more of the methods of the disclosure.
As defined herein, “sample” and its derivatives, is used in its broadest sense and includes any specimen, culture and the like that is suspected of including a target. In some embodiments, the sample comprises cDNA, RNA, PNA, LNA, chimeric, hybrid, or multiplex-forms of nucleic acids. The sample can include any biological, clinical, surgical, agricultural, atmospheric or aquatic-based specimen containing one or more nucleic acids. The term also includes any isolated nucleic acid sample such a expressed RNA, fresh-frozen or formalin-fixed paraffin-embedded nucleic acid specimen.
As used herein, “contacting” and its derivatives, when used in reference to two or more components, refers to any process whereby the approach, proximity, mixture or commingling of the referenced components is promoted or achieved without necessarily requiring physical contact of such components, and includes mixing of solutions containing any one or more of the referenced components with each other. The referenced components may be contacted in any particular order or combination and the particular order of recitation of components is not limiting. For example, “contacting A with B and C” encompasses embodiments where A is first contacted with B then C, as well as embodiments where C is contacted with A then B, as well as embodiments where a mixture of A and C is contacted with B, and the like. Furthermore, such contacting does not necessarily require that the end result of the contacting process be a mixture including all of the referenced components, as long as at some point during the contacting process all of the referenced components are simultaneously present or simultaneously included in the same mixture or solution. Where one or more of the referenced components to be contacted includes a plurality (e.g., “contacting a target sequence with a plurality of target-specific primers and a polymerase”), then each member of the plurality can be viewed as an individual component of the contacting process, such that the contacting can include contacting of any one or more members of the plurality with any other member of the plurality and/or with any other referenced component (e.g., some but not all of the plurality of target specific primers can be contacted with a target sequence, then a polymerase, and then with other members of the plurality of target-specific primers) in any order or combination.
As used herein, the term “primer” and its derivatives refer to any polynucleotide that can hybridize to a target sequence of interest. In some embodiments, the primer can also serve to prime nucleic acid synthesis. Typically, the primer functions as a substrate onto which nucleotides can be polymerized by a polymerase; in some embodiments, however, the primer can become incorporated into the synthesized nucleic acid strand and provide a site to which another primer can hybridize to prime synthesis of a new strand that is complementary to the synthesized nucleic acid molecule. The primer may be comprised of any combination of nucleotides or analogs thereof, which may be optionally linked to form a linear polymer of any suitable length. In some embodiments, the 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 any difference in length between the two). In some embodiments, the primer is single-stranded but it can also be double-stranded. The primer optionally occurs naturally, as in a purified restriction digest, or can be produced synthetically. In some embodiments, the primer acts as a point of initiation for amplification or synthesis when exposed to amplification or synthesis conditions; such amplification or synthesis can occur in a template-dependent fashion and optionally results in formation of a primer extension product that is complementary to at least a portion of the target sequence. Exemplary amplification or synthesis conditions can include contacting the primer with a polynucleotide template (e.g., a template including a target sequence), nucleotides and an inducing agent such as a polymerase at a suitable temperature and pH to induce polymerization of nucleotides onto an end of the target-specific primer. If double-stranded, the primer can optionally be treated to separate its strands before being used to prepare primer extension products. In some embodiments, the primer is an oligodeoxyribonucleotide or an oligoribonucleotide. In some embodiments, the primer can include one or more nucleotide analogs. The exact length and/or composition, including sequence, of the target-specific primer can influence many properties, including melting temperature (Tm), GC content, formation of secondary structures, repeat nucleotide motifs, length of predicted primer extension products, extent of coverage across a nucleic acid molecule of interest, number of primers present in a single amplification or synthesis reaction, presence of nucleotide analogs or modified nucleotides within the primers, and the like. In some embodiments, a primer can be paired with a compatible primer within an amplification or synthesis reaction to form a primer pair consisting or a forward primer and a reverse primer. In some embodiments, the forward primer of the primer pair includes a sequence that is substantially complementary to at least a portion of a strand of a nucleic acid molecule, and the reverse primer of the primer of the primer pair includes a sequence that is substantially identical to at least of portion of the strand. In some embodiments, the forward primer and the reverse primer are capable of hybridizing to opposite strands of a nucleic acid duplex. Optionally, the forward primer primes synthesis of a first nucleic acid strand, and the reverse primer primes synthesis of a second nucleic acid strand, wherein the first and second strands are substantially complementary to each other, or can hybridize to form a double-stranded nucleic acid molecule. In some embodiments, one end of an 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, where the amplification or synthesis of lengthy primer extension products is required, such as amplifying an exon, coding region, or gene, several primer pairs can be created than span the desired length to enable sufficient amplification of the region. In some embodiments, a primer can include one or more cleavable groups. In some embodiments, primer lengths are in the range of about 10 to about 60 nucleotides, about 12 to about 50 nucleotides and about 15 to about 40 nucleotides in length. Typically, a primer is capable of hybridizing to a corresponding target sequence and undergoing primer extension when exposed to amplification conditions in the presence of dNTPS and a polymerase. In some instances, the particular nucleotide sequence or a portion of the primer is known at the outset of the amplification reaction or can be determined by one or more of the methods disclosed herein. In some embodiments, the primer includes one or more cleavable groups at one or more locations within the primer.
As used herein, “target-specific primer” and its derivatives, refers to a single stranded or double-stranded polynucleotide, typically an oligonucleotide, that includes 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 includes a target sequence. In such instances, the target-specific primer and target sequence are described as “corresponding” to each other. In some embodiments, the target-specific primer is capable of hybridizing to at least a portion of its corresponding target sequence (or to a complement of the target sequence); such hybridization can optionally be performed under standard hybridization conditions or under stringent hybridization conditions. In some embodiments, the target-specific primer is not capable of hybridizing to the target sequence, or to its complement, but is capable of hybridizing to a portion of a nucleic acid strand including the target sequence, or to its complement. In some embodiments, the target-specific primer includes 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; in other embodiments, the target-specific primer includes 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 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; 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 the sample that do not include or correspond to a target sequence (or to a complement of the target sequence) are referred to as “non-specific” sequences or “non-specific nucleic acids”. In some embodiments, the target-specific primer is designed to include 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, across its entire length to at least a portion of a nucleic acid molecule that includes 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, across 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 the target sequence via template-dependent primer extension. Typically, each primer of a target-specific primer pair includes at least one sequence that is substantially complementary to at least a portion of a nucleic acid molecule including a corresponding target sequence but that is less than 50% complementary to at least one other target sequence in the sample. In some embodiments, amplification can be performed using multiple target-specific primer pairs in a single amplification reaction, wherein each primer pair includes a forward target-specific primer and a reverse target-specific primer, each including at least one sequence that 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 an amplification reaction. In some embodiments, the target-specific primer can include minimal cross hybridization to other target-specific primers in the amplification reaction. In some embodiments, target-specific primers include minimal cross-hybridization to non-specific sequences in the amplification reaction mixture. In some embodiments, the target-specific primers include minimal self-complementarity. In some embodiments, the target-specific primers can include one or more cleavable groups located at the 3′ end. In some embodiments, the target-specific primers can include one or more cleavable groups located near or about a central nucleotide of the target-specific primer. In some embodiments, one of more targets-specific primers includes only non-cleavable nucleotides at the 5′ end of the target-specific primer. In some embodiments, a target specific primer includes minimal nucleotide sequence overlap at the 3′end or the 5′ end of the primer as 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 include one or more of the above embodiments. In some embodiments, substantially all of the plurality of target-specific primers in a single reaction mixture includes one or more of the above embodiments.
As used herein, “polymerase” and its derivatives, refers to any enzyme that can catalyze the polymerization of nucleotides (including analogs thereof) into a nucleic acid strand. Typically but not necessarily, such nucleotide polymerization can occur in a template-dependent fashion. Such polymerases can include without limitation naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fusion 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 can be a mutant polymerase comprising one or more mutations involving the replacement 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 linkage of parts of two or more polymerases. Typically, the polymerase comprises one or more active sites at which nucleotide binding and/or catalysis of nucleotide polymerization can occur. Some exemplary polymerases include without limitation DNA polymerases and RNA polymerases. The term “polymerase” and its variants, as used herein, also refers to fusion proteins comprising at least two portions linked to each other, where the first portion comprises a peptide that can catalyze the polymerization of nucleotides into a nucleic acid strand and is linked to a second portion that comprises a second polypeptide. In some embodiments, the second polypeptide can include a reporter enzyme or a processivity-enhancing domain. Optionally, the polymerase can possess 5′ exonuclease activity or terminal transferase activity. In some embodiments, the polymerase can be optionally reactivated, for example through the use of heat, chemicals or re-addition of new amounts of polymerase into a reaction mixture. In some embodiments, the polymerase can include a hot-start polymerase or an aptamer based polymerase that optionally can be reactivated.
As used herein, the term “nucleotide” and its variants comprises any compound, including without limitation any naturally occurring nucleotide or analog thereof, which can bind selectively to, or can be polymerized by, a polymerase. Typically, but not necessarily, selective binding of the nucleotide to the polymerase is followed by polymerization of the nucleotide into a nucleic acid strand by the polymerase; occasionally however the nucleotide may dissociate from the polymerase without becoming incorporated into the nucleic acid strand, an event referred to herein as a “non-productive” event. Such nucleotides include not only naturally occurring nucleotides but also any analogs, regardless of their structure, that can bind selectively to, or can be polymerized by, a polymerase. While naturally occurring nucleotides typically comprise base, sugar and phosphate moieties, the nucleotides of the present disclosure can include compounds lacking any one, some or all of such moieties. In some embodiments, the nucleotide can optionally include a chain of phosphorus atoms comprising three, four, five, six, seven, eight, nine, ten or more phosphorus atoms. In some embodiments, the phosphorus chain can be attached to any carbon of a sugar ring, such as the 5′ carbon. The phosphorus chain can be linked to the sugar with an intervening O or S. In one embodiment, one or more phosphorus atoms in the chain can be part of a phosphate group having P and O. In another embodiment, the phosphorus atoms in the chain can be linked together with intervening O, NH, S, methylene, substituted methylene, ethylene, substituted ethylene, CNH2, C(O), C(CH2), CH2CH2, or C(OH)CH2R (where R can be a 4-pyridine or 1-imidazole). In one embodiment, the phosphorus atoms in the chain can have side groups having O, BH3, or S. In the phosphorus chain, a phosphorus atom with a side group other than O can be a substituted phosphate group. In the phosphorus chain, phosphorus atoms with an intervening atom other than O can be a substituted phosphate group. Some examples of nucleotide analogs are described in Xu, U.S. Pat. No. 7,405,281. In some embodiments, the nucleotide comprises a label and referred to herein as a “labeled nucleotide”; the label of the labeled nucleotide is referred to herein as a “nucleotide label”. In some embodiments, the label can be in the form of a fluorescent dye attached to the terminal phosphate group, i.e., the phosphate group most distal from the sugar. Some examples of nucleotides that can be used in the disclosed methods and compositions include, but are not limited to, ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, ribonucleotide polyphosphates, deoxyribonucleotide polyphosphates, modified ribonucleotide polyphosphates, modified deoxyribonucleotide polyphosphates, peptide nucleotides, modified peptide nucleotides, metallonucleosides, phosphonate nucleosides, and modified phosphate-sugar backbone nucleotides, analogs, derivatives, or variants of the foregoing compounds, and the like. In some embodiments, the nucleotide can comprise non-oxygen moieties such as, for example, thio- or borano-moieties, in place of the oxygen moiety bridging the alpha phosphate and the sugar of the nucleotide, or the alpha and beta phosphates of the nucleotide, or the beta and gamma phosphates of the nucleotide, or between any other two phosphates of the nucleotide, or any combination thereof. “Nucleotide 5′-triphosphate” refers to a nucleotide with a triphosphate ester group at the 5′ position, and are sometimes denoted as “NTP”, or “dNTP” and “ddNTP” to particularly point out the structural features of the ribose sugar. The triphosphate ester group can include sulfur substitutions for the various oxygens, e.g. .alpha.-thio-nucleotide 5′-triphosphates. For a review of nucleic acid chemistry, see: Shabarova, Z. and Bogdanov, A. Advanced Organic Chemistry of Nucleic Acids, VCH, New York, 1994.
The term “extension” and its variants, as used herein, when used in reference to a given primer, comprises any in vivo or in vitro enzymatic activity characteristic of a given polymerase that relates to polymerization of one or more nucleotides onto an end of an existing nucleic acid molecule. Typically but not necessarily such primer extension occurs in a template-dependent fashion; during template-dependent extension, the order and selection of bases is driven by established base pairing rules, which can include Watson-Crick type base pairing rules or alternatively (and especially in the case of extension reactions involving nucleotide analogs) by some other type of base pairing paradigm. In one non-limiting example, extension occurs via polymerization of nucleotides on the 3′OH end of the nucleic acid molecule by the polymerase.
The term “portion” and its variants, as used herein, when used in reference to a given nucleic acid molecule, for example a primer or a template nucleic acid molecule, comprises any number of contiguous nucleotides within the length of the nucleic acid molecule, including the partial or entire length of the nucleic acid molecule.
The terms “identity” and “identical” and their variants, as used herein, when used in reference to two or more nucleic acid sequences, refer to similarity in sequence of the two or more sequences (e.g., nucleotide or polypeptide sequences). In the context of two or more homologous sequences, the percent identity or homology of the sequences or subsequences thereof indicates 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 percent identity can be over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. Sequences are said to be “substantially identical” when there is at least 85% identity at the amino acid level or at the nucleotide level. Preferably, the identity exists over a region that is at least about 25, 50, or 100 residues in length, or across the entire length of at least one compared sequence. A typical algorithm 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 the algorithms of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), and Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), etc. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent hybridization conditions.
The terms “complementary” and “complement” and their variants, as used herein, refer to any two or more nucleic acid sequences (e.g., portions or entireties of template nucleic acid molecules, target sequences and/or primers) that can undergo cumulative base pairing at two or more individual corresponding positions in antiparallel orientation, as in a hybridized duplex. Such base pairing can proceed according to any set of established rules, for example according to Watson-Crick base pairing rules or according to some other base pairing paradigm. Optionally there can be “complete” or “total” complementarity between a first and second nucleic acid sequence where each nucleotide in the first nucleic acid sequence can undergo a stabilizing base pairing interaction with a nucleotide in the corresponding antiparallel position on the second nucleic acid sequence. “Partial” complementarity describes nucleic acid sequences in which at least 20%, but less than 100%, of the residues of one nucleic acid sequence are complementary to 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 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 residues in the other nucleic acid sequence. Sequences are said to be “substantially complementary” when at least 85% of the residues of one nucleic acid sequence are complementary to residues in the other nucleic acid sequence. In some embodiments, two complementary or substantially complementary sequences are capable of hybridizing to each other under standard or stringent hybridization conditions. “Non-complementary” describes nucleic acid sequences in which less than 20% of the residues of one nucleic acid sequence are complementary to residues in the other nucleic acid sequence. Sequences are said to be “substantially non-complementary” when less than 15% of the residues of one nucleic acid sequence are complementary to 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” is present at any position in the two opposed nucleotides are not complementary. Complementary nucleotides include nucleotides that are efficiently incorporated by DNA polymerases opposite each other during DNA replication under physiological conditions. In a typical embodiment, complementary nucleotides can form base pairs with each other, such as the A-T/U and G-C base pairs formed through specific Watson-Crick type hydrogen bonding, or base pairs formed through some other type of base pairing paradigm, between the nucleobases of nucleotides and/or polynucleotides in positions antiparallel to each other. The complementarity of other artificial base pairs can be based on other types of hydrogen bonding and/or hydrophobicity of bases and/or shape complementarity between bases.
As used herein, “amplified target sequences” and its derivatives, refers to a nucleic acid sequence produced by the amplification of/amplifying the target sequences using target-specific primers and the methods provided herein. The amplified target sequences may be either of the same sense (the positive strand produced in the second round and subsequent even-numbered rounds of amplification) or antisense (i.e., the negative strand produced during the first and subsequent odd-numbered rounds of amplification) with respect to the target sequences. For the purposes of this disclosure, the amplified target sequences are typically less than 50% complementary to any portion of another amplified target sequence in the reaction.
As used herein, the terms “ligating”, “ligation” and their derivatives refer to the act or process for covalently linking two or more molecules together, for example, covalently linking two or more nucleic acid molecules to each other. In some embodiments, ligation includes joining nicks between adjacent nucleotides of nucleic acids. In some embodiments, ligation includes forming a covalent bond between an end of a first and an end of a second nucleic acid molecule. In some embodiments, for example embodiments wherein the nucleic acid molecules to be ligated include conventional nucleotide residues, the litigation can include forming a covalent bond between a 5′ phosphate group of one nucleic acid and a 3′ hydroxyl group of a second nucleic acid thereby forming a ligated nucleic acid molecule. In some embodiments, any means for joining nicks or bonding a 5′phosphate to a 3′ hydroxyl between adjacent nucleotides can be employed. In an exemplary embodiment, an enzyme such as a ligase can be used. For the purposes of this disclosure, an amplified target sequence can be ligated to an adapter to generate an adapter-ligated amplified target sequence.
As used herein, “ligase” and its derivatives, refers to any agent capable of catalyzing the ligation of two substrate molecules. In some embodiments, the ligase includes an enzyme capable of catalyzing the joining of nicks between adjacent nucleotides of a nucleic acid. In some embodiments, the ligase includes an enzyme capable of catalyzing the formation of a covalent bond between a 5′ phosphate of one nucleic acid molecule to a 3′ hydroxyl of another nucleic acid molecule thereby forming a ligated nucleic acid molecule. Suitable ligases may include, but not limited to, T4 DNA ligase, T4 RNA ligase, and E. coli DNA ligase.
As used herein, “ligation conditions” and its derivatives, refers to conditions suitable for ligating two molecules to each other. In some embodiments, the ligation conditions are suitable for sealing nicks or gaps between nucleic acids. As defined herein, a “nick” or “gap” refers to a nucleic acid molecule that lacks a directly bound 5′ phosphate of a mononucleotide pentose ring to a 3′ hydroxyl of a neighboring mononucleotide pentose ring within internal nucleotides of a nucleic acid sequence. As used herein, the term nick or gap is consistent with the use of the term in the art. Typically, a nick or gap can be ligated in the presence of an enzyme, such as ligase at an appropriate temperature and pH. In some embodiments, T4 DNA ligase can join a nick between nucleic acids at a temperature of about 70-72° C.
As used herein, “blunt-end ligation” and its derivatives, refers to ligation of two blunt-end double-stranded nucleic acid molecules to each other. A “blunt end” refers to an end of a double-stranded nucleic acid molecule wherein substantially all of the nucleotides in the end of one strand of the nucleic acid molecule are base paired with opposing nucleotides in the other strand of the same nucleic acid molecule. A nucleic acid molecule is not blunt ended if it has an end that includes a single-stranded portion greater than two nucleotides in length, referred to herein as an “overhang”. In some embodiments, the end of nucleic acid molecule does not include any single stranded portion, such that every nucleotide in one strand of the end is based paired with opposing nucleotides in the other strand of the same nucleic acid molecule. In some embodiments, the ends of the two blunt ended nucleic acid molecules that become ligated to each other do not include any overlapping, shared or complementary sequence. Typically, blunted-end ligation excludes the use of additional oligonucleotide adapters to assist in the ligation of the double-stranded amplified target sequence to the double-stranded adapter, such as patch oligonucleotides as described in Mitra and Varley, US2010/0129874, published May 27, 2010. In some embodiments, blunt-ended ligation includes a nick translation reaction to seal a nick created during the ligation process.
As used herein, the terms “adapter” or “adapter and its complements” and their derivatives, refers to any linear oligonucleotide which can be ligated to a nucleic acid molecule of the disclosure. Optionally, the adapter includes a nucleic acid sequence that is not substantially complementary to the 3′ end or the 5′ end of at least one target sequences within the sample. In some embodiments, the adapter is substantially non-complementary to the 3′ end or the 5′ end of any target sequence present in the sample. In some embodiments, the adapter includes any single stranded or double-stranded linear oligonucleotide that is not substantially complementary to an amplified target sequence. In some embodiments, the adapter is substantially non-complementary to at least one, some or all of the nucleic acid molecules of the sample. In some embodiments, suitable adapter lengths are in the range of about 10-100 nucleotides, about 12-60 nucleotides and about 15-50 nucleotides in length. An adapter can include any combination of nucleotides and/or nucleic acids. In some aspects, the adapter can include one or more cleavable groups at one or more locations. In another aspect, the adapter can include a sequence that is substantially identical, or substantially complementary, to at least a portion of a primer, for example a universal primer. In some embodiments, the adapter can include a barcode or tag to assist with downstream cataloguing, identification or sequencing. In some embodiments, a single-stranded adapter can act as a substrate for amplification when ligated to an amplified target sequence, particularly in the presence of a polymerase and dNTPs under suitable temperature and pH.
As used herein, “reamplifying” or “reamplification” and their derivatives refer to any process whereby at least a portion of an amplified nucleic acid molecule is further amplified via any suitable amplification process (referred to in some embodiments as a “secondary” amplification or “reamplification”, thereby producing a reamplified nucleic acid molecule. The secondary amplification need not be identical to the original amplification process whereby the amplified nucleic acid molecule was produced; nor need the reamplified nucleic acid molecule be completely identical or completely complementary to the amplified nucleic acid molecule; all that is required is that the reamplified nucleic acid molecule include at least a portion of the amplified nucleic acid molecule or its complement. For example, the reamplification can involve the use of different amplification conditions and/or different primers, including different target-specific primers than the primary amplification.
As defined herein, a “cleavable group” refers to any moiety that once incorporated into a nucleic acid can be cleaved under appropriate conditions. For example, a cleavable group can be incorporated into a target-specific primer, an amplified sequence, an adapter or a nucleic acid molecule of the sample. In an exemplary embodiment, a target-specific primer can include a cleavable group that becomes incorporated into the amplified product and is subsequently cleaved after amplification, thereby removing a portion, or all, of the target-specific primer from the amplified product. The cleavable group can be cleaved or otherwise removed from a target-specific primer, an amplified sequence, an adapter or a nucleic acid molecule of the 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 the sample by enzymatic, thermal, photo-oxidative or chemical treatment. In one aspect, a cleavable group can include a nucleobase that is not naturally occurring. For example, an oligodeoxyribonucleotide can include one or more RNA nucleobases, such as uracil that can be removed by a uracil glycosylase. In some embodiments, a cleavable group can include one or more modified nucleobases (such as 7-methylguanine, 8-oxo-guanine, xanthine, hypoxanthine, 5,6-dihydrouracil or 5-methylcytosine) or one or more modified nucleosides (i.e., 7-methylguanosine, 8-oxo-deoxyguanosine, 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, a cleavable group can include a moiety that can be removed from a primer after amplification (or synthesis) upon exposure to ultraviolet light (i.e., bromodeoxyuridine). In another embodiment, a cleavable group can include methylated cytosine. Typically, methylated cytosine can be cleaved from a primer for example, after induction of amplification (or synthesis), upon sodium bisulfite treatment. In some embodiments, a cleavable moiety can include a restriction site. For example, a primer or target sequence can include a nucleic acid sequence that is specific to one or more restriction enzymes, and following amplification (or synthesis), the primer or target sequence can be treated with the one or more restriction enzymes such that the cleavable group is removed. Typically, one or more cleavable groups can be included at one or more locations with a target-specific primer, an amplified sequence, an adapter or a nucleic acid molecule of the sample.
As used herein, “cleavage step” and its derivatives, refers to any process by which a cleavable group is cleaved or otherwise removed from a target-specific primer, an amplified sequence, an adapter or a nucleic acid molecule of the sample. In some embodiments, the cleavage steps involves a chemical, thermal, photo-oxidative or digestive process.
As used herein, the term “hybridization” is consistent with its use in the art, and refers to the process whereby two nucleic acid molecules undergo base pairing interactions. Two nucleic acid molecule 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; it is not necessarily required that the two nucleic acid molecules be hybridized across their entire respective lengths and in some embodiments, at least one of the nucleic acid molecules can include portions that are not hybridized to the other nucleic acid molecule. The phrase “hybridizing under stringent conditions” and its variants refers to conditions under which hybridization of a target-specific primer to a target sequence occurs in the presence of high hybridization temperature and low ionic strength. In one exemplary embodiment, stringent hybridization conditions include an aqueous environment containing about 30 mM magnesium sulfate, about 300 mM Tris-sulfate at pH 8.9, and about 90 mM ammonium sulfate at about 60-68° C., or equivalents thereof. As used herein, the phrase “standard hybridization conditions” and its variants refers to conditions under which hybridization of a primer to an oligonucleotide (i.e., a target sequence), occurs in the presence of low hybridization temperature 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 thereof.
As used herein, “GC content” and its derivatives, refers to the cytosine and guanine content of a nucleic acid molecule. The GC content of a target-specific primer (or adapter) of the disclosure is 85% or lower. More typically, the GC content of a target-specific primer or adapter of the disclosure is between 15-85%.
As used herein, the term “end” and its variants, when used in reference to a nucleic acid molecule, for example a target sequence or amplified target sequence, can include the terminal 30 nucleotides, the terminal 20 and even more typically the terminal 15 nucleotides of the nucleic acid molecule. A linear nucleic acid molecule comprised of linked series of contiguous nucleotides typically includes at least two ends. In some embodiments, one end of the 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 a 5′ phosphate group of a mononucleotide pentose ring. Typically, the 3′ end includes one or more 5′ linked nucleotides located adjacent to the nucleotide including the unlinked 3′ hydroxyl group, typically the nucleotides located adjacent to the 3′ hydroxyl, typically the terminal 20 and even more typically the terminal 15 nucleotides. One or more linked nucleotides can be represented as a percentage of the nucleotides present in the oligonucleotide or can be provided as a number of linked nucleotides adjacent to the unlinked 3′ hydroxyl. For example, the 3′ end can include less than 50% of the nucleotide length of the oligonucleotide. In some embodiments, the 3′ end does not include any unlinked 3′ hydroxyl group but can include any moiety capable of serving as a site for attachment of nucleotides via primer extension and/or nucleotide polymerization. In some embodiments, the term “3′ end” for example when referring to a target-specific primer, can include 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 include nucleotides located at nucleotide positions 10 or fewer from the 3′ terminus.
As used herein, “5′ end”, and its derivatives, refers to an end of a nucleic acid molecule, for example a target sequence or amplified target sequence, which includes a free 5′ phosphate group or its equivalent. In some embodiments, the 5′ end includes a 5′ phosphate group that is not linked to a 3′ hydroxyl of a neighboring mononucleotide pentose ring. Typically, the 5′ end includes to one or more linked nucleotides located adjacent to the 5′ phosphate, typically the 30 nucleotides located adjacent to the nucleotide including the 5′ phosphate group, typically the terminal 20 and even more typically the terminal 15 nucleotides. One or more linked nucleotides can be represented 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 an oligonucleotide. In another exemplary embodiment, the 5′ end can include about 15 nucleotides adjacent to the nucleotide including the terminal 5′ phosphate. In some embodiments, the 5′ end does not include any unlinked 5′ phosphate group but can include any moiety capable of serving as a site of attachment to a a 3′ hydroxyl group, or to the 3′end of another nucleic acid molecule. In some embodiments, the term “5′ end” for example when referring to a target-specific primer, can include the terminal 10 nucleotides, the terminal 5 nucleotides, the terminal 4, 3, 2 or fewer nucleotides at the 5′end. In some embodiments, the term “5′ end” when referring to a target-specific primer can include nucleotides located at positions 10 or fewer from the 5′ terminus. In some embodiments, the 5′ end of a target-specific primer can include only non-cleavable nucleotides, for example nucleotides that do not contain one or more cleavable groups as disclosed herein, or a cleavable nucleotide as would be readily determined by one of ordinary skill in the art.
As used herein, “DNA barcode” or “DNA tagging sequence” and its derivatives, refers to a unique short (6-14 nucleotide) nucleic acid sequence within an adapter that can act as a ‘key’ to distinguish or separate a plurality of amplified target sequences in a sample. For the purposes of this disclosure, a DNA barcode or DNA tagging sequence can be incorporated into the nucleotide sequence of an adapter.
As used herein, the phrases “two rounds of target-specific hybridization” or “two rounds of target-specific selection” and their derivatives refers to any process whereby the same target sequence is subjected to two consecutive rounds of hybridization-based target-specific selection, wherein a target sequence is hybridized to a target-specific sequence. Each round of hybridization based target-specific selection can include multiple target-specific hybridizations to at least some portion of a target-specific sequence. In one exemplary embodiment, a round of target-specific selection includes a first target-specific hybridization involving a first region of the target sequence and a second target-specific hybridization involving a second region of the target sequence. The first and second regions can be the same or different. In some embodiments, each round of hybridization-based target-specific selection can include use of two target specific oligonucleotides (e.g., a forward target-specific primer and a reverse target-specific primer), such that each round of selection includes two target-specific hybridizations.
As used herein, “comparable maximal minimum melting temperatures” and its derivatives, refers to the melting temperature (Tm) of each nucleic acid fragment for a single adapter or target-specific primer after cleavage of the cleavable groups. The hybridization temperature of each nucleic acid fragment generated by a single adapter or target-specific primer is compared to determine the maximal minimum temperature required preventing hybridization of any nucleic acid fragment from the target-specific primer or adapter to the target sequence. Once the maximal hybridization temperature is known, it is possible to manipulate the adapter or target-specific primer, for example by moving the location of the cleavable group along the length of the primer, to achieve a comparable maximal minimum melting temperature with respect to each nucleic acid fragment.
As used herein, “addition only” and its derivatives, refers 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 the removal of the reaction mixture from a first vessel to a second vessel in order to complete the series of steps. An addition only process excludes the manipulation of the reaction mixture outside the vessel containing the reaction mixture. Typically, an addition-only process is amenable to automation and high-throughput.
As used herein, “synthesizing” and its derivatives, refers to a reaction involving nucleotide polymerization by a polymerase, optionally in a template-dependent fashion. Polymerases synthesize an oligonucleotide via transfer of a nucleoside monophosphate from a nucleoside triphosphate (NTP), deoxynucleoside triphosphate (dNTP) or dideoxynucleoside triphosphate (ddNTP) to the 3′ hydroxyl of an extending oligonucleotide chain. For the purposes of this disclosure, synthesizing includes to the serial extension of a hybridized adapter or a target-specific primer via transfer of a nucleoside monophosphate from a deoxynucleoside triphosphate.
As used herein, “polymerizing conditions” and its derivatives, refers to conditions suitable for nucleotide polymerization. In typical embodiments, such nucleotide polymerization is catalyzed by a polymerase. In some embodiments, polymerizing conditions include conditions for primer extension, optionally in a template-dependent manner, resulting in the generation of a synthesized nucleic acid sequence. In some embodiments, the polymerizing conditions include polymerase chain reaction (PCR). Typically, the polymerizing conditions include use of a reaction mixture that is sufficient to synthesize nucleic acids and includes a polymerase and nucleotides. The polymerizing 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, polymerizing conditions can be practiced using thermocycling. Additionally, polymerizing conditions can include a plurality of cycles where the steps of annealing, extending, and separating the two nucleic strands are repeated. Typically, the polymerizing conditions include a cation such as MgCl2. Polymerization of one or more nucleotides to form a nucleic acid strand includes that the nucleotides be linked to each other via phosphodiester bonds, however, alternative linkages may be possible in the context of particular nucleotide analogs.
As used herein, the term “nucleic acid” refers to natural nucleic acids, artificial nucleic acids, analogs thereof, or combinations thereof, including polynucleotides and oligonucleotides. As used herein, the terms “polynucleotide” and “oligonucleotide” are used interchangeably and mean single-stranded and double-stranded polymers of nucleotides including, but not limited to, 2′-deoxyribonucleotides (nucleic acid) and ribonucleotides (RNA) linked by internucleotide phosphodiester bond linkages, e.g. 3′-5′ and 2′-5′, inverted linkages, e.g. 3′-3′ and 5′-5′, branched structures, or analog nucleic acids. Polynucleotides have associated counter ions, such as H+, NH4+, trialkylammonium, Mg2+, Na+ and the like. An oligonucleotide can be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof. Oligonucleotides can be comprised of nucleobase and sugar analogs. Polynucleotides typically range in size from a few monomeric units, e.g. 5-40, when they are more commonly frequently referred to in the art as oligonucleotides, to several thousands of monomeric nucleotide units, when they are more commonly referred to in the art as polynucleotides; for purposes of this disclosure, however, both oligonucleotides and polynucleotides may be of any suitable length. Unless denoted otherwise, whenever a oligonucleotide sequence is represented, it will be understood that the nucleotides are in 5′ to 3′ order from left to right and that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, “T” denotes thymidine, and “U” denotes deoxyuridine. Oligonucleotides are said to have “5′ ends” and “3′ ends” because mononucleotides are typically reacted to form oligonucleotides via attachment of the 5′ phosphate or equivalent group of one nucleotide to the 3′ hydroxyl or equivalent group of its neighboring nucleotide, optionally via a phosphodiester or other suitable linkage.
As defined herein, the term “nick translation” and its variants comprise the translocation of one or more nicks or gaps within a nucleic acid strand to a new position along the nucleic acid strand. In some embodiments, a nick can be formed when a double stranded adapter is ligated to a double stranded amplified target sequence. In one example, the primer can include at its 5′ end, a phosphate group that can ligate to the double stranded amplified target sequence, leaving a nick between the adapter and the amplified target sequence in the complementary strand. In some embodiments, nick translation results in the movement of the nick to the 3′ end of the nucleic acid strand. In some embodiments, moving the nick can include performing a nick translation reaction on the adapter-ligated amplified target sequence. In some embodiments, the nick translation reaction can be a coupled 5′ to 3′ DNA polymerization/degradation reaction, or coupled to a 5′ to 3′ DNA polymerization/strand displacement reaction. In some embodiments, moving the nick can include performing a DNA strand extension reaction at the nick site. In some embodiments, moving the nick can include performing a single strand exonuclease reaction on the nick to form a single stranded portion of the adapter-ligated amplified target sequence and performing a DNA strand extension reaction on the single stranded portion of the adapter-ligated amplified target sequence to a new position. In some embodiments, a nick is formed in the nucleic acid strand opposite the site of ligation.
As used herein, the term “polymerase chain reaction” (“PCR”) refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby incorporated by reference, which describe a method for increasing the concentration of a segment of a polynucleotide of interest in a mixture of expressed RNA or cDNA without cloning or purification. This process for amplifying the polynucleotide of interest consists of introducing a large excess of two oligonucleotide primers to the 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 effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the polynucleotide of interest 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”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired polynucleotide of interest. The length of the amplified segment of the desired polynucleotide of interest (amplicon) is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of repeating the process, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified segments of the polynucleotide of interest become the predominant nucleic acid sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified”. As defined herein, target nucleic acid molecules within a sample including a plurality of target nucleic acid molecules are amplified via PCR. In a modification to the method discussed above, the target nucleic acid molecules can be PCR amplified using a plurality of different primer pairs, in some cases, one or more primer pairs 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 amplified target sequences. It is also possible to detect the amplified target sequences by several different methodologies (e.g., quantitation with a bioanalyzer or qPCR, hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified target sequence). Any oligonucleotide sequence can be amplified with the appropriate set of primers, thereby allowing for the amplification of target nucleic acid molecules from RNA, cDNA, formalin-fixed paraffin-embedded DNA, fine-needle biopsies and various other sources. In particular, the amplified target sequences created by the multiplex PCR process as disclosed herein, are themselves efficient substrates for subsequent PCR amplification or various downstream assays or manipulations.
As defined herein “multiplex amplification” refers to selective and non-random amplification of two or more target sequences within a sample using at least one target-specific primer. In some embodiments, multiplex amplification is performed such that some or all of the target sequences are amplified within a single reaction vessel. The “plexy” or “plex” of a given multiplex amplification refers to the number of different target-specific sequences that are amplified during that single multiplex amplification. In some embodiments, the plexy can be about 12-plex, 24-plex, 48-plex, 74-plex, 96-plex, 120-plex, 144-plex, 168-plex, 192-plex, 216-plex, 240-plex, 264-plex, 288-plex, 312-plex, 336-plex, 360-plex, 384-plex, or 398-plex.
The practice of the present subject matter may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant techniques), cell biology, and biochemistry, which are within the skill of the art. 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 can be used by reference to the examples provided herein. Other equivalent conventional procedures can also be used. Such conventional techniques and descriptions 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); and the like.
We have developed a multiplex next generation sequencing workflow to measure expression of genes involved in immune response, e.g., checkpoint pathways, T cell related signaling pathways, markers of different tumor infiltrating lymphocyte (TIL) subsets, and tumor markers, in order to determine immune response in a sample. The immune response assay compositions and methods of the invention offer a specific and robust solution for biomarker screening and for understanding mechanisms involved with tumor immune response. Thus, provided are multiplex gene expression compositions for multiplex library preparation and use in conjunction with next generation sequencing technologies and workflow solutions (e.g., Ion Torrent™ NGS workflow), manual or automated, to evaluate pathways that shield tumors from the immune response.
Thus, provided are compositions for a single stream multiplex determination of an immune response in a sample. In some embodiments, the composition consists of a plurality of sets of primer pair reagents directed to a plurality of target sequences to measure the expression levels of the targets in the sample, wherein the target genes are selected from immune response genes consisting of the following function: checkpoint pathways, T cell related signaling pathways, markers of tumor infiltrating lymphocytes (TILS), tumor markers, and housekeeping genes. In some embodiments, the target genes are selected from immune response genes consisting of one or more function of Table A. In some embodiments, the target genes are selected from immune response genes consisting of the following function: immune checkpoint pathways and targets; T and B cell signaling genes, markers of lymphocyte subsets, interferon signaling genes, cytokine signaling genes; tumor markers, tumor antigens, proliferation markers; and housekeeping genes. In some embodiments, the target genes are selected from immune response genes that elucidate T and B lymphocyte functions, from activation to antigen processing, including, e.g., genes consisting of the following function: antigen presentation, antigen processing, innate immune response, leukocyte inhibition, leukocyte migration, lymphocyte activation, lymphocyte development, lymphocyte infiltrate, B cell receptor signaling, T cell receptor signaling, T cell regulation and TCR coexpression. In some embodiments, the target genes are selected from immune response genes that elucidate the level of inflammation and activation and co-op by helper cells, including, e.g., genes consisting of the following function: chemokine signaling, cytokine signaling, interferon signaling, Type I interferon signaling, and Type II interferon signaling. In some embodiments, the target genes identify the presence of various relevant cell types, including, e.g., genes selected from immune response genes consisting of the following function: B cell marker, dendritic cell, dendritic cell/macrophage, helper T cell, macrophage, myeloid, neutrophil, NK activation, NK cell, and T cell differentiation. In some embodiments, the target genes elucidate the mechanism of action, including, e.g., genes selected from immune response genes consisting of the following function: checkpoint pathway, PD-signaling, and drug target. In some embodiments, the target genes demonstrate the proliferative activity and/or stemness characteristics of tumor cells, including, e.g., genes selected from immune response genes consisting of the following function: adhesion/migration, apoptosis, proliferation, tumor antigen, and tumor marker. In total, the various functions of genes comprising the provided multiplex panel of the invention provide a comprehensive picture of complex activities of the tumor microenvironment.
In some embodiments, immune response target sequences are selected from immune response genes consisting of the following function: antigen presentation, antigen processing, innate immune response, leukocyte inhibition, leukocyte migration, lymphocyte activation, lymphocyte development, lymphocyte infiltrate, B cell receptor signaling, T cell receptor signaling, T cell regulation, TCR coexpression, chemokine signaling, cytokine signaling, interferon signaling, Type I interferon signaling, Type II interferon signaling, B cell marker, dendritic cell, dendritic cell/macrophage, helper T cell, macrophage, myeloid, neutrophil, NK activation, NK cell, T cell differentiation, checkpoint pathway, PD-signaling, drug target, adhesion/migration, apoptosis, proliferation, tumor antigen, and tumor marker.
In some embodiments, immune response target sequences are selected from immune response genes consisting of the following functions: antigen presentation, antigen processing, innate immune response, leukocyte inhibition, leukocyte migration, lymphocyte activation, lymphocyte development, lymphocyte infiltrate, B cell receptor signaling, T cell receptor signaling, T cell regulation, TCR coexpression chemokine signaling, cytokine signaling, interferon signaling, Type I interferon signaling, Type II interferon signaling, B cell marker, dendritic cell, dendritic cell/macrophage, helper T cell, macrophage, myeloid, neutrophil, NK activation, NK cell, T cell differentiation, checkpoint pathway, PD-signaling, and drug target. In some embodiments, immune response target sequences are selected from immune response genes consisting of the following function: antigen presentation, antigen processing, innate immune response, leukocyte inhibition, leukocyte migration, lymphocyte activation, lymphocyte development, lymphocyte infiltrate, B cell receptor signaling, T cell receptor signaling, T cell regulation, TCR coexpression, chemokine signaling, cytokine signaling, interferon signaling, Type I interferon signaling, Type II interferon signaling, B cell marker, dendritic cell, dendritic cell/macrophage, helper T cell, macrophage, myeloid, neutrophil, NK activation, NK cell, T cell differentiation, adhesion/migration, apoptosis, proliferation, tumor antigen, and tumor marker. In some embodiments, immune response target sequences are selected from immune response genes consisting of the following function: antigen presentation, antigen processing, innate immune response, leukocyte inhibition, leukocyte migration, lymphocyte activation, lymphocyte development, lymphocyte infiltrate, B cell receptor signaling, T cell receptor signaling, T cell regulation, TCR coexpression, checkpoint pathway, PD-signaling, drug target, adhesion/migration, apoptosis, proliferation, tumor antigen, and tumor marker. In some embodiments, immune response target sequences are selected from immune response genes consisting of the following function: antigen presentation, antigen processing, innate immune response, leukocyte inhibition, leukocyte migration, lymphocyte activation, lymphocyte development, lymphocyte infiltrate, B cell receptor signaling, T cell receptor signaling, T cell regulation, TCR coexpression, chemokine signaling, cytokine signaling, interferon signaling, Type I interferon signaling, Type II interferon signaling, B cell marker, dendritic cell, dendritic cell/macrophage, helper T cell, macrophage, myeloid, neutrophil, NK activation, NK cell, T cell differentiation, checkpoint pathway, PD-signaling, drug target, adhesion/migration, apoptosis, proliferation, tumor antigen, and tumor marker. In some embodiments, immune response target sequences are selected from immune response genes consisting of the following function: chemokine signaling, cytokine signaling, interferon signaling, Type I interferon signaling, Type II interferon signaling, B cell marker, dendritic cell, dendritic cell/macrophage, helper T cell, macrophage, myeloid, neutrophil, NK activation, NK cell, T cell differentiation, checkpoint pathway, PD-signaling, drug target, adhesion/migration, apoptosis, proliferation, tumor antigen, and tumor marker.
In certain embodiments, target immune response sequences are directed to sequences having aberrant expression associated with cancer. In some embodiments, the target sequences or amplified target sequences are directed to sequences having aberrant expression associated with one or more solid tumor cancers selected from the group consisting of head and neck cancers (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 cancer, cervical cancer, endometrial cancer, fallopian cancer), colorectal cancer, gallbladder cancer, esophageal cancer, gastrointestinal cancer, gastric cancer, bladder cancer, prostate cancer, testicular cancer, urothelial 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, bile duct cancer, pituitary tumor, wilms tumor, kaposi sarcoma, hairy cell carcinoma, osteosarcoma, thymus cancer, skin cancer, melanoma, heart cancer, oral and larynx cancer, neuroblastoma, mesothelioma, and other solid tumors (thymic, bone, soft tissue, oral SCC, myelofibrosis, synovial sarcoma). In one embodiment, the mutations can include substitutions, insertions, inversions, point mutations, deletions, mismatches and translocations. In some embodiments, the target sequences or amplified target sequences are directed to sequences having aberrant expression associated with one or more blood/hematologic cancers selected from the group consisting of multiple myeloma, diffuse large B cell lymphoma (DLBCL), lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, follicular lymphoma, leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), myelodisplastic syndrome. In one embodiment, the aberrant expression associated with cancer are located in at least one of the genes provided in Table 1.
In some embodiments, one or more aberrant expression immune response sequences are located in at least one of the genes selected from CD63, CD69, CXCL1, KLRD1, HLA-DOB, CXCR5, IL12B, PTK7, CEACAM1, CXCL9, IL13, NT5E, VEGFA, ABCF1, CD38, JAML, S100A8, MYC, IRF1, CCL22, CXCR2, IFIT1, IFIT2, CD68, M6PR, SH2D1A, ISG20, GBP1, TBP, STAT6, ID3, CX3CL1, KLRB1, TNFSF4, CD52, IL10RA, HLA-DOA, IFNB1, CCR5, IKZF3, STAT1, CD6, BRCA1, CORO1A, TBX21, KLRK1, CXCR6, PTEN, PMEL, DMBT1, IFI44L, LAPTM5, CD226, TNFSF13B, ICOS, CD160, TRIM29, LST1, ZBTB46, VTCN1, KREMEN1, PDCD1LG2, TUBB, CLEC4C, CD86, HAVCR2, GZMH, NFATC1, CD8B, BCL2, GADD45GIP1, CBLB, ITGA1, CD8A, IL2RA, EIF2AK2, MADCAM1, PTPN6, LRG1, ADGRE5, SH2D1B, ITGB2, HLA-DPA1, DGAT2, IGF1R, TAGAP, LMNA, NCAM1, TIGIT, IL17F, HLA-F-AS1, CD247, CD79B, IDO2, IL4, TYROBP, BTLA, AKT1, IL2RG, POLR2A, ITGAX, IL1B, CSF2RB, DDX58, KIAA0101, CD274, LAMP3, TNFAIP8, FOXP3, IL12A, SAMHD1, SIT1, CD3E, ICOSLG, HGF, MELK, IGSF6, GNLY, TDO2, KRT7, HLA-E, HLA-DM, LAMP1, NTN3, CD28, TARP, EGFR, CCR4, MAGEA3, BATF, KLRG1, IRS1, CSF1R, CTLA4, TNFSF18, POU2AF1, GZMA, PIK3CA, ITK, IFI27, EOMES, LCN2, CD80, CD83, CXCL13, MTOR, FCER1G, TFRC, RORC, MMP9, BST2, PIK3CD, FCGR2B, TNFRSF14, OAS3, GRAP2, CCNB2, MLANA, MAGEA12, VCAM1, CDKN3, NCR1, FAS, GZMB, IRF9, IFITM2, TNFSF14, HLA-B, SDHA, NRP1, EBI3, EFNA4, PVR, BUB1, SKAP2, PRF1, CCL20, TNFRSF18, CTSS, NKG7, ISG15, PDCD1, SNAI1, CXCL11, CIITA, IFI35, TNFSF9, TNFSF10, MMP2, EGR3, MAGEA1, CD163, IL6, KLRF1, B3GAT1, C1QA, OAS1, IKZF2, TLR9, KLF2, GUSB, NFKBIA, IL23A, HERC6, SLAMF8, IL15, TLR7, OAS2, HLA-DR, CRTAM, MAGEC2, ICAM1, CD4, MAPK14, C1QB, NOTCH3, NCR3, STAT3, TLR8, CYBB, IKZF4, IFIH1, LCK, BCL2L11, ITGAM, ITGB7, JCHAIN, CD209, SLAMF7, IL10, IL1A, FCGR3A, IFNA17, EGR2, TOP2A, C10orf54, FOXM1, AXL, MS4A1, IFI6, CD3D, GPR18, CD3G, ZAP70, HMBS, IL7, IFIT3, RB1, PTGS2, TGFB1, NCF1, TWIST1, CA4, SELL, LILRB1, CD14, ALOX15B, PECAM1, NOS2, FASLG, CD44, ENTPD1, CMKLR1, CD53, TNF, CXCL8, CD40LG, HLA-F, GATA3, LYZ, ARG1, IL2RB, NECTIN2, MPO, CCR2, BRCA2, ADORA2A, G6PD, TAP1, MX1, HLA-DQB2, CD27, CD276, STAT4, PTPN7, PTPRC, PSMB9, CD244, CXCR4, MAPK1, TP63, IRF4, CCL3, CCL18, IL7R, HLA-DRB1, CEACAM8, CXCL10, CCL2, SRGN, CD19, ITGB1, IFITM1, CCL21, MRC1, PGF, ITGAL, ID2, CD22, CCL17, ITGAE, IL3RA, CCR7, CD1C, MAD2L1, PYGL, CD40, LY9, HLA-G, TLR3, CD48, STAT5A, FCRLA, BCL6, ZEB1, CCL5, IDO1, IL18, TNFRSF9, HIF1A, HLA-DPB1, FOXO1, CD33, S100A9, HLA-DMB, HLA-A, SNAI2, TNFRSF17, LRP1, MAGEA4, HLA-DQA1, CD1D, RPS6, MKI67, GZMK, CD79A, CD37, FUT4, AIF1, CCR1, PRDM1, CD47, CD74, LAG3, TNFRSF4, CD2, CCL4, BAGE, LEXM, CCR6, CD70, CDK1, CTAG1B, CTAG2, CX3CR1, CX3CR1, CX3CR1, CX3CR1, GAGE1, GAGE12I, GAGE12F, GAGE12J, GAGE2C, GAGE2A, GAGE2E, GAGE10, GAGE13, IKZF1, IL17A, IL2, IL21, IL22, KIR2DL2, KIR2DL3, MAGEA10, MIF, PTPRCAP, SSX2, TCF7, XAGE1B, CEACAM8, CXCR3, FCGR1A, FCGR3B, FYB, HLA-C, HLA-DQA2, IFNG, KIR2DL1, KRT5, LMNA, and PTPN11. In some embodiments the one or more aberrant expression sequences indicate cancer activity.
In some embodiments the one or more aberrant expression sequences indicate a patient's likelihood to response to a therapeutic agent. In some embodiments, the one or more aberrant expression sequences indication a patient's likelihood to not be responsive to a therapeutic agent. In certain embodiments, relevant therapeutic agents can be immunotherapies including but not limited to checkpoint blockades, T cell therapies, and therapeutic vaccines. In some embodiments a therapeutic agent may modify an immune response gene selected from PD1 (e.g., nivolumab, pembrolizumab, AMP-244, MEDI0680, AMP-514, pidlizumab), CTLA4 (e.g., ipilimumab, tremelimumab), PD-L1 (e.g., atezolizumab, MDX1105-01, MEDI4736, avelumab), MR (e.g., lirilumab, NCC0141-0000-0100), CD9/NKG2A (e.g., IPH2201), LAG3 (e.g., BMS986016), GITR (e.g., TRX518), OX40 (e.g., MEDI6383, MEDI6489, MOXR0916), IDO (e.g., indoximod (NLG8189), INCB024360, F001287, NLG919), TGFbeta (e.g., sotaracept, fresolumimab, trabedersen, lucanix), TGFbetaR (e.g., LY2157299, ACE536), CD137 (e.g., urelumab), CD137/41BB (e.g., pf05082566), CD289/TLR9 (e.g., MGN1703), MUC1/CD227 (e.g., OINT-10, ASN-004), CD27 (e.g., varlilumab), CD27L (e.g., AMG172), SLAMF7/CD1 (e.g., elotuzumab), CD20 (e.g., DI-Leu16-IL2), talimogene laherparepvec, CD70 (e.g., ARGX110), IL10 (e.g., AM0010), PSA (e.g., PROSTVAC), GP100 (e.g., MDX1379), STAT3 (e.g., AZD9150), CVAC, IL12 (e.g., veledimex, INXN2001, MSB0010360N, IMMUNOPULSE, GEN-1, INO-9012; IL2: MSB0010445, RG7813/R06895882), IL33 (e.g., alarmin IL33), ICT140, CNDO-109, hTERT (e.g., INO-1400), dysplastic tissues (e.g., ADXS-HPV), SMAC-mimetic (e.g., birinapant), ImmTACs (e.g., IMCgp100), and CD40 (e.g., RO7009789. In certain embodiments, relevant therapeutic agent is an immunotherapy selected from checkpoint blockade, T cell therapy, and therapeutic vaccine. In some embodiments a therapeutic agent modifies an immune response gene selected from any of PD1 (e.g., nivolumab, pembrolizumab, AMP-244, MEDI0680, AMP-514, pidlizumab), CTLA4 (e.g., ipilimumab, tremelimumab), PD-L1 (e.g., atezolizumab, MDX1105-01, MEDI4736, avelumab), MR (e.g., lirilumab, NCC0141-0000-0100), CD9/NKG2A (e.g., IPH2201), LAG3 (e.g., BMS986016), GITR (e.g., TRX518), OX40 (e.g., MEDI6383, MEDI6489, MOXR0916), IDO (e.g., indoximod (NLG8189), INCB024360, F001287, NLG919), TGFbeta (e.g., sotaracept, fresolumimab, trabedersen, lucanix), TGFbetaR (e.g., LY2157299, ACE536), CD137 (e.g., urelumab), CD137/41BB (e.g., pf05082566), CD289/TLR9 (e.g., MGN1703), MUC1/CD227 (e.g., OINT-10, ASN-004), CD27 (e.g., varlilumab), CD27L (e.g., AMG172), SLAMF7/CD1 (e.g., elotuzumab), CD20 (e.g., DI-Leu16-IL2), talimogene laherparepvec, CD70 (e.g., ARGX110), IL10 (e.g., AM0010), PSA (e.g., PROSTVAC), GP100 (e.g., MDX1379), STAT3 (e.g., AZD9150), CVAC, IL12 (e.g., veledimex, INXN2001, MSB0010360N, IMMUNOPULSE, GEN-1, INO-9012; IL2: MSB0010445, RG7813/R06895882), IL33 (e.g., alarmin IL33), ICT140, CNDO-109, hTERT (e.g., INO-1400), dysplastic tissues (e.g., ADXS-HPV), SMAC-mimetic (e.g., birinapant), ImmTACs (e.g., IMCgp100), and CD40 (e.g., RO7009789.
In some embodiments, target sequences or amplified target sequences are directed to mutations associated with cancer. In some embodiments, the target sequences or amplified target sequences are directed to mutations associated with one or more solid tumor cancers selected from the group consisting of head and neck cancers (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 cancer, cervical cancer, endometrial cancer, fallopian cancer), colorectal cancer, gallbladder cancer, esophageal cancer, gastrointestinal cancer, gastric cancer, bladder cancer, prostate cancer, testicular cancer, urothelial 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, bile duct cancer, pituitary tumor, wilms tumor, kaposi sarcoma, hairy cell carcinoma, osteosarcoma, thymus cancer, skin cancer, melanoma, heart cancer, oral and larynx cancer, neuroblastoma, mesothelioma, and other solid tumors (thymic, bone, soft tissue, oral SCC, myelofibrosis, synovial sarcoma). In one embodiment, the mutations can include substitutions, insertions, inversions, point mutations, deletions, mismatches and translocations. In one embodiment, the mutations can include variation in copy number. In one embodiment, the mutations can include germline or somatic mutations. In some embodiments, the target sequences or amplified target sequences are directed to sequences having mutations associated with one or more blood/hematologic cancers selected from the group consisting of multiple myeloma, diffuse large B cell lymphoma (DLBCL), lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, follicular lymphoma, leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), myelodisplastic syndrome. In one embodiment, the mutations associated with cancer are located in at least one of the genes provided in Table 1.
In some embodiments, one or more mutations in immune response sequences associated with cancer are located in at least one of the genes selected from CD63, CD69, CXCL1, KLRD1, HLA-DOB, CXCR5, IL12B, PTK7, CEACAM1, CXCL9, IL13, NT5E, VEGFA, ABCF1, CD38, JAML, S100A8, MYC, IRF1, CCL22, CXCR2, IFIT1, IFIT2, CD68, M6PR, SH2D1A, ISG20, GBP1, TBP, STAT6, ID3, CX3CL1, KLRB1, TNFSF4, CD52, IL10RA, HLA-DOA, IFNB1, CCR5, IKZF3, STAT1, CD6, BRCA1, CORO1A, TBX21, KLRK1, CXCR6, PTEN, PMEL, DMBT1, IFI44L, LAPTM5, CD226, TNFSF13B, ICOS, CD160, TRIM29, LST1, ZBTB46, VTCN1, KREMEN1, PDCD1LG2, TUBB, CLEC4C, CD86, HAVCR2, GZMH, NFATC1, CD8B, BCL2, GADD45GIP1, CBLB, ITGA1, CD8A, IL2RA, EIF2AK2, MADCAM1, PTPN6, LRG1, ADGRE5, SH2D1B, ITGB2, HLA-DPA1, DGAT2, IGF1R, TAGAP, LMNA, NCAM1, TIGIT, IL17F, HLA-F-AS1, CD247, CD79B, IDO2, IL4, TYROBP, BTLA, AKT1, IL2RG, POLR2A, ITGAX, IL1B, CSF2RB, DDX58, KIAA0101, CD274, LAMP3, TNFAIP8, FOXP3, IL12A, SAMHD1, SIT1, CD3E, ICOSLG, HGF, MELK, IGSF6, GNLY, TDO2, KRT7, HLA-E, HLA-DM, LAMP1, NTN3, CD28, TARP, EGFR, CCR4, MAGEA3, BATF, KLRG1, IRS1, CSF1R, CTLA4, TNFSF18, POU2AF1, GZMA, PIK3CA, ITK, IFI27, EOMES, LCN2, CD80, CD83, CXCL13, MTOR, FCER1G, TFRC, RORC, MMP9, BST2, PIK3CD, FCGR2B, TNFRSF14, OAS3, GRAP2, CCNB2, MLANA, MAGEA12, VCAM1, CDKN3, NCR1, FAS, GZMB, IRF9, IFITM2, TNFSF14, HLA-B, SDHA, NRP1, EBI3, EFNA4, PVR, BUB1, SKAP2, PRF1, CCL20, TNFRSF18, CTSS, NKG7, ISG15, PDCD1, SNAI1, CXCL11, CIITA, IFI35, TNFSF9, TNFSF10, MMP2, EGR3, MAGEA1, CD163, IL6, KLRF1, B3GAT1, C1QA, OAS1, IKZF2, TLR9, KLF2, GUSB, NFKBIA, IL23A, HERC6, SLAMF8, IL15, TLR7, OAS2, HLA-DR, CRTAM, MAGEC2, ICAM1, CD4, MAPK14, C1QB, NOTCH3, NCR3, STAT3, TLR8, CYBB, IKZF4, IFIH1, LCK, BCL2L11, ITGAM, ITGB7, JCHAIN, CD209, SLAMF7, IL10, IL1A, FCGR3A, IFNA17, EGR2, TOP2A, C10orf54, FOXM1, AXL, MS4A1, IFI6, CD3D, GPR18, CD3G, ZAP70, HMBS, IL7, IFIT3, RB1, PTGS2, TGFB1, NCF1, TWIST1, CA4, SELL, LILRB1, CD14, ALOX15B, PECAM1, NOS2, FASLG, CD44, ENTPD1, CMKLR1, CD53, TNF, CXCL8, CD40LG, HLA-F, GATA3, LYZ, ARG1, IL2RB, NECTIN2, MPO, CCR2, BRCA2, ADORA2A, G6PD, TAP1, MX1, HLA-DQB2, CD27, CD276, STAT4, PTPN7, PTPRC, PSMB9, CD244, CXCR4, MAPK1, TP63, IRF4, CCL3, CCL18, IL7R, HLA-DRB1, CEACAM8, CXCL10, CCL2, SRGN, CD19, ITGB1, IFITM1, CCL21, MRC1, PGF, ITGAL, ID2, CD22, CCL17, ITGAE, IL3RA, CCR7, CD1C, MAD2L1, PYGL, CD40, LY9, HLA-G, TLR3, CD48, STAT5A, FCRLA, BCL6, ZEB1, CCL5, IDO1, IL18, TNFRSF9, HIF1A, HLA-DPB1, FOXO1, CD33, S100A9, HLA-DMB, HLA-A, SNAI2, TNFRSF17, LRP1, MAGEA4, HLA-DQA1, CD1D, RPS6, MKI67, GZMK, CD79A, CD37, FUT4, AIF1, CCR1, PRDM1, CD47, CD74, LAG3, TNFRSF4, CD2, CCL4, BAGE, LEXM, CCR6, CD70, CDK1, CTAG1B, CTAG2, CX3CR1, CX3CR1, CX3CR1, CX3CR1, GAGE1, GAGE12I, GAGE12F, GAGE12J, GAGE2C, GAGE2A, GAGE2E, GAGE10, GAGE13, IKZF1, IL17A, IL2, IL21, IL22, KIR2DL2, KIR2DL3, MAGEA10, MIF, PTPRCAP, SSX2, TCF7, XAGE1B, CEACAM8, CXCR3, FCGR1A, FCGR3B, FYB, HLA-C, HLA-DQA2, IFNG, KIR2DL1, KRT5, LMNA, and PTPN11
In some embodiments, amplified target sequences are directed to any one of more of the genes provided in Table 1. In some embodiments, amplified target sequences comprise any one or more amplicon sequences provided in Table 1. In some embodiments, amplified target sequences consist of any one or more amplicon sequences provided in Table 1. In some embodiments, amplified target sequences include each amplicon sequence provided in Table 1.
In some embodiments, compositions comprise any one or more of the immune response target-specific primer pairs provided in Table 2. In some embodiments, compositions comprise all of the immune response target-specific primer pairs provided in Table 2. In some embodiments, any one or more of the immune response target-specific primer pairs provided in Table 2 can be used to amplify a target sequence present in a sample as disclosed by the methods described herein.
In some embodiments, the immune response target-specific primers from Table 2 can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 40, 60, 80, 100, 150, 200, 250, 300, 350, 398 or more, target-specific primer pairs. In some embodiments, the amplified target sequences can include any one or more of the amplified target sequences provided in Table 1 (e.g., using amplicon ID target-specific primers provided in Table 2). In some embodiments, at least one of the target-specific primers associated with cancer is at least 90% identical to at least one nucleic acid sequence selected from SEQ ID NOs: 399-1194. In some embodiments, at least one of the target-specific primers associated with immune response is complementary across its entire length to at least one target sequence in a sample. In some embodiments, at least one of the target-specific primers associated with immune response includes a non-cleavable nucleotide at the 3′ end. In some embodiments, the non-cleavable nucleotide at the 3′ end includes the terminal 3′ nucleotide. In one embodiment, the amplified target sequences are directed to one or more individual exons having aberrant expression associated with cancer. In one embodiment, the amplified target sequences are directed to individual exons having a mutation associated with cancer. In some embodiments, provided methods comprise selective amplification of more than one target sequences in a sample and the detection and/or identification of mutations associated with cancer. In some embodiments, the amplified target sequences include two or more nucleotide sequences provided in Table 2. In some embodiments, the amplified target sequences can include any one or more the amplified target sequences generated using the target-specific primers provided in Table 2. In one embodiment, the amplified target sequences include 10, 50, 100, 150, 200, 250, 300, 350 or more amplicons from Table 1. In some embodiments, methods comprise detection and optionally, the 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 can be associated by one of ordinary skill in the art with, but not limited to, prognosis for the treatment of cancer. In one embodiment, prognosis for the treatment of cancer includes the identification of mutations and/or expression patterns associated with responsiveness or non-responsiveness of a cancer to a drug, drug combination, or treatment regime. In one embodiment, methods comprise amplification of a plurality of target sequences from a population of nucleic acid molecules linked to, or correlated with, the onset, progression or remission of cancer.
In some embodiments, housekeeping genes are comprised in the immune response assay. In certain embodiments, one or more housekeeping gene sequences are included in the assay. In particular embodiments the one or more housekeeping genes are selected from ABCF1, G6PD, GUSB, HMBS, LMNA, LRP1, POLR2A, SDHA, TBP, TFRC, and TUBB.
Provided herein are methods for determining immune response activity in a sample. In some embodiments, the method comprises multiplex amplification of a plurality of target expression sequences from a biological sample, wherein amplifying comprises contacting at least a portion of the sample with a plurality of sets of primer pair reagents directed to the plurality of target sequences, and a polymerase under amplification conditions, to thereby produce amplified target expression sequences. The method further comprises detecting the levels of expression of the target sequences in the sample, wherein a change in the level of expression of one or more immune response markers as compared with a control determines a change in immune response activity in the sample. In some embodiments the target expression sequences of the methods are selected from immune response genes consisting of the following function: checkpoint pathways, T cell related signaling pathways, markers of tumor infiltrating lymphocytes (TILS), tumor markers, and housekeeping genes. In some embodiments, the target genes are selected from immune response genes consisting of the following function: immune checkpoint pathways and targets; T and B cell signaling genes, markers of lymphocyte subsets, interferon signaling genes, cytokine signaling genes; tumor markers, tumor antigens, proliferation markers; and housekeeping genes. In some embodiments, the target genes of the methods are selected from immune response genes consisting of the following function: antigen presentation, antigen processing, innate immune response, leukocyte inhibition, leukocyte migration, lymphocyte activation, lymphocyte development, lymphocyte infiltrate, B cell receptor signaling, T cell receptor signaling, T cell regulation and TCR coexpression. In some embodiments, the target genes of the methods are selected from immune response genes consisting of the following function: chemokine signaling, cytokine signaling, interferon signaling, Type I interferon signaling, and Type II interferon signaling. In some embodiments, the target genes of the methods are selected from immune response genes consisting of the following function of markers: B cell marker, dendritic cell, dendritic cell/macrophage, helper T cell, macrophage, myeloid, neutrophil, NK activation, NK cell, and T cell differentiation. In some embodiments, the target genes of the methods are selected from immune response genes consisting of the following function of marker: checkpoint pathway, PD-signaling, and drug target. In some embodiments, the target genes of the methods are selected from immune response genes consisting of the following function of markers: adhesion/migration, apoptosis, proliferation, tumor antigen, and tumor marker.
In some embodiments, immune response target sequences of the methods are selected from immune response genes consisting of the following function: antigen presentation, antigen processing, innate immune response, leukocyte inhibition, leukocyte migration, lymphocyte activation, lymphocyte development, lymphocyte infiltrate, B cell receptor signaling, T cell receptor signaling, T cell regulation, TCR coexpression, chemokine signaling, cytokine signaling, interferon signaling, Type I interferon signaling, Type II interferon signaling, B cell marker, dendritic cell, dendritic cell/macrophage, helper T cell, macrophage, myeloid, neutrophil, NK activation, NK cell, T cell differentiation, checkpoint pathway, PD-signaling, drug target, adhesion/migration, apoptosis, proliferation, tumor antigen, and tumor marker.
In some embodiments, immune response target sequences of the methods are selected from immune response genes consisting of the following function: antigen presentation, antigen processing, innate immune response, leukocyte inhibition, leukocyte migration, lymphocyte activation, lymphocyte development, lymphocyte infiltrate, B cell receptor signaling, T cell receptor signaling, T cell regulation, TCR coexpression chemokine signaling, cytokine signaling, interferon signaling, Type I interferon signaling, Type II interferon signaling, B cell marker, dendritic cell, dendritic cell/macrophage, helper T cell, macrophage, myeloid, neutrophil, NK activation, NK cell, T cell differentiation, checkpoint pathway, PD-signaling, and drug target. In some embodiments, immune response target sequences of the methods are selected from immune response genes consisting of the following function: antigen presentation, antigen processing, innate immune response, leukocyte inhibition, leukocyte migration, lymphocyte activation, lymphocyte development, lymphocyte infiltrate, B cell receptor signaling, T cell receptor signaling, T cell regulation, TCR coexpression, chemokine signaling, cytokine signaling, interferon signaling, Type I interferon signaling, Type II interferon signaling, B cell marker, dendritic cell, dendritic cell/macrophage, helper T cell, macrophage, myeloid, neutrophil, NK activation, NK cell, T cell differentiation, adhesion/migration, apoptosis, proliferation, tumor antigen, and tumor marker. In some embodiments, immune response target sequences of the methods are selected from immune response genes consisting of the following function: antigen presentation, antigen processing, innate immune response, leukocyte inhibition, leukocyte migration, lymphocyte activation, lymphocyte development, lymphocyte infiltrate, B cell receptor signaling, T cell receptor signaling, T cell regulation, TCR coexpression, checkpoint pathway, PD-signaling, drug target, adhesion/migration, apoptosis, proliferation, tumor antigen, and tumor marker. In some embodiments, immune response target sequences of the methods are selected from immune response genes consisting of the following function: antigen presentation, antigen processing, innate immune response, leukocyte inhibition, leukocyte migration, lymphocyte activation, lymphocyte development, lymphocyte infiltrate, B cell receptor signaling, T cell receptor signaling, T cell regulation, TCR coexpression, chemokine signaling, cytokine signaling, interferon signaling, Type I interferon signaling, Type II interferon signaling, B cell marker, dendritic cell, dendritic cell/macrophage, helper T cell, macrophage, myeloid, neutrophil, NK activation, NK cell, T cell differentiation, checkpoint pathway, PD-signaling, drug target, adhesion/migration, apoptosis, proliferation, tumor antigen, and tumor marker. In some embodiments, immune response target sequences of the methods are selected from immune response genes consisting of the following function: chemokine signaling, cytokine signaling, interferon signaling, Type I interferon signaling, Type II interferon signaling, B cell marker, dendritic cell, dendritic cell/macrophage, helper T cell, macrophage, myeloid, neutrophil, NK activation, NK cell, T cell differentiation, checkpoint pathway, PD-signaling, drug target, adhesion/migration, apoptosis, proliferation, tumor antigen, and tumor marker.
In certain embodiments, target immune response sequences of the methods are directed to sequences having aberrant expression associated with cancer. In some embodiments, the target sequences or amplified target sequences of the methods are directed to sequences having aberrant expression associated with associated with one or more solid tumor cancers selected from the group consisting of head and neck cancers (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 cancer, cervical cancer, endometrial cancer, fallopian cancer), colorectal cancer, gallbladder cancer, esophageal cancer, gastrointestinal cancer, gastric cancer, bladder cancer, prostate cancer, testicular cancer, urothelial 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, bile duct cancer, pituitary tumor, wilms tumor, kaposi sarcoma, hairy cell carcinoma, osteosarcoma, thymus cancer, skin cancer, melanoma, heart cancer, oral and larynx cancer, neuroblastoma, mesothelioma, and other solid tumors (thymic, bone, soft tissue, oral SCC, myelofibrosis, synovial sarcoma). In certain embodiments, the mutations can include any of substitutions, insertions, inversions, point mutations, deletions, mismatches and translocations. In some embodiments, the target sequences or amplified target sequences are directed to sequences having aberrant expression associated with one or more blood/hematologic cancers selected from the group consisting of multiple myeloma, diffuse large B cell lymphoma (DLBCL), lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, follicular lymphoma, leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), myelodisplastic syndrome. In certain embodiments, aberrant expression associated with cancer involves in at least one of the genes provided in Table 1.
In some embodiments, one or more aberrant expression immune response sequences of the methods are at least one of the genes selected from CD63, CD69, CXCL1, KLRD1, HLA-DOB, CXCR5, IL12B, PTK7, CEACAM1, CXCL9, IL13, NT5E, VEGFA, ABCF1, CD38, JAML, S100A8, MYC, IRF1, CCL22, CXCR2, IFIT1, IFIT2, CD68, M6PR, SH2D1A, ISG20, GBP1, TBP, STAT6, ID3, CX3CL1, KLRB1, TNFSF4, CD52, IL10RA, HLA-DOA, IFNB1, CCR5, IKZF3, STAT1, CD6, BRCA1, CORO1A, TBX21, KLRK1, CXCR6, PTEN, PMEL, DMBT1, IFI44L, LAPTM5, CD226, TNFSF13B, ICOS, CD160, TRIM29, LST1, ZBTB46, VTCN1, KREMEN1, PDCD1LG2, TUBB, CLEC4C, CD86, HAVCR2, GZMH, NFATC1, CD8B, BCL2, GADD45GIP1, CBLB, ITGA1, CD8A, IL2RA, EIF2AK2, MADCAM1, PTPN6, LRG1, ADGRE5, SH2D1B, ITGB2, HLA-DPA1, DGAT2, IGF1R, TAGAP, LMNA, NCAM1, TIGIT, IL17F, HLA-F-AS1, CD247, CD79B, IDO2, IL4, TYROBP, BTLA, AKT1, IL2RG, POLR2A, ITGAX, IL1B, CSF2RB, DDX58, KIAA0101, CD274, LAMP3, TNFAIP8, FOXP3, IL12A, SAMHD1, SIT1, CD3E, ICOSLG, HGF, MELK, IGSF6, GNLY, TDO2, KRT7, HLA-E, HLA-DM, LAMP1, NTN3, CD28, TARP, EGFR, CCR4, MAGEA3, BATF, KLRG1, IRS1, CSF1R, CTLA4, TNFSF18, POU2AF1, GZMA, PIK3CA, ITK, IFI27, EOMES, LCN2, CD80, CD83, CXCL13, MTOR, FCER1G, TFRC, RORC, MMP9, BST2, PIK3CD, FCGR2B, TNFRSF14, OAS3, GRAP2, CCNB2, MLANA, MAGEA12, VCAM1, CDKN3, NCR1, FAS, GZMB, IRF9, IFITM2, TNFSF14, HLA-B, SDHA, NRP1, EBI3, EFNA4, PVR, BUB1, SKAP2, PRF1, CCL20, TNFRSF18, CTSS, NKG7, ISG15, PDCD1, SNAI1, CXCL11, CIITA, IFI35, TNFSF9, TNFSF10, MMP2, EGR3, MAGEA1, CD163, IL6, KLRF1, B3GAT1, C1QA, OAS1, IKZF2, TLR9, KLF2, GUSB, NFKBIA, IL23A, HERC6, SLAMF8, IL15, TLR7, OAS2, HLA-DR, CRTAM, MAGEC2, ICAM1, CD4, MAPK14, C1QB, NOTCH3, NCR3, STAT3, TLR8, CYBB, IKZF4, IFIH1, LCK, BCL2L11, ITGAM, ITGB7, JCHAIN, CD209, SLAMF7, IL10, IL1A, FCGR3A, IFNA17, EGR2, TOP2A, C10orf54, FOXM1, AXL, MS4A1, IFI6, CD3D, GPR18, CD3G, ZAP70, HMBS, IL7, IFIT3, RB1, PTGS2, TGFB1, NCF1, TWIST1, CA4, SELL, LILRB1, CD14, ALOX15B, PECAM1, NOS2, FASLG, CD44, ENTPD1, CMKLR1, CD53, TNF, CXCL8, CD40LG, HLA-F, GATA3, LYZ, ARG1, IL2RB, NECTIN2, MPO, CCR2, BRCA2, ADORA2A, G6PD, TAP1, MX1, HLA-DQB2, CD27, CD276, STAT4, PTPN7, PTPRC, PSMB9, CD244, CXCR4, MAPK1, TP63, IRF4, CCL3, CCL18, IL7R, HLA-DRB1, CEACAM8, CXCL10, CCL2, SRGN, CD19, ITGB1, IFITM1, CCL21, MRC1, PGF, ITGAL, ID2, CD22, CCL17, ITGAE, IL3RA, CCR7, CD1C, MAD2L1, PYGL, CD40, LY9, HLA-G, TLR3, CD48, STAT5A, FCRLA, BCL6, ZEB1, CCL5, IDO1, IL18, TNFRSF9, HIF1A, HLA-DPB1, FOXO1, CD33, S100A9, HLA-DMB, HLA-A, SNAI2, TNFRSF17, LRP1, MAGEA4, HLA-DQA1, CD1D, RPS6, MKI67, GZMK, CD79A, CD37, FUT4, AIF1, CCR1, PRDM1, CD47, CD74, LAG3, TNFRSF4, CD2, CCL4, BAGE, LEXM, CCR6, CD70, CDK1, CTAG1B, CTAG2, CX3CR1, CX3CR1, CX3CR1, CX3CR1, GAGE1, GAGE12I, GAGE12F, GAGE12J, GAGE2C, GAGE2A, GAGE2E, GAGE10, GAGE13, IKZF1, IL17A, IL2, IL21, IL22, KIR2DL2, KIR2DL3, MAGEA10, MIF, PTPRCAP, SSX2, TCF7, XAGE1B, CEACAM8, CXCR3, FCGR1A, FCGR3B, FYB, HLA-C, HLA-DQA2, IFNG, KIR2DL1, KRT5, LMNA, and PTPN11. In some embodiments the one or more aberrant expression sequences indicate cancer activity. In some embodiments the one or more aberrant expression sequences indicate a patient's likelihood to response to a therapeutic agent. In some embodiments, the one or more aberrant expression sequences indication a patient's likelihood to not be responsive to a therapeutic agent. In certain embodiments, relevant therapeutic agents can be immunotherapies including but not limited to checkpoint blockades, T cell therapies, and therapeutic vaccines. In some embodiments a therapeutic agent may modify an immune response gene selected from PD1 (e.g., nivolumab, pembrolizumab, AMP-244, MEDI0680, AMP-514, pidlizumab), CTLA4 (e.g., ipilimumab, tremelimumab), PD-L1 (e.g., atezolizumab, MDX1105-01, MEDI4736, avelumab), MR (e.g., lirilumab, NCC0141-0000-0100), CD9/NKG2A (e.g., IPH2201), LAG3 (e.g., BMS986016), GITR (e.g., TRX518), OX40 (e.g., MEDI6383, MEDI6489, MOXR0916), IDO (e.g., indoximod (NLG8189), INCB024360, F001287, NLG919), TGFbeta (e.g., sotaracept, fresolumimab, trabedersen, lucanix), TGFbetaR (e.g., LY2157299, ACE536), CD137 (e.g., urelumab), CD137/41BB (e.g., pf05082566), CD289/TLR9 (e.g., MGN1703), MUC1/CD227 (e.g., OINT-10, ASN-004), CD27 (e.g., varlilumab), CD27L (e.g., AMG172), SLAMF7/CD1 (e.g., elotuzumab), CD20 (e.g., DI-Leu16-IL2), talimogene laherparepvec, CD70 (e.g., ARGX110), IL10 (e.g., AM0010), PSA (e.g., PROSTVAC), GP100 (e.g., MDX1379), STAT3 (e.g., AZD9150), CVAC, IL12 (e.g., veledimex, INXN2001, MSB0010360N, IMMUNOPULSE, GEN-1, INO-9012; IL2: MSB0010445, RG7813/R06895882), IL33 (e.g., alarmin IL33), ICT140, CNDO-109, hTERT (e.g., INO-1400), dysplastic tissues (e.g., ADXS-HPV), SMAC-mimetic (e.g., birinapant), ImmTACs (e.g., IMCgp100), and CD40 (e.g., RO7009789. In certain embodiments, relevant therapeutic agent is an immunotherapy selected from checkpoint blockade, T cell therapy, and therapeutic vaccine. In some embodiments a therapeutic agent modifies an immune response gene selected from any of PD1 (e.g., nivolumab, pembrolizumab, AMP-244, MEDI0680, AMP-514, pidlizumab), CTLA4 (e.g., ipilimumab, tremelimumab), PD-L1 (e.g., atezolizumab, MDX1105-01, MEDI4736, avelumab), MR (e.g., lirilumab, NCC0141-0000-0100), CD9/NKG2A (e.g., IPH2201), LAG3 (e.g., BMS986016), GITR (e.g., TRX518), OX40 (e.g., MEDI6383, MEDI6489, MOXR0916), IDO (e.g., indoximod (NLG8189), INCB024360, F001287, NLG919), TGFbeta (e.g., sotaracept, fresolumimab, trabedersen, lucanix), TGFbetaR (e.g., LY2157299, ACE536), CD137 (e.g., urelumab), CD137/41BB (e.g., pf05082566), CD289/TLR9 (e.g., MGN1703), MUC1/CD227 (e.g., OINT-10, ASN-004), CD27 (e.g., varlilumab), CD27L (e.g., AMG172), SLAMF7/CD1 (e.g., elotuzumab), CD20 (e.g., DI-Leu16-IL2), talimogene laherparepvec, CD70 (e.g., ARGX110), IL10 (e.g., AM0010), PSA (e.g., PROSTVAC), GP100 (e.g., MDX1379), STAT3 (e.g., AZD9150), CVAC, IL12 (e.g., veledimex, INXN2001, MSB0010360N, IMMUNOPULSE, GEN-1, INO-9012; IL2: MSB0010445, RG7813/R06895882), IL33 (e.g., alarmin IL33), ICT140, CNDO-109, hTERT (e.g., INO-1400), dysplastic tissues (e.g., ADXS-HPV), SMAC-mimetic (e.g., birinapant), ImmTACs (e.g., IMCgp100), and CD40 (e.g., RO7009789.
In some embodiments, target sequences or amplified target sequences of the methods are directed to mutations associated with cancer. In some embodiments, the target sequences or amplified target sequences are directed to mutations associated with one or more solid tumor cancers selected from the group consisting of head and neck cancers (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 cancer, cervical cancer, endometrial cancer, fallopian cancer), colorectal cancer, gallbladder cancer, esophageal cancer, gastrointestinal cancer, gastric cancer, bladder cancer, prostate cancer, testicular cancer, urothelial 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, bile duct cancer, pituitary tumor, wilms tumor, kaposi sarcoma, hairy cell carcinoma, osteosarcoma, thymus cancer, skin cancer, melanoma, heart cancer, oral and larynx cancer, neuroblastoma, mesothelioma, and other solid tumors (thymic, bone, soft tissue, oral SCC, myelofibrosis, synovial sarcoma). In one embodiment, the mutations can include substitutions, insertions, inversions, point mutations, deletions, mismatches and translocations. In one embodiment, the mutations can include variation in copy number. In one embodiment, mutations can include germline or somatic mutations. In some embodiments, the target sequences or amplified target sequences are directed to sequences having mutations associated with one or more blood/hematologic cancers selected from the group consisting of multiple myeloma, diffuse large B cell lymphoma (DLBCL), lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, follicular lymphoma, leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), myelodisplastic syndrome. In one embodiment, mutations associated with cancer are located in at least one of the genes provided in Table 1.
In some embodiments, one or more mutations in immune response sequences associated with cancer are located in at least one of the genes selected from CD63, CD69, CXCL1, KLRD1, HLA-DOB, CXCR5, IL12B, PTK7, CEACAM1, CXCL9, IL13, NT5E, VEGFA, ABCF1, CD38, JAML, S100A8, MYC, IRF1, CCL22, CXCR2, IFIT1, IFIT2, CD68, M6PR, SH2D1A, ISG20, GBP1, TBP, STAT6, ID3, CX3CL1, KLRB1, TNFSF4, CD52, IL10RA, HLA-DOA, IFNB1, CCR5, IKZF3, STAT1, CD6, BRCA1, CORO1A, TBX21, KLRK1, CXCR6, PTEN, PMEL, DMBT1, IFI44L, LAPTM5, CD226, TNFSF13B, ICOS, CD160, TRIM29, LST1, ZBTB46, VTCN1, KREMEN1, PDCD1LG2, TUBB, CLEC4C, CD86, HAVCR2, GZMH, NFATC1, CD8B, BCL2, GADD45GIP1, CBLB, ITGA1, CD8A, IL2RA, EIF2AK2, MADCAM1, PTPN6, LRG1, ADGRE5, SH2D1B, ITGB2, HLA-DPA1, DGAT2, IGF1R, TAGAP, LMNA, NCAM1, TIGIT, IL17F, HLA-F-AS1, CD247, CD79B, IDO2, IL4, TYROBP, BTLA, AKT1, IL2RG, POLR2A, ITGAX, IL1B, CSF2RB, DDX58, KIAA0101, CD274, LAMP3, TNFAIP8, FOXP3, IL12A, SAMHD1, SIT1, CD3E, ICOSLG, HGF, MELK, IGSF6, GNLY, TDO2, KRT7, HLA-E, HLA-DM, LAMP1, NTN3, CD28, TARP, EGFR, CCR4, MAGEA3, BATF, KLRG1, IRS1, CSF1R, CTLA4, TNFSF18, POU2AF1, GZMA, PIK3CA, ITK, IFI27, EOMES, LCN2, CD80, CD83, CXCL13, MTOR, FCER1G, TFRC, RORC, MMP9, BST2, PIK3CD, FCGR2B, TNFRSF14, OAS3, GRAP2, CCNB2, MLANA, MAGEA12, VCAM1, CDKN3, NCR1, FAS, GZMB, IRF9, IFITM2, TNFSF14, HLA-B, SDHA, NRP1, EBI3, EFNA4, PVR, BUB1, SKAP2, PRF1, CCL20, TNFRSF18, CTSS, NKG7, ISG15, PDCD1, SNAI1, CXCL11, CIITA, IFI35, TNFSF9, TNFSF10, MMP2, EGR3, MAGEA1, CD163, IL6, KLRF1, B3GAT1, C1QA, OAS1, IKZF2, TLR9, KLF2, GUSB, NFKBIA, IL23A, HERC6, SLAMF8, IL15, TLR7, OAS2, HLA-DR, CRTAM, MAGEC2, ICAM1, CD4, MAPK14, C1QB, NOTCH3, NCR3, STAT3, TLR8, CYBB, IKZF4, IFIH1, LCK, BCL2L11, ITGAM, ITGB7, JCHAIN, CD209, SLAMF7, IL10, IL1A, FCGR3A, IFNA17, EGR2, TOP2A, C10orf54, FOXM1, AXL, MS4A1, IFI6, CD3D, GPR18, CD3G, ZAP70, HMBS, IL7, IFIT3, RB1, PTGS2, TGFB1, NCF1, TWIST1, CA4, SELL, LILRB1, CD14, ALOX15B, PECAM1, NOS2, FASLG, CD44, ENTPD1, CMKLR1, CD53, TNF, CXCL8, CD40LG, HLA-F, GATA3, LYZ, ARG1, IL2RB, NECTIN2, MPO, CCR2, BRCA2, ADORA2A, G6PD, TAP1, MX1, HLA-DQB2, CD27, CD276, STAT4, PTPN7, PTPRC, PSMB9, CD244, CXCR4, MAPK1, TP63, IRF4, CCL3, CCL18, IL7R, HLA-DRB1, CEACAM8, CXCL10, CCL2, SRGN, CD19, ITGB1, IFITM1, CCL21, MRC1, PGF, ITGAL, ID2, CD22, CCL17, ITGAE, IL3RA, CCR7, CD1C, MAD2L1, PYGL, CD40, LY9, HLA-G, TLR3, CD48, STAT5A, FCRLA, BCL6, ZEB1, CCL5, ID01, IL18, TNFRSF9, HIF1A, HLA-DPB1, FOXO1, CD33, S100A9, HLA-DMB, HLA-A, SNAI2, TNFRSF17, LRP1, MAGEA4, HLA-DQA1, CD1D, RPS6, MKI67, GZMK, CD79A, CD37, FUT4, AIF1, CCR1, PRDM1, CD47, CD74, LAG3, TNFRSF4, CD2, CCL4, BAGE, LEXM, CCR6, CD70, CDK1, CTAG1B, CTAG2, CX3CR1, CX3CR1, CX3CR1, CX3CR1, GAGE1, GAGE12I, GAGE12F, GAGE12J, GAGE2C, GAGE2A, GAGE2E, GAGE10, GAGE13, IKZF1, IL17A, IL2, IL21, IL22, KIR2DL2, KIR2DL3, MAGEA10, MIF, PTPRCAP, SSX2, TCF7, XAGE1B, CEACAM8, CXCR3, FCGR1A, FCGR3B, FYB, HLA-C, HLA-DQA2, IFNG, KIR2DL1, KRT5, LMNA, and PTPN11.
In one aspect, compositions and methods of the invention are used to identify biomarkers of response, sensitivity and/or non-response. In some embodiments, provided compositions and methods are used to compare absolute or relative gene expression changes between samples derived from a patients responding to treatment of immunotherapy and samples derived from non-responding patients. Any one or more gene(s) that demonstrate significantly different expression levels (e.g., high in responders group, low in non-responders group) is considered biomarker(s) predictive of drug response. In certain embodiments, such biomarkers of response are used for stratification of patients for immune response therapy. In some embodiments, provided compositions and methods are used to identify biomarkers of immune response monitoring after treatment. In certain embodiments, provided compositions and methods are used to compare absolute or relative gene expression changes correlating with immunohistochemistry readouts of an established clinical makers (e.g., PD-L1, NY-ESO-1, MAGE). In certain embodiments, provided compositions and methods are used to compare absolute or relative gene expression changes correlating with changes in any therapeutic target, pharmacodynamics markers, or immunogenic antigen. Any genes or combination of genes that show correlated up or down expression with these readouts could be considered as predictive markers of drug response. In some embodiments, provided compositions and methods are used to compare absolute or relative gene expression changes correlating with samples containing high level of tumor infiltrating lymphocytes (TIL) and samples containing low level of TIL, as reported by pathology review. Any genes or combination of genes significantly differ in expression between these two groups could be considered predictors of drug response. In some embodiments, provided compositions and methods are used to compare absolute or relative gene expression changes correlating with samples of different molecular subtypes (e.g., examples for colon-rectal cancer include but are not limited to microsatellite instability immune; hypermutated, microsatellite unstable and strong immune activation; epithelial and evident metabolic dysregulation; mesenchymal [http://www.nature.com/nm/journal/vaop/ncurrent/full/nm.3967.html]). Any genes or combination of genes significantly differ in expression between any of these groups could be considered predictors of drug response. In certain embodiments, provided compositions and methods are used to compare absolute or relative gene expression changes correlating with samples of different histopathology subtypes (e.g., examples for non-small cell lung include but are not limited to adenocarcinoma, squamous cell carcinoma, not-otherwise specified; examples for colon rectal). Any genes or combination of genes significantly differ in expression between these two groups could be considered predictors of drug response. In certain embodiments, provided compositions and methods are used to evaluate absolute or relative gene expression levels of different pathways and functions such as the T cell signaling pathway between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. Any genes that show significantly different expression levels (high in one group, low in the other) could be considered potential biomarkers that are predictive of drug response.
In certain embodiments, provided compositions and methods are used to evaluate absolute or relative gene expression levels of different pathways and functions such as the T cell regulation pathway between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. Any genes that show significantly different expression levels (high in one group, low in the other) could be considered potential biomarkers that are predictive of drug response. In certain embodiments, provided compositions and methods are used to evaluate absolute or relative gene expression levels of different pathways and functions such as antigen processing and/or antigen presentation between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. Any genes that show significantly different expression levels (high in one group, low in the other) could be considered potential biomarkers that are predictive of drug response. In some embodiments, provided compositions and methods are used to evaluate absolute or relative gene expression levels of different pathways and functions such as the cytokine signaling pathway between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. Any genes that show significantly different expression levels (high in one group, low in the other) could be considered potential biomarkers that are predictive of drug response. In some embodiments, provided compositions and methods are used to evaluate absolute or relative gene expression levels of different pathways and functions such as type I or type II interferon pathways between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. Any genes that show significantly different expression levels (high in one group, low in the other) could be considered potential biomarkers that are predictive of drug response. In certain embodiments, provided compositions and methods are used to evaluate absolute or relative gene expression levels of different pathways and functions such as lymphocyte development and/or lymphocyte migration between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. Any genes that show significantly different expression levels (high in one group, low in the other) could be considered potential biomarkers that are predictive of drug response. In certain embodiments, provided compositions and methods are used to evaluate absolute or relative gene expression levels of different pathways and functions such as the immune checkpoint pathway between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. Any genes that show significantly different expression levels (high in one group, low in the other) could be considered potential biomarkers that are predictive of drug response. In certain embodiments, provided compositions and methods are used to evaluate the expression level of any genes contained on the panel or combination thereof (e.g., PD-1, PD-L1, CTLA4, etc.) between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. Any genes that show significantly different expression levels (high in one group, low in the other) could be considered potential biomarkers of drug response. In certain embodiments, provided compositions and methods are used to evaluate the expression level of genes or isoforms of antigens (e.g., cancer testis antigens (GAGE), melanoma antigens (MAGE)) between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. Any genes that show significantly different expression levels (high in one group, low in the other) could be considered potential biomarkers of drug response. In some embodiments, provided compositions and methods are used to evaluate different mathematical, quantitative or qualitative transformations of raw read count or normalized read count of any genes contained on the panel or combination thereof (for example, PD-1, PD-L1, CTLA4, etc.) between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. Any genes that show significantly different expression levels (high in one group, low in the other) could be considered potential biomarkers of drug response. In certain embodiments, provided compositions and methods are used to profile the inflammatory level and abundance of different immune subsets (for example: cytotoxic T cells, helper T cells, macrophages, neutrophils, natural killer cells, myeloid derived suppressor cells) in the tumor micro-environment. Any differences in expression levels of these markers between samples derived responders and non-responders of treatment could be considered biomarkers predictive of response. In certain embodiments, provided compositions and methods are used to determine HLA type and its correlation with antigen presentation, T cell and B cell activation and drug response. In certain embodiments, provided compositions and methods are used to classify tumor samples as likely responder or non-responders to a therapeutic agent based on a classifier defined by another assay. In particular embodiments, provided compositions and methods are used to identify a subset of genes from which another panel can be derived to be used for purposes described herein. In a particular embodiment, additional primer sets could be added to the current panel to create an additional panel which can be used for purposes described herein.
In another aspect, provided compositions and methods are used to evaluate the expression levels of genes up or down regulated by immune checkpoint modulators. See, e.g., https://en.wikipedia.org/wiki/Immune_checkpoint. The identity of one or more gene(s) modulated by any specific therapeutic agent could be used to elucidate the mechanism of action of that agent. In some embodiments, provided compositions and methods are used to evaluate the expression levels of genes involved in the immune checkpoint pathway (including, e.g., PD-L1 signaling pathway). The identities of one or more genes modulated by any specific therapeutic agent could be used to elucidate the mechanism of action of that agent. In certain embodiments, provided compositions and methods are used to evaluate the expression levels of genes involved chemokine signaling, cytokine signaling, and/or interferon signaling. The identities of one or more genes modulated by any specific therapeutic agent can be used to elucidate the mechanism of action of that agent. In certain embodiments, provided compositions and methods are used to evaluate the expression levels of genes involved tumor cell adhesion, tumor cell migration, tumor cell proliferation or cancer stemness. The pattern of expression associating with each therapeutic agent could be used to infer the mechanism of action of that agent. In particular embodiments, provided compositions and methods are used to detect post-treatment changes in expression of any one or more genes in the panel. The high or low expression levels of such genes would be used to elucidate the mechanism of action of treated agent. In some embodiments, provided compositions and methods are used to detect post-treatment changes in expression of any intracellular or extracellular protein encoding genes contained on the panel. The high or low expression levels of such genes could be used to elucidate the mechanism of action of that agent. In some embodiments, provided compositions and methods are used to study the changed expression of intracellular protein-encoding genes in response to treatment of therapeutic agent or agents modulating extracellular proteins. High or low expression levels of such genes could be used to elucidate the mechanism of action of such agents. In still additional embodiments, provided compositions and methods are used to study the expression of any genes contained on the panel at different time points or treatment conditions. High or low expression levels of such genes could be used to elucidate the mechanism of action of that agent. In particular embodiments, provided compositions and methods are used to study the expression of any genes contained on the panel at different time points or treatment conditions with different therapeutic agents. The patterns of expression that are considered complementary could provide rationales to combine the corresponding agents for greater benefit. In particular embodiments, provided compositions and methods are used to study the expression of any genes contained on the panel at different time points or treatment conditions with different therapeutic agents. Patterns of expression that are considered complementary could provide rationales to combine the corresponding agents for reduced risk of adverse events. In certain embodiments, provided compositions and methods are used to study the expression of any genes modulated by any radiotherapy, chemotherapy, targeted therapy or immunotherapy. Complementary patterns of expression complementary could provide rationales to combine the corresponding agents for greater benefit or reduced risk of adverse events. In still other embodiments, provided compositions and methods are used to study expression of any genes modulated by a T cell therapy and immunosuppressive therapy. Complementary patterns of expression could provide rationales to combine the corresponding agents for greater benefit or reduced risk of adverse events.
In some embodiments, amplified target sequences of the methods are directed to any one of more of the genes provided in Table 1. In some embodiments, amplified target sequences of the methods comprise any one or more amplicon sequences provided in Table 1. In some embodiments, amplified target sequences of the methods consist of any one or more amplicon sequences provided in Table 1. In some embodiments, amplified target sequences of the methods include each amplicon sequence provided in Table 1.
In some embodiments, compositions used in provided methods comprise any one or more of the immune response target-specific primer pairs provided in Table 2. In some embodiments, compositions used in connection with provided methods comprise all of the immune response target-specific primer pairs provided in Table 2. In some embodiments, any one or more of the immune response target-specific primer pairs provided in Table 2 can be used to amplify a target sequence present in a sample as disclosed by the methods described herein.
In some embodiments, the methods utilize immune response target-specific primers from Table 2 and include 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 40, 60, 80, 100, 150, 200, 250, 300, 350, 398 or more, target-specific primer pairs. In some embodiments, methods include targeting amplified target sequences including one or more of the amplified target sequences generated in Table 1 (e.g., using amplicon target-specific primers provided in Table 2). In some embodiments, methods include use of at least one target-specific primer pair wherein each primer is at least 90% identical to at least one nucleic acid sequence selected from SEQ ID NOs: 399-1194. In some embodiments, methods include use of at least one of the target-specific primers associated with immune response is complementary across its entire length to at least one target sequence in a sample. In some embodiments, methods include use of at least one of the target-specific primers associated with immune response includes a non-cleavable nucleotide at the 3′ end. In some embodiments, methods include use of target specific primers wherein non-cleavable nucleotide at the 3′ end includes the terminal 3′ nucleotide. In one embodiment, methods include use of amplified target sequences directed to one or more individual exons having aberrant expression associated with cancer. In one embodiment, methods include use of amplified target sequences directed to individual exons having a mutation associated with cancer. In some embodiments, provided methods comprise selective amplification of more than one target sequences in a sample and the detection and/or identification of mutations associated with cancer. In some embodiments, methods include use of amplified target sequences including two or more nucleotide sequences provided in Table 2. In some embodiments, methods include use of amplified target sequences including any one or more the amplified target sequences generated using the target-specific primers provided in Table 2. In one embodiment, methods include use of amplified target sequences including 10, 50, 100, 150, 200, 250, 300, 350 or more amplicons from Table 1. In some embodiments, methods comprise detection and optionally, the identification of clinically actionable marker(s). As defined herein, the term “clinically actionable marker” includes clinically actionable mutations and/or clinically actionable expression patterns that are known or can be associated by one of ordinary skill in the art with, but not limited to, prognosis for the treatment of cancer. In one embodiment, prognosis for the treatment of cancer includes the identification of mutations and/or expression patterns associated with responsiveness or non-responsiveness of a cancer to a drug, drug combination, or treatment regime. In one embodiment, methods comprise amplification of a plurality of target sequences from a population of nucleic acid molecules linked to, or correlated with, the onset, progression or remission of cancer.
In some embodiments, provided compositions and methods are used to compare absolute or relative gene expression changes between samples derived from patients with good prognosis (defined by durable response, overall survival or 5-year survival) and those with poor prognosis. For example, gene(s) that show significantly different expression levels (e.g., high in one group, low in the other) could be considered potential prognostic biomarkers. In some embodiments, provided compositions and methods are used to compare absolute or relative gene expression changes correlating with samples containing high level of tumor infiltrating lymphocytes (TIL) and samples containing low level of TIL, as reported by pathology review. For example, gene(s) or combination of genes significantly differ in expression between these two groups could be considered potential prognostic biomarkers. In some embodiments, provided compositions and methods are used to compare absolute or relative gene expression changes correlating with samples of different histopathology subtypes molecular subtypes (examples for breast cancer include luminal A, luminal B, basal-like, HER2 positive). For example, gene(s) or combination of genes that significantly differ in expression between any of these groups could be considered potential prognostic biomarkers. In some embodiments, provided compositions and methods are used to compare absolute or relative gene expression changes correlating with samples of different histopathology subtypes (e.g., for breast cancer include, for example, ER+, PR+, HER2+, triple negative). For example, gene(s) or combination of genes that significantly differ in expression between these two groups could be considered prognostic biomarkers. In some embodiments, provided compositions and methods are used to evaluate absolute or relative gene expression levels of different pathways and functions such as the T cell signaling pathway between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. For example, gene(s) that show significantly different expression levels (e.g., high in one group, low in another) could be considered potential prognostic biomarkers. In some embodiments, provided compositions and methods are used to evaluate gene expression levels of different pathways and functions such as the T cell regulation pathway between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. For example, gene(s) that show significantly different expression levels (high in one group, low in the other) could be considered potential prognostic biomarkers. In some embodiments, provided compositions and methods are used to evaluate gene expression levels of different pathways and functions such as the expression of genes in antigen processing and/or antigen presentation between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. For example, gene(s) that show significantly different expression levels (high in one group, low in the other) could be considered potential prognostic biomarkers. In some embodiments, provided compositions and methods are used to evaluate absolute or relative gene expression levels of different pathways and functions such as the expression of genes in cytokine signaling pathway between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. For example, gene(s) that show significantly different expression levels (high in one group, low in the other) could be considered potential prognostic biomarkers. In some embodiments, provided compositions and methods are used to evaluate absolute or relative gene expression levels of different pathways and functions such as type I or type II interferon pathways between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. For example, gene(s) that show significantly different expression levels (high in one group, low in the other) could be considered potential prognostic biomarkers. In some embodiments, provided compositions and methods are used to evaluate absolute or relative gene expression levels of different pathways and functions such as lymphocyte development and migration between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. For example, gene(s) that show significantly different expression levels (high in one group, low in the other) could be considered potential prognostic biomarkers. In some embodiments, provided compositions and methods are used to evaluate absolute or relative gene expression levels of different pathways and functions such as the immune checkpoint pathway between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. For example, gene(s) that show significantly different expression levels (high in one group, low in the other) could be considered potential prognostic biomarkers. In some embodiments, provided compositions and methods are used to evaluate the expression level of any genes contained on the panel or combination thereof (e.g., for example, PD-1, PD-L1, CTLA4, etc.) between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. For example, gene(s) that show significantly different expression levels (high in one group, low in the other) could be considered potential prognostic biomarkers. In some embodiments, provided compositions and methods are used to evaluate different mathematical, quantitative or qualitative transformations of raw read count or normalized read count of any genes contained on the panel or combination thereof (e.g., for example, PD-1, PD-L1, CTLA4, etc.) between samples derived from patients responding to treatment of immunotherapy and samples derived from non-responders. For example, gene(s) that show significantly different expression levels (high in one group, low in the other) could be considered potential prognostic biomarkers. In some embodiments, provided compositions and methods are used to profile the inflammatory level and presence of different immune subsets (for example: cytotoxic T cells, helper T cells, macrophages, neutrophils, natural killer cells, myeloid derived suppressor cells) in the tumor micro-environment. Any differences in expression levels of these markers between samples derived responder and non-responder of treatment could be considered potential prognostic biomarkers.
In some embodiments, housekeeping genes are utilized in conjunction with provided methods in the immune response assay. In certain embodiments, one or more housekeeping gene sequences are included in the assay methods. In particular method embodiments the one or more housekeeping genes are selected from ABCF1, G6PD, GUSB, HMBS, LMNA, LRP1, POLR2A, SDHA, TBP, TFRC, and TUBB.
In some embodiments, the disclosure provides for amplification of multiple target-specific sequences from a population of expressed nucleic acid molecules. In some embodiments, the method comprises hybridizing one or more target-specific primer pairs to the target sequence, extending a first primer of the primer pair, denaturing the extended first primer product from the population of nucleic acid molecules, hybridizing to the extended first primer product the second primer of the primer pair, extending the second primer to form a double stranded product, and digesting the target-specific primer pair away from the double stranded product to generate a plurality of amplified target sequences. In some embodiments, the digesting includes partial digesting of one or more of the target-specific primers from the amplified target sequence. In some embodiments, the amplified target sequences can be ligated to one or more adapters. In some embodiments, adapters can include one or more DNA barcodes or tagging sequences. In some embodiments, amplified target sequences once ligated to an adapter can undergo a nick translation reaction and/or further amplification to generate a library of adapter-ligated amplified target sequences.
In some embodiments, provided methods comprise preparation and formation of multiple immune response target-specific amplicons. In some embodiments, the method comprises hybridizing one or more target-specific primer pairs to a nucleic acid molecule, extending a first primer of the primer, pair, denaturing the extended first primer from the nucleic acid molecule, hybridizing to the extended first primer product, a second primer of the primer pair and extending the second primer, digesting the target-specific primer pairs to generate a plurality of target-specific amplicons. In some embodiments, adapters can be ligated to the ends of the target-specific amplicons prior to performing a nick translation reaction to generate a plurality of target-specific amplicons suitable for nucleic acid sequencing. In some embodiments, the one or more target specific amplicons can be amplified using bridge amplification or emPCR to generate a plurality of clonal templates suitable for nucleic acid sequencing. In some embodiments, the disclosure provides methods for preparing a target-specific amplicon library, for use in a variety of downstream processes or assays such as nucleic acid sequencing or clonal amplification. In one embodiment, the disclosure provides a method of performing target-specific multiplex PCR on a nucleic acid sample having a plurality of expressed target sequences using primers having a cleavable group.
In certain embodiments, library and/or template preparation to be sequenced can be prepared automatically from a population of nucleic acid samples using the compositions provided herein using an automated systems, e.g., the Ion Chef™ system.
In one embodiment, nucleic acid templates to be sequenced using the Ion Torrent PGM 318™ or Ion Torrent S5 520™ or Ion Torrent S5 530™ system can be prepared from a population of nucleic acid molecules using the target-specific amplification techniques as outlined herein. Optionally, following target-specific amplification a secondary and/or tertiary amplification process including, but not limited to, a library amplification step and/or a clonal amplification step such as emPCR can be performed.
In some embodiments, provided are compositions comprising a plurality of target-specific primer pairs, each containing a forward primer and a reverse primer having at least one cleavable group located at either a) the 3′ end or the 5′ end, and/or b) at about the central nucleotide position of the target-specific primer, and wherein the target-specific primer pairs can be substantially non-complementary to other primer pairs in the composition. In some embodiments, the composition comprises at least 50, 100, 150, 200, 250, 300, 350, 398, or more target-specific primer pairs. In some embodiments, the target-specific primer pairs comprise about 15 nucleotides to about 40 nucleotides in length, wherein at least one nucleotide is replaced with a cleavable group. In some embodiments the cleavable group can be a uridine nucleotide. In some embodiments, the target-specific primer sets are designed to amplify an exon, gene, exome or region of the genome associated with a clinical or pathological condition, e.g., the amplification of one or more expressed sequences associated with cancer, wherein an increase and/or decrease in expression is associated with a change in immune response. In some embodiments, the target-specific primer sets are designed to amplify an exon, gene, exome or region of the genome associated with a change in immune response wherein an increase and/or decrease in expression is indicative of a cancer patient's likelihood to respond to one or more therapeutic agent(s). In some embodiments, the target-specific primer sets are designed to amplify an exon, gene, exome or region of the genome associated with a clinical or pathological condition, e.g., the amplification of one or more mutations associated with cancer. In some embodiments, the target-specific primer pairs when hybridized to a target sequence and amplified as outlined herein can generate a library of adapter-ligated amplified target sequences that are about 100 to about 500 base pairs in length. In some embodiments, no one adapter-ligated amplified target sequence is overexpressed in the library by more than 30% as compared to the remainder of the adapter-ligated amplified target sequences in the library. In some embodiments, the adapter-ligated amplified target sequence library is substantially homogenous with respect to GC content, amplified target sequence length or melting temperature (Tm).
In some embodiments, a kit is provided for performing multiplex PCR comprising a plurality of target-specific primers having a cleavable group, a DNA polymerase, an adapter, dATP, dCTP, dGTP and dTTP. In some embodiments, the cleavable group can be a uracil nucleotide. The kit can further include one or more antibodies, nucleic acid barcodes, purification solutions or columns.
In some embodiments, provided is a kit for generating a target-specific amplicon library comprising a plurality of target-specific primers having a cleavable group, a DNA polymerase, an adapter, dATP, dCTP, dGTP, dTTP, and a cleaving reagent. In some embodiments, the kit further comprises one or more antibodies, nucleic acid barcodes, purification solutions or columns.
In one embodiment, methods are provided for amplification of multiple target-specific sequences from a single nucleic acid source or sample. In another embodiment, methods are provided for target-specific amplification of two or more target sequences from two or more nucleic acid sources, samples or species. For example, it is envisioned by the disclosure that a single nucleic acid sample can include expressed RNA or fixed-formalin paraffin-embedded (FFPE) RNA. It is also envisioned that the sample can 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 (matched) from a single individual such as a tumor sample and normal tissue sample, or genetic material from a single source that contains two distinct forms of genetic material such as maternal and fetal RNA obtained from a maternal subject, or the presence of contaminating bacteria RNA in a sample that contains plant or animal nucleic acid. In some embodiments, the source of nucleic acid material can include nucleic acids obtained from a newborn, for example as typically procured as a blood sample for newborn screening.
The nucleic acid sample can include high molecular weight material such as expressed RNA or cDNA. The sample can include low molecular weight material such as nucleic acid molecules obtained from FFPE or archived RNA samples. In another embodiment, low molecular weight material includes enzymatically or mechanically sheared nucleic acid sample. The sample can include cell-free circulating RNA such as material obtained from a maternal subject. In some embodiments, the sample can include nucleic acid molecules obtained from biopsies, tumors, scrapings, swabs, blood, mucus, urine, plasma, semen, hair, laser capture micro-dissections, surgical resections, and other clinical or laboratory obtained samples. In some embodiments, the sample can include nucleic acid molecules obtained from an animal such as a human or mammalian source. In another embodiment, the sample can include 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 molecules may be an archived or extinct sample or species.
In some embodiments, provided methods comprise selective amplification of at least one target sequence in a normal or diseased containing tissue, biopsy, core, tumor, fine needle aspirate, fine needle biopsy, tumor microenvironment, blood, serum, or other sample. In some embodiments, provided methods comprise selective amplification of at least one target sequence and the detection and/or identification of mutations in the diseased tissue, biopsy, core, tumor, fine needle aspirate, fine needle biopsy, tumor microenvironment, blood, serum, or other sample. In some embodiments, the diseased or normal sample can include whole transcriptomic RNA, formalin-fixed paraffin-embedded tissue (FFPE), sheared or enzymatically treated RNA. In some embodiments, the disclosure is directed to the selective amplification of at least one target sequence and detection and/or identification of clinically actionable mutations or expression patterns. In some embodiments, the disclosure is directed to the detection and/or identification of mutations or expression associated with drug resistance or drug susceptibility.
In some embodiments, provided methods comprise selective amplification of at least one target sequence in cell-free circulating RNA. In some embodiments, the selective amplification of at least one target sequence in a sample includes a mixture of different nucleic acid molecules. The selective amplification can optionally be accompanied by detection and/or identification of mutations observed in circulating RNA. In some embodiments, selective amplification can optionally be accompanied by detection and/or identification of mutations associated with cancer or an inherited disease such as metabolic, neuromuscular, developmental, cardiovascular, autoimmune or other inherited disorder.
In some embodiments, the target-specific primers and primer pairs are target-specific sequences that can amplify specific regions of a nucleic acid molecule. In some embodiments, the target-specific primers can amplify expressed RNA or cDNA. In some embodiments, the target-specific primers can amplify mammalian RNA, such as human RNA or cDNA prepared therefrom. In some embodiments, the amount of RNA required for selective amplification can be from about 1 ng to 1 microgram. In some embodiments, the amount of RNA required for selective amplification of one or more target sequences can be about 1 ng, about 5 ng or about 10 ng. In some embodiments, the amount of RNA required for selective amplification of target sequence is about 10 ng to about ng.
In some embodiments, selective amplification of at least one target sequence further includes nucleic acid sequencing of the amplified target sequence. Optionally, the method further includes detecting and/or identifying mutations present in the sample identified through nucleic acid sequencing of the amplified target sequence.
In one embodiment, a sample containing one or more target sequences can be amplified using any one or more of the target-specific primers disclosed herein. In another embodiment, amplified target sequences obtained using the methods (and associated compositions, systems, apparatuses and kits) disclosed herein, can be coupled to a downstream process, such as but not limited to, nucleic acid sequencing. For example, once the nucleic acid sequence of an amplified target sequence is known, the nucleic acid sequence can be compared to one or more reference samples such as Hg19 genome. The Hg19 genome is commonly used in the genomics field as a reference genome sample for humans.
It is envisaged that one of ordinary skill in the art can readily prepare one or more target-specific primers using the primer criteria disclosed herein without undue experimentation. It is also envisaged that one of ordinary skill in the art can readily prepare one or more target-specific primers using the criteria disclosed herein, to identify at least one medically relevant polymorphism. In some instances, a medically relevant polymorphism can be used in forensic or human identification purposes. A medically relevant mutation includes a mutation that is associated with at least one disease state in multiple populations (e.g., a European Caucasian population). In some embodiments, a medically relevant polymorphism includes any one or more of the polymorphisms outlined below.
In some embodiments, provided are methods (and associated compositions, systems, apparatuses and kits) for reducing the formation of amplification artifacts in a multiplex PCR. In some embodiments, primer-dimers or non-specific amplification products are obtained in lower number or yield as compared to standard multiplex PCR of the prior art. In some embodiments, the reduction in amplification artifacts is in part, governed by the use of target-specific primer pairs in the multiplex PCR reaction. In one embodiment, the number of target-specific primer pairs in the multiplex PCR reaction can be greater than 50, 100, 150, 200, 250, 300 or more. In some embodiments, provided are methods (and associated compositions, systems, apparatuses and kits) for performing multiplex PCR using target-specific primers that contain a cleavable group. In one embodiment, target-specific primers containing a cleavable group can include one or more cleavable moieties per primer of each primer pair. In some embodiments, a target-specific primer containing a cleavable group includes a nucleotide neither normally present in a non-diseased sample nor native to the population of nucleic acids undergoing multiplex PCR. For example, a target-specific primer can include one or more non-native nucleic acid molecules such as, but not limited to thymine dimers, 8-oxo-2′-deoxyguanosine, inosine, deoxyuridine, bromodeoxyuridine, apurinic nucleotides, and the like.
In some embodiments, provided methods (and associated compositions, systems, etc.,) involve performing a primary amplification of target sequences from a population of nucleic acids, optionally using target-specific primers. In some embodiments, provided methods involve amplifying target sequences using target-specific forward and reverse primer pairs. The target-specific forward and reverse primer pairs can optionally include one or more intron-specific and/or exon specific forward and reverse primer pairs. In some embodiments, each primer pair is directed to a single or discrete exon. In some embodiments, provided methods involve amplifying target sequences using exon-specific forward and reverse primer pairs containing at least one cleavable group. In some embodiments, the target-specific forward and reverse primer pairs contain a uracil nucleotide as the one or more cleavable groups. In one embodiment, a target-specific primer pair can include a uracil nucleotide in each of the forward and reverse primers of each primer pair. In one embodiment, a target-specific forward or reverse primer contains one, two, three or more uracil nucleotides. In some embodiments, provided methods involve amplifying at least 10, 50, 100, 150, 200, 250, 300, 350, 398 or more, target sequences from a population of nucleic acids having a plurality of target sequences using target-specific forward and reverse primer pairs containing at least two uracil nucleotides.
In some embodiments, target-specific primers (including but not limited to intron-specific and exon-specific primers, which can be forward and/or reverse primers) can be designed de novo using algorithms that generate oligonucleotide sequences according to specified design criteria. For example, the primers may be selected according to any one or more of criteria specified herein. In some embodiments, one or more of the target-specific primers are selected or designed to satisfy any one or more of the following criteria: (1) inclusion of two or more modified nucleotides within the primer sequence, at least one of which is included near or at the termini of the primer and at least one of which is included at, or about the center nucleotide position of the primer sequence; (2) primer length of about 15 to about 40 bases in length; (3) Tm of from about 60° C. to about 70° C.; (4) low cross-reactivity with non-target sequences present in the target genome or sample of interest; (5) for each primer in a given reaction, the sequence of at least the first four nucleotides (going from 3′ to 5′ direction) are not complementary to any sequence within any other primer present in the same reaction; and (6) no amplicon includes any consecutive stretch of at least 5 nucleotides that is complementary to any sequence within any other amplicon.
In some embodiments, the target-specific primers include one or more primer pairs designed to amplify target sequences from the sample that are about 100 base pairs to about 500 base pairs in length. In some embodiments, the target-specific primers include a plurality of primer pairs designed to amplify target sequences, where the amplified target sequences are predicted to vary in length from each other by no more than 50%, typically no more than 25%, even more typically by no more than 10%, or 5%. For example, if one target-specific primer pair is selected or predicted to amplify a product that is 100 nucleotides in length, then other primer pairs are selected or predicted to amplify products that are between 50-150 nucleotides in length, typically between 75-125 nucleotides in length, even more typically between 90-110 nucleotides, or 95-105 nucleotides, or 99-nucleotides in length.
In some embodiments, at least one primer pair in the amplification reaction is not designed de novo according to any predetermined selection criteria. For example, at least one primer pair can be an oligonucleotide sequence selected or generated at random, or previously selected or generated for other applications. In one exemplary embodiment, the amplification reaction can include at least one primer pair selected from the TaqMan® probe reagents (Roche Molecular Systems). The TaqMan® reagents include labeled probes and can be useful, inter alia, for measuring the amount of target sequence present in the sample, optionally in real time. Some examples of TaqMan technology are disclosed in U.S. Pat. Nos. 5,210,015, 5,487,972, 5,804,375, 6,214,979, 7,141,377 and 7,445,900, hereby incorporated by reference in their entireties.
In some embodiments, at least one primer within the amplification reaction can be labeled, for example with an optically detectable label, to facilitate a particular application of interest. For example, labeling may facilitate quantification of target template and/or amplification product, isolation of the target template and/or amplification, product, and the like.
In some embodiments, one or more of the primers within the amplification reaction can be useful in genotyping of a nucleic acid sample.
In some embodiments, the target-specific primers can be provided as a set of target-specific primer pairs in a single amplification vessel. In some embodiments, the target-specific primers can be provided in one or more aliquots of target-specific primer pairs that can be pooled prior to performing the multiplex PCR reaction in a single amplification vessel or reaction chamber. In one embodiment, the target-specific primers can be provided as a pool of target-specific forward primers and a separate pool of target-specific reverse primers. In another embodiment, target-specific primer pairs can be pooled into subsets such as non-overlapping target-specific primer pairs. In some embodiments, the pool of target-specific primer pairs can be provided in a single reaction chamber or microwell, for example on a PCR plate to perform multiplex PCR using a thermocycler. In some embodiments, the target-specific forward and reverse primer pairs can be substantially complementary to the target sequences.
In some embodiments, the method of performing multiplex PCR amplification includes contacting a plurality of target-specific primer pairs having a forward and reverse primer, with a population of target sequences to form a plurality of template/primer duplexes; adding a DNA polymerase and a mixture of dNTPs to the plurality of template/primer duplexes for sufficient time and at sufficient temperature to extend either (or both) the forward or reverse primer in each target-specific primer pair via template-dependent synthesis thereby generating a plurality of extended primer product/template duplexes; denaturing the extended primer product/template duplexes; annealing to the extended primer product the complementary primer from the target-specific primer pair; and extending the annealed primer in the presence of a DNA polymerase and dNTPs to form a plurality of target-specific double-stranded nucleic acid molecules. In some embodiments, the steps of the amplification PCR method can be performed in any order. In some instances, the methods disclosed herein can be further optimized to remove one or more steps and still obtain sufficient amplified target sequences to be used in a variety of downstream processes. For example, the number of purification or clean-up steps can be modified to include more or less steps than disclose herein, providing the amplified target sequences are generated in sufficient yield.
In some embodiments, the target-specific primer pairs do not contain a common extension (tail) at the 3′ or 5′ end of the primer. In another embodiment, the target-specific primers do not contain a Tag or universal sequence. In some embodiments, the target-specific primer pairs are designed to eliminate or reduce interactions that promote the formation of non-specific amplification.
In one embodiment, the target-specific primer pairs comprise at least one cleavable group per forward and reverse target-specific primer. In one embodiment, the cleavable group can be a uracil nucleotide. In one embodiment, the target-specific primer pairs are partially or substantially removed after generation of the amplified target sequence. In one embodiment, the removal can include enzymatic, heat or alkali treatment of the target-specific primer pairs as part of the amplified target sequence. In some embodiments, the amplified target sequences are further treated to form blunt-ended amplification products, referred to herein as, blunt-ended amplified target sequences.
There is a need for new methods, computer readable media, and systems for identifying or designing products or kits that use PCR to enrich one or more genomic regions of interest (which may be, for example, cumulative regions of 1 kb to 1 Mb) for subsequent sequencing.
There is a need for new methods, computer readable media, and systems for identifying or designing products or kits including primers or assays that maximize coverage of one or more genomic regions or targets of interest while minimizing one or more of off-target hybridization, a number of primers, and a number of primer pools.
In accordance with the teachings and principles embodied in this application, new methods, computer readable media, and systems are provided that identify or design products or kits that use PCR to enrich one or more genomic regions or targets of interest for subsequent sequencing and/or that include primers or assays that maximize coverage of one or more genomic regions or targets of interest while minimizing one or more of off-target hybridization, a number of primers, and a number of primer pools.
According to an exemplary embodiment, there is provided a method comprising: (1) receiving or providing as inputs a genomic target region and a set of candidate amplicons for the genomic target region; (2) generating a graph comprising a source vertex, a set of amplicon vertices arranged in correspondence with the set of candidate amplicons, and a sink vertex; (3) determining a cost associated with one or more paths across the graph from the source vertex to the sink vertex via amplicon vertices; and (4) extracting the amplicon vertices from the one of the one or more paths across the graph having a least cost associated therewith.
In various embodiments, the one or more paths may comprise a sequence of amplicons wherein an ending portion of an insert of a first amplicon in the sequence of amplicons overlaps a beginning portion of an insert of a second amplicon in the sequence of amplicons. An ending portion of an insert of the second amplicon in the sequence of amplicons may overlap a beginning portion of an insert of a third amplicon in the sequence of amplicons. An ending portion of an insert of the third amplicon in the sequence of amplicons may overlap a beginning portion of an insert of a fourth amplicon in the sequence of amplicons.
In various embodiments, the one or more paths may comprise a sequence of N amplicons, N being a positive integer, wherein an ending portion of an insert of an amplicon amp in the sequence of amplicons overlaps a beginning portion of an insert of an amplicon amp+1 in the sequence of amplicons, wherein amp is an integer taking values 1, . . . , N−1. The one or more paths may comprise a sequence of L=N+M amplicons, N and M being positive integers, wherein an ending portion of an insert of an amplicon amp in the sequence of amplicons overlaps (which may include merely touching) a beginning portion of an insert of an amplicon amp+1 in the sequence of amplicons where amp is an integer taking values 1, . . . , N−1; wherein an ending portion of an insert of an amplicon amp in the sequence of amplicons overlaps (which may include merely touching) a beginning portion of an insert of an amplicon amp+1 in the sequence of amplicons where amp is an integer taking values N+1, . . . , N+M−1; and wherein there is a gap between an ending portion of an insert of amplicon amp=N and a beginning portion of an insert of amplicon amp=N+1.
In various embodiments, the cost associated with each of the one or more paths may be a sum of the cost of every edge of the path linking two amplicon vertices. The cost associated with every edge of the path linking two amplicon vertices may be a sum of a first term related to the cost of the edge's destination amplicon vertex and a second term related to the cost of an overlap between an insert of the edge's destination amplicon and an insert of the edge's origin amplicon. The first term and the second term may be weighed by a blending factor such that the first term is multiplied by the blending factor or a function thereof and the second term is multiplied by one minus the blending factor or a function thereof. The cost of an amplicon vertex may be a numerical value along a scale between a first value representing a lower level of one or more undesirable characteristics selected from a group comprising at least a level of off-target amplification and a level of primer-dimer propensity and a second value representing a higher level of the one or more undesirable characteristics. The cost of an overlap between an insert of the edge's destination amplicon and an insert of the edge's origin amplicon may be determined based on a redundancy introduced by overlapping inserts. The cost of an overlap between an insert of the edge's destination amplicon and an insert of the edge's origin amplicon may be a function of a quotient between a number of base pairs in an overlap between the insert of the edge's destination amplicon and the insert of the edge's origin amplicon and a number of base pairs in a union of the insert of the edge's destination amplicon and the insert of the edge's origin amplicon.
According to an exemplary embodiment, there is provided a non-transitory machine-readable storage medium comprising instructions which, when executed by a processor, cause the processor to perform a method comprising: (1) receiving or providing as inputs a genomic target region and a set of candidate amplicons for the genomic target region; (2) generating a graph comprising a source vertex, a set of amplicon vertices arranged in correspondence with the set of candidate amplicons, and a sink vertex; (3) determining a cost associated with one or more paths across the graph from the source vertex to the sink vertex via amplicon vertices; and (4) extracting the amplicon vertices from the one of the one or more paths across the graph having a least cost associated therewith. In some embodiments, such a method may be extended to a method for pooling amplicons across a plurality of pools by using as input the amplicons corresponding to the extracted vertices.
According to an exemplary embodiment, there is provided a system, comprising: (1) a machine-readable memory; and (2) a processor configured to execute machine-readable instructions, which, when executed by the processor, cause the system to perform steps including: (a) receiving or providing as inputs a genomic target region and a set of candidate amplicons for the genomic target region; (b) generating a graph comprising a source vertex, a set of amplicon vertices arranged in correspondence with the set of candidate amplicons, and a sink vertex; (c) determining a cost associated with one or more paths across the graph from the source vertex to the sink vertex via amplicon vertices; and (d) extracting the amplicon vertices from the one of the one or more paths across the graph having a least cost associated therewith. In some embodiments, such a system may be extended to a system for pooling amplicons across a plurality of pools by using as input the amplicons corresponding to the extracted vertices.
According to various exemplary embodiments, one or more features of any one or more of the above-discussed teachings and/or exemplary embodiments may be performed or implemented using appropriately configured and/or programmed hardware and/or software elements. Determining whether an embodiment is implemented using hardware and/or software elements may be based on any number of factors, such as desired computational rate, power levels, heat tolerances, processing cycle budget, input data rates, output data rates, memory resources, data bus speeds, etc., and other design or performance constraints.
Examples of hardware elements may include processors, microprocessors, input(s) and/or output(s) (I/O) device(s) (or peripherals) that are communicatively coupled via a local interface circuit, circuit elements (e.g., transistors, resistors, capacitors, inductors, and so forth), integrated circuits, application specific integrated circuits (ASIC), programmable logic devices (PLD), digital signal processors (DSP), field programmable gate array (FPGA), logic gates, registers, semiconductor device, chips, microchips, chip sets, and so forth. The local interface may include, for example, one or more buses or other wired or wireless connections, controllers, buffers (caches), drivers, repeaters and receivers, etc., to allow appropriate communications between hardware components. A processor is a hardware device for executing software, particularly software stored in memory. The processor can be any custom made or commercially available processor, a central processing unit (CPU), an auxiliary processor among several processors associated with the computer, a semiconductor based microprocessor (e.g., in the form of a microchip or chip set), a macroprocessor, or any device for executing software instructions. A processor can also represent a distributed processing architecture. The I/O devices can include input devices, for example, a keyboard, a mouse, a scanner, a microphone, a touch screen, an interface for various medical devices and/or laboratory instruments, a bar code reader, a stylus, a laser reader, a radio-frequency device reader, etc. Furthermore, the I/O devices also can include output devices, for example, a printer, a bar code printer, a display, etc. Finally, the I/O devices further can include devices that communicate as both inputs and outputs, for example, a modulator/demodulator (modem; for accessing another device, system, or network), a radio frequency (RF) or other transceiver, a telephonic interface, a bridge, a router, etc.
Examples of software may include software components, programs, applications, computer programs, application programs, system programs, machine programs, operating system software, middleware, firmware, software modules, routines, subroutines, functions, methods, procedures, software interfaces, application program interfaces (API), instruction sets, computing code, computer code, code segments, computer code segments, words, values, symbols, or any combination thereof. A software in memory may include one or more separate programs, which may include ordered listings of executable instructions for implementing logical functions. The software in memory may include a system for identifying data streams in accordance with the present teachings and any suitable custom made or commercially available operating system (O/S), which may control the execution of other computer programs such as the system, and provides scheduling, input-output control, file and data management, memory management, communication control, etc.
According to various exemplary embodiments, one or more features of any one or more of the above-discussed teachings and/or exemplary embodiments may be performed or implemented using appropriately configured and/or programmed non-transitory machine-readable medium or article that may store an instruction or a set of instructions that, if executed by a machine, may cause the machine to perform a method and/or operations in accordance with the exemplary embodiments. Such a machine may include, for example, any suitable processing platform, computing platform, computing device, processing device, computing system, processing system, computer, processor, scientific or laboratory instrument, etc., and may be implemented using any suitable combination of hardware and/or software. The machine-readable medium or article may include, for example, any suitable type of memory unit, memory device, memory article, memory medium, storage device, storage article, storage medium and/or storage unit, for example, memory, removable or non-removable media, erasable or non-erasable media, writeable or re-writeable media, digital or analog media, hard disk, floppy disk, read-only memory compact disc (CD-ROM), recordable compact disc (CD-R), rewriteable compact disc (CD-RW), optical disk, magnetic media, magneto-optical media, removable memory cards or disks, various types of Digital Versatile Disc (DVD), a tape, a cassette, etc., including any medium suitable for use in a computer. Memory can include any one or a combination of volatile memory elements (e.g., random access memory (RAM, such as DRAM, SRAM, SDRAM, etc.)) and nonvolatile memory elements (e.g., ROM, EPROM, EEROM, Flash memory, hard drive, tape, CDROM, etc.). Moreover, memory can incorporate electronic, magnetic, optical, and/or other types of storage media. Memory can have a distributed architecture where various components are situated remote from one another, but are still accessed by the processor. The instructions may include any suitable type of code, such as source code, compiled code, interpreted code, executable code, static code, dynamic code, encrypted code, etc., implemented using any suitable high-level, low-level, object-oriented, visual, compiled and/or interpreted programming language.
According to various exemplary embodiments, one or more features of any one or more of the above-discussed teachings and/or exemplary embodiments may be performed or implemented at least partly using a distributed, clustered, remote, or cloud computing resource.
According to various exemplary embodiments, one or more features of any one or more of the above-discussed teachings and/or exemplary embodiments may be performed or implemented using a source program, executable program (object code), script, or any other entity comprising a set of instructions to be performed. When a source program, the program can be translated via a compiler, assembler, interpreter, etc., which may or may not be included within the memory, so as to operate properly in connection with the O/S. The instructions may be written using (a) an object oriented programming language, which has classes of data and methods, or (b) a procedural programming language, which has routines, subroutines, and/or functions, which may include, for example, C, C++, Pascal, Basic, Fortran, Cobol, Perl, Java, and Ada.
According to various exemplary embodiments, one or more of the above-discussed exemplary embodiments may include transmitting, displaying, storing, printing or outputting to a user interface device, a computer readable storage medium, a local computer system or a remote computer system, information related to any information, signal, data, and/or intermediate or final results that may have been generated, accessed, or used by such exemplary embodiments. Such transmitted, displayed, stored, printed or outputted information can take the form of searchable and/or filterable lists of runs and reports, pictures, tables, charts, graphs, spreadsheets, correlations, sequences, and combinations thereof, for example.
Various additional exemplary embodiments may be derived by repeating, adding, or substituting any generically or specifically described features and/or components and/or substances and/or steps and/or operating conditions set forth in one or more of the above-described exemplary embodiments. Further, it should be understood that an order of steps or order for performing certain actions is immaterial so long as the objective of the steps or action remains achievable, unless specifically stated otherwise. Furthermore, two or more steps or actions can be conducted simultaneously so long as the objective of the steps or action remains achievable, unless specifically stated otherwise. Moreover, any one or more feature, component, aspect, step, or other characteristic mentioned in one of the above-discussed exemplary embodiments may be considered to be a potential optional feature, component, aspect, step, or other characteristic of any other of the above-discussed exemplary embodiments so long as the objective of such any other of the above-discussed exemplary embodiments remains achievable, unless specifically stated otherwise.
In some embodiments, the amplified target sequences generated by the methods disclosed herein represent at least 60%, 70%, 80%, 90%, or more, of one or more exons amplified from the plurality of target sequences. In one embodiment, amplified target sequences of the present invention are about 90 to about 140 base pairs in length, about 100 to about 200 base pairs in length, about 100 to about 300 base pairs in length, or about 100 to about 400 base pairs in length. In one embodiment, the amplified target sequence includes the length of the forward primer and the length of the complementary reverse primer for each primer pair. In another embodiment, the amplified target sequence length includes the length of the reverse primer and the length of the complementary forward primer. In some embodiments, the length of the amplified target sequence minus the forward and reverse primer lengths is about 40 base pairs to about 350 base pairs. In some embodiments, the length of the amplified target sequences generated in the multiplex PCR reaction is substantially the same. As defined herein, “substantially the same” with respect to length of amplified target sequences generated via the methods disclosed herein refers to no more than 30% deviation in nucleotide length across the total number of amplified target sequences. In one embodiment, the percent GC content of an amplicon is less than 85%, less than 75%, less than 65%, less than 60%, or less than 50%. In one embodiment, substantially all amplified target sequences within a reaction contain between 30% and less than 85% GC content. In one embodiment, where the nucleic acid molecules are obtained from an archived or FFPE RNA sample, the length of the amplified target sequence is typically about 100 to about 200 base pairs in length. In one embodiment, if the nucleic acid sample is derived or obtained from expressed RNA or cDNA, the length of the amplified target sequence can be about 100 to about 500 base pairs in length.
In some embodiments, the amplified target sequences of provided methods can be used in various downstream analysis or assays with, or without, further purification or manipulation. For example, the amplified target sequences can be clonally amplified by techniques known in the art, such a bridge amplification or emPCR to generate a template library that can be used in next generation sequencing. In some embodiments, the amplified target sequences of provided methods or the resulting template libraries can be used for single nucleotide polymorphism (SNP) analysis, genotyping or epigenetic analysis, copy number variation analysis, gene expression analysis, analysis of gene mutations including but not limited to detection, prognosis and/or diagnosis, detection and analysis of rare or low frequency allele mutations, nucleic acid sequencing including but not limited to de novo sequencing, targeted resequencing and synthetic assembly analysis. In one embodiment, amplified target sequences can be used to detect mutations at less than 5% allele frequency. In some embodiments, the methods disclosed herein can be used to detect mutations in a population of nucleic acids at less than 4%, 3%, 2% or at about 1% allele frequency. In another embodiment, amplified target sequences prepared as described herein can be sequenced to detect and/or identify germline or somatic mutations from a population of nucleic acid molecules.
In some embodiments, the forward and/or reverse target-specific primers in the target-specific primer pairs can be “complementary” or “substantially complementary” to the population of nucleic acid molecules. As termed herein “substantially complementary to the population of nucleic acid molecules” refers to percentage complementarity between the primer and the nucleic acid molecule to which the primer will hybridize. The term “substantially complementary” as used herein refers to at least 70% complementarity. Therefore, substantially complementary refers to a range of complementarity of at least 70% but less than 100% complementarity between the primer and the nucleic acid molecule. A complementary primer is one that possesses 100% complementarity to the nucleic acid molecule. In one embodiment, each target-specific primer pair is designed to minimize cross-hybridization to another primer (or primer pair) in the same multiple PCR reaction (i.e., reduce the prevalence of primer-dimers). In another embodiment, each target-specific primer pair is designed to minimize cross-hybridization to non-specific nucleic acid sequences in the population of nucleic acid molecules (i.e., minimize off-target hybridization). In one embodiment, each target-specific primer is designed to minimize self-complementarity, formation of hairpin structures or other secondary structures.
In some embodiments, the amplified target sequences are formed via polymerase chain reaction. Extension of target-specific primers can be accomplished using one or more DNA polymerases. In one embodiment, the polymerase can be any Family A DNA polymerase (also known as pol I family) or any Family B DNA polymerase. In some embodiments, the DNA polymerase can be a recombinant form capable of extending target-specific primers with superior accuracy and yield as compared to a non-recombinant DNA polymerase. For example, the polymerase can include a high-fidelity polymerase or thermostable polymerase. In some embodiments, conditions for extension of target-specific primers can include ‘Hot Start’ conditions, for example Hot Start polymerases, such as Amplitaq Gold® DNA polymerase (Applied Biosciences), Platinum® Taq DNA Polymerase High Fidelity (Invitrogen) or KOD Hot Start DNA polymerase (EMD Biosciences). A ‘Hot Start’ polymerase includes a thermostable polymerase and one or more antibodies that inhibit DNA polymerase and 3′-5′ exonuclease activities at ambient temperature. In some instances, ‘Hot Start’ conditions can include an aptamer.
In some embodiments, the polymerase can be an enzyme such as Taq polymerase (from Thermus aquaticus), Tfi polymerase (from Thermus filiformis), Bst polymerase (from Bacillus stearothermophilus), Pfu polymerase (from Pyrococcus furiosus), Tth polymerase (from Thermus thermophilus), Pow polymerase (from Pyrococcus woesei), Tli polymerase (from Thermococcus litoralis), Ultima polymerase (from Thermotoga maritima), KOD polymerase (from Thermococcus kodakaraensis), Pol I and II polymerases (from Pyrococcus abyssi) and Pab (from Pyrococcus abyssi). In some embodiments, the DNA polymerase can include at least one polymerase such as Amplitaq Gold® DNA polymerase (Applied Biosciences), Stoffel fragment of Amplitaq® DNA Polymerase (Roche), KOD polymerase (EMD Biosciences), KOD Hot Start polymerase (EMD Biosciences), Deep Vent™ DNA polymerase (New England Biolabs), Phusion polymerase (New England Biolabs), Klentaql polymerase (DNA Polymerase Technology, Inc), Klentaq Long Accuracy polymerase (DNA Polymerase Technology, Inc), Omni KlenTaq™ DNA polymerase (DNA Polymerase Technology, Inc), Omni KlenTaq™ LA DNA polymerase (DNA Polymerase Technology, Inc), Platinum® Taq DNA Polymerase (Invitrogen), Hemo Klentaq™ (New England Biolabs), Platinum® Taq DNA Polymerase High Fidelity (Invitrogen), Platinum® Pfx (Invitrogen), Accuprime™ Pfx (Invitrogen), or Accuprime™ Taq DNA Polymerase High Fidelity (Invitrogen).
In some embodiments, the DNA polymerase can be a thermostable DNA polymerase. In some embodiments, the mixture of dNTPs can be applied concurrently, or sequentially, in a random or defined order. In some embodiments, the amount of DNA polymerase present in the multiplex reaction is significantly higher than the amount of DNA polymerase used in a corresponding single plex PCR reaction. As defined herein, the term “significantly higher” refers to an at least 3-fold greater concentration of DNA polymerase present in the multiplex PCR reaction as compared to a corresponding single plex PCR reaction.
In some embodiments, the amplification reaction does not include a circularization of amplification product, for example as disclosed by rolling circle amplification.
In some embodiments, the methods of the disclosure include selectively amplifying target sequences in a sample containing a plurality of nucleic acid molecules and ligating the amplified target sequences to at least one adapter and/or barcode. Adapters and barcodes for use in molecular biology library preparation techniques are well known to those of skill in the art. The definitions of adapters and barcodes as used herein are consistent with the terms used in the art. For example, the use of barcodes allows for the detection and analysis of multiple samples, sources, tissues or populations of nucleic acid molecules per multiplex reaction. A barcoded and amplified target sequence contains a unique nucleic acid sequence, typically a short 6-15 nucleotide sequence, that identifies and distinguishes one amplified nucleic acid molecule from another amplified nucleic acid molecule, even when both nucleic acid molecules minus the barcode contain the same nucleic acid sequence. The use of adapters allows for the amplification of each amplified nucleic acid molecule in a uniformed manner and helps reduce strand bias. Adapters can include universal adapters or propriety adapters both of which can be used downstream to perform one or more distinct functions. For example, amplified target sequences prepared by the methods disclosed herein can be ligated to an adapter that may be used downstream as a platform for clonal amplification. The adapter can function as a template strand for subsequent amplification using a second set of primers and therefore allows universal amplification of the adapter-ligated amplified target sequence. In some embodiments, selective amplification of target nucleic acids to generate a pool of amplicons can further comprise ligating one or more barcodes and/or adapters to an amplified target sequence. The ability to incorporate barcodes enhances sample throughput and allows for analysis of multiple samples or sources of material concurrently. In one example, amplified target nucleic acid molecules prepared by provided methods can be ligated to Ion Torrent™ Sequencing Adapters (A and P1 adapters, sold as a component of the Ion Fragment Library Kit, Life Technologies, Part No. 4466464) or Ion Torrent™ DNA Barcodes (Life Technologies, Part No. 4468654).
The methods disclosed herein are directed to the amplification of multiple target sequences via polymerase chain reaction (PCR). In some embodiments the multiplex PCR comprises hybridizing one or more target-specific primer pairs to a nucleic acid molecule, extending the primers of the target-specific primer pairs via template dependent synthesis in the presence of a DNA polymerase and dNTPs; repeating the hybridization and extension steps for sufficient time and sufficient temperature there generating a plurality of amplified target sequences. In some embodiments, the steps of the multiplex amplification reaction method can be performed in any order.
The amount of nucleic acid material required for successful multiplex amplification can be about 1 ng. In some embodiments, the amount of nucleic acid material can be about 10 ng to about 50 ng, about 10 ng to about 100 ng, or about 1 ng to about 200 ng of nucleic acid material. Higher amounts of input material can be used, however one aspect of the disclosure is to selectively amplify a plurality of target sequence from a low (ng) about of starting material.
The multiplex PCR amplification reactions disclosed herein can include a plurality of “cycles” typically performed on a thermocycler. Each cycle includes at least one annealing step and at least one extension step. In one embodiment, a multiplex PCR amplification reaction is performed wherein target-specific primer pairs are hybridized to a target sequence; the hybridized primers are extended generating an extended primer product/nucleic acid duplex; the extended primer product/nucleic acid duplex is denatured allowing the complementary primer to hybridize to the extended primer product, wherein the complementary primer is extended to generate a plurality of amplified target sequences. In one embodiment, the methods disclosed herein have about 5 to about cycles per preamplification reaction. The annealing temperature and/or annealing duration per cycle can be identical; can include incremental increases or decreases, or a combination of both. The extension temperature and/or extension duration per cycle can be identical; can include incremental increases or decreases, or a combination of both. For example, the annealing temperature or extension temperature can remain constant per cycle. In some embodiments, the annealing temperature can remain constant each cycle and the extension duration can incrementally increase per cycle. In some embodiments, increases or decreases in duration can occur in 15 second, 30 second, 1 minute, 2 minute or 4 minute increments. In some embodiments, increases or decrease in temperature can occur as 0.5, 1, 2, 3, or 4 Celsius deviations. In some embodiments, the amplification reaction can be conducted using hot-start PCR techniques. These techniques include the use of a heating step (>60° C.) before polymerization begins to reduce the formation of undesired PCR products. Other techniques such as the reversible inactivation or physical separation of one or more critical reagents of the reaction, for example the magnesium or DNA polymerase can be sequestered in a wax bead, which melts as the reaction is heated during the denaturation step, releasing the reagent only at higher temperatures. The DNA polymerase can also be kept in an active state by binding to an aptamer or an antibody. This binding is disrupted at higher temperatures, releasing the functional DNA polymerase that can proceed with the PCR unhindered.
In some embodiments, the disclosed methods can optionally include destroying one or more primer-containing amplification artifacts, e.g., primer-dimers, dimer-dimers or superamplicons. In some embodiments, the destroying can optionally include treating the primer and/or amplification product so as to cleave specific cleavable groups present in the primer and/or amplification product. In some embodiments, the treating can include partial or complete digestion of one or more target-specific primers. In one embodiment, the treating can include removing at least 40% of the target specific primer from the amplification product. The cleavable treatment can include enzymatic, acid, alkali, thermal, photo or chemical activity. The cleavable treatment can result in the cleavage or other destruction of the linkages between one or more nucleotides of the primer, or between one or more nucleotides of the amplification product. The primer and/or the amplification product can optionally include one or more modified nucleotides or nucleobases. In some embodiments, the cleavage can selectively occur at these sites, or adjacent to the modified nucleotides or nucleobases. In some embodiments, the cleavage or treatment of the amplified target sequence can result in the formation of a phosphorylated amplified target sequence. In some embodiments, the amplified target sequence is phosphorylated at the 5′ terminus.
In some embodiments, the template, primer and/or amplification product includes nucleotides or nucleobases that can be recognized by specific enzymes. In some embodiments, the nucleotides or nucleobases can be bound by specific enzymes. Optionally, the specific enzymes can also cleave the template, primer and/or amplification product at one or more sites. In some embodiments, such cleavage can occur at specific nucleotides within the template, primer and/or amplification product. For example, the template, primer and/or amplification product can include one or more nucleotides or nucleobases including uracil, which can be recognized and/or cleaved by enzymes such as uracil DNA glycosylase (UDG, also referred to as UNG) or formamidopyrimidine DNA glycosylase (Fpg). The template, primer and/or amplification product can include one or more nucleotides or nucleobases including RNA-specific bases, which can be recognized and/or cleaved by enzymes such as RNAseH. In some embodiments, the template, primer and/or amplification product can include one or more abasic sites, which can be recognized and/or cleaved using various proofreading polymerases or apyrase treatments. In some embodiments, the template, primer and/or amplification product can include 7,8-dihydro-8-oxoguanine (8-oxoG) nucleobases, which can be recognized or cleaved by enzymes such as Fpg. In some embodiments, one or more amplified target sequences can be partially digested by a FuPa reagent.
In some embodiments, the primer and/or amplification product includes one or more modified nucleotides including bases that bind, e.g., base pair, with other nucleotides, for example nucleotides in a complementary nucleic acid strand, via chemical linkages. In some embodiments, the chemical linkages are subject to specific chemical attack that selectively cleaves the modified nucleotides (or selectively cleaves one or more covalent linkages between the modified nucleotides and adjacent nucleotides within the primer and/or amplification product) but leaves the other nucleotides unaffected. For example, in some embodiments modified nucleotides can form disulfite linkages with other nucleotides in a complementary strand. Such disulfite linkages can be oxidized via suitable treatments. Similarly, certain modified nucleotides can base pair with other nucleotides in a complementary nucleic acid strand through linkages that can be selectively disrupted via alkali treatment. In some embodiments, the primer and/or amplification product includes one or more modified nucleotides that bind, e.g., base pair, with other nucleotides in a complementary nucleic acid strand through linkages exhibiting decreased thermal stability relative to typical base pairing linkages formed between natural bases. Such reduced-thermal stability linkages can be selectively disrupted through exposure of the primer and/or amplification product to elevated temperatures following amplification.
In an exemplary embodiment amplification primers are bisulfite in design, with either a 5′ universal forward amplification sequence linked to a 3′ target-specific forward primer, or a 5′ universal reverse amplification sequence linked to a 3′ target-specific reverse primer. Both primers contain modified nucleotides.
In some embodiments, primers are synthesized that are complementary to, and can hybridize with, discrete segments of a nucleic acid template strand, including: a primer that can hybridize to the 5′ region of the template, which encompasses a sequence that is complementary to either the forward or reverse amplification primer. In some embodiments, the forward primers, reverse primers, or both, share no common nucleic acid sequence, such that they hybridize to distinct nucleic acid sequences. For example, target-specific forward and reverse primers can be prepared that do not compete with other primer pairs within the primer pool to amplify the same nucleic acid sequence. In this example, primer pairs that do not compete with other primer pairs in the primer pool assist in the reduction of non-specific or spurious amplification products. In some embodiments, the forward and reverse primers of each primer pair are unique, in that the nucleotide sequence for each primer is non-complementary and non-identical to the other primer in the primer pair. In some embodiments, the primer pair can differ by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% nucleotide identity. In some embodiments, the forward and reverse primers in each primer pair are non-complementary or non-identical to other primer pairs in the primer pool or multiplex reaction. For example, the primer pairs within a primer pool or multiplex reaction can differ by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% nucleotide identity to other primer pairs within the primer pool or multiplex reaction. Primers are designed to minimize the formation of primer-dimers, dimer-dimers or other non-specific amplification products. Typically, primers are optimized to reduce GC bias and low melting temperatures (Tm) during the amplification reaction. In some embodiments, the primers are designed to possess a Tm of about 55° C. to about 72° C. In some embodiments, the primers of a primer pool can possess a Tm of about 59° C. to about 70° C., 60° C. to about 68° C., or 60° C. to about 65° C. In some embodiments, the primer pool can possess a Tm that does not deviate by more than 5° C.
In some embodiments, the target-specific primers do not contain a carbon-spacer or terminal linker. In some embodiments, the target-specific primers or amplified target sequences do not contain an enzymatic, magnetic, optical or fluorescent label.
The template can include a 3′ region that contains the sequence for either the upstream or downstream regions surrounding a particular gene or region of interest, such that the region of interest is bracketed by a forward amplification/upstream gene-specific fusion, and a reverse amplification/downstream region of interest fusion primer. In some embodiments, an internal separator sequence can separate the template regions that can hybridize to the amplification and gene-specific primers, and this may act as a key or barcode for subsequent downstream applications such as sequencing, etc. In some embodiments, a barcode or key can be incorporated into each of the amplification products to assist with data analysis and for example, cataloging. In some embodiments, the barcodes can be Ion Torrent™ DNA barcodes (Life Technologies).
In some embodiments, the primer includes a sufficient number of modified nucleotides to allow functionally complete degradation of the primer by the cleavage treatment, but not so many as to interfere with the primer's specificity or functionality prior to such cleavage treatment, for example in the amplification reaction. In some embodiments, the primer includes at least one modified nucleotide, but no greater than 75% of nucleotides of the primer are modified.
In some embodiments, multiple different primers including at least one modified nucleotide can be used in a single amplification reaction. For example, multiplexed primers including modified nucleotides can be added to the amplification reaction mixture, where each primer (or set of primers) selectively hybridizes to, and promotes amplification of different target nucleic acid molecules within the nucleic acid population. In some embodiments, different primer combinations can be added to the amplification reaction at plexy of at least about about 12-plex, 24-plex, 48-plex, 74-plex, 96-plex, 120-plex, 144-plex, 168-plex, 192-plex, 216-plex, 240-plex, 264-plex, 288-plex, 312-plex, 336-plex, 360-plex, 384-plex, or 398-plex. In some embodiments, the modified primers contain at least one modified nucleotide near or at the termini of the primer. In some embodiments, the modified primers contain two or more modified nucleotides within the primer sequence. In an exemplary embodiment, the primer sequence contains a uracil near, or at, the termini of the primer sequence. For the purposes of this disclosure “near” or “at the termini” of the primer sequences refers up to 10 nucleotides from the termini of the primer sequence. In some embodiments, the primer sequence contains a uracil located at, or about, the center nucleotide position of the primer sequence. For the purposes of this disclosure “at, or about the center nucleotide position of the primer sequence” refers to the incorporation of a uracil moiety at the center nucleotide of the primer sequence or within eight nucleotides, in either a 3′ or 5′ direction flanking the center nucleotide. In one embodiment, the target-specific primer sequence can contain a modified nucleobase at or about the center nucleotide position and contain a modified nucleobase at the 3′ and/or 5′ terminus. In some embodiments, the length of the forward or reverse primer sequence can be about 15 to about 40 bases in length. In some embodiments, the Tm of the primer sequence used in the multiplex reaction can be about 55° C. to about 72° C. In some embodiments, the primer pairs are designed to amplify sequences from the target nucleic acid molecules or amplicons that are about 100 base pairs to about 500 base pairs in length.
In some embodiments, the amplification reactions are conducted in parallel within a single reaction phase (for example, within the same amplification reaction mixture within a single tube). In some instances, an amplification reaction can generate a mixture of products including both the intended amplicon product as well as unintended, unwanted, nonspecific amplification artifacts such as primer-dimers. Post amplification, the reactions are then treated with any suitable agent that will selectively cleave or otherwise selectively destroy the nucleotide linkages of the modified nucleotides within the excess unincorporated primers and the amplification artifacts without cleaving or destroying the specification amplification products. For example, the primers can include uracil-containing nucleobases that can be selectively cleaved using UNG/UDG (optionally with heat and/or alkali). In some embodiments, the primers can include uracil-containing nucleotides that can be selectively cleaved using UNG and Fpg. In some embodiments, the cleavage treatment includes exposure to oxidizing conditions for selective cleavage of dithiols, treatment with RNAseH for selective cleavage of modified nucleotides including RNA-specific moieties (e.g., ribose sugars, etc.), and the like. This cleavage treatment can effectively fragment the original amplification primers and non-specific amplification products into small nucleic acid fragments that include relatively few nucleotides each. Such fragments are typically incapable of promoting further amplification at elevated temperatures. Such fragments can also be removed relatively easily from the reaction pool through the various post-amplification cleanup procedures known in the art (e.g., spin columns, NaEtOH precipitation, etc).
In some embodiments, amplification products following cleavage or other selective destruction of the nucleotide linkages of the modified nucleotides are optionally treated to generate amplification products that possess a phosphate at the 5′ termini. In some embodiments, the phosphorylation treatment includes enzymatic manipulation to produce 5′ phosphorylated amplification products. In one embodiment, enzymes such as polymerases can be used to generate 5′ phosphorylated amplification products. For example, T4 polymerase can be used to prepare 5′ phosphorylated amplicon products. Klenow can be used in conjunction with one or more other enzymes to produce amplification products with a 5′ phosphate. In some embodiments, other enzymes known in the art can be used to prepare amplification products with a 5′ phosphate group. For example, incubation of uracil nucleotide containing amplification products with the enzyme UDG, Fpg and T4 polymerase can be used to generate amplification products with a phosphate at the 5′ termini. It will be apparent to one of skill in the art that other techniques, other than those specifically described herein, can be applied to generate phosphorylated amplicons. It is understood that such variations and modifications that are applied to practice the methods, systems, kits, compositions and apparatuses disclosed herein, without resorting to undue experimentation are considered within the scope of the disclosure.
In some embodiments, primers that are incorporated in the intended (specific) amplification products, these primers are similarly cleaved or destroyed, resulting in the formation of “sticky ends” (e.g., 5′ or 3′ overhangs) within the specific amplification products. Such “sticky ends” can be addressed in several ways. For example, if the specific amplification products are to be cloned, the overhang regions can be designed to complement overhangs introduced into the cloning vector, thereby enabling sticky ended ligations that are more rapid and efficient than blunt ended ligations. Alternatively, the overhangs may need to be repaired (as with several next-generation sequencing methods). Such repair can be accomplished either through secondary amplification reactions using only forward and reverse amplification primers (e.g., correspond to A and P1 primers) comprised of only natural bases. In this manner, subsequent rounds of amplification rebuild the double-stranded templates, with nascent copies of the amplicon possessing the complete sequence of the original strands prior to primer destruction. Alternatively, the sticky ends can be removed using some forms of fill-in and ligation processing, wherein the forward and reverse primers are annealed to the templates. A polymerase can then be employed to extend the primers, and then a ligase, optionally a thermostable ligase, can be utilized to connect the resulting nucleic acid strands. This could obviously be also accomplished through various other reaction pathways, such as cyclical extend-ligation, etc. In some embodiments, the ligation step can be performed using one or more DNA ligases.
In some embodiments, provided are methods for single-tube multiplex PCR. In some embodiments, the method for single-tube multiplex PCR can include target-specific or exon-specific primers. In some embodiments, the exon-specific or target-specific primers can include at least one uracil nucleotide. In some embodiments, single-tube multiplex PCR can include selective amplification of at least 10, 50, 100, 150, 200, 250, 300, 350, 398 or more target nucleic acid molecules using target-specific or exon-specific uracil based primers.
In some embodiments, provided are methods for generating a target-specific or exon-specific amplicon library. In some embodiments, the amplicon library generated using target-specific or exon-specific primers can be associated with aberrant expression in human cancers. In some embodiments, the amplicon library generated using target-specific or exon-specific primers can be associated with mutations of human cancers. In some embodiments, the amplicon library can be generated from expressed RNA or cDNA or formalin-fixed, paraffin-embedded (FFPE) tissue. In some embodiments, the amplicons of the amplicon library prepared using the methods disclosed herein can be about 100 to about 300 base pairs in length, about 100 to about 250 base pairs in length, about 120 to about 220 base pairs in length or about 135 to about 205 base pairs in length. In some embodiments, the amplicon library can be prepared using primer pairs that are targeted to cancer specific mutations. In some embodiments, the primer pairs can be used to generate amplicons that once sequenced by any sequencing platform, including semi-conductor sequencing technology can be used to detect genetic mutations such as inversion, deletions, point mutations and variations in copy number.
In some embodiments, the primer pairs used to produce an amplicon library can result in the amplification of target-specific nucleic acid molecules possessing one or more of the following metrics: greater than 97% target coverage at 20× if normalized to 100× average coverage depth; greater than 97% of bases with greater than 0.2× mean; greater than 90% base without strand bias; greater than 95% of all reads on target; greater than 99% of bases with greater than 0.01× mean; and greater than 99.5% per base accuracy.
In some embodiments, the amplicon library can be used to detect and/or identify known mutations or de novo mutations in a sample.
In some embodiments, the amplicon library prepared using target—specific primer pairs can be used in downstream enrichment applications such as emulsion PCR or bridge PCR. In some embodiments, the amplicon library can be used in an enrichment application and a sequencing application. For example, an amplicon library can be sequenced using any suitable DNA sequencing platform. In some embodiments, an amplicon library can be sequenced using an Ion Torrent PGM Sequencer (Life Technologies). In some embodiments, a PGM sequencer can be coupled to server that applies parameters or software to determine the sequence of the amplified target nucleic acid molecules. In some embodiments, the amplicon library can be prepared, enriched and sequenced in less than 24 hours. In some embodiments, the amplicon library can be prepared, enriched and sequenced in approximately 9 hours. In some embodiments, an amplicon library can be a paired library, that is, a library that contains amplicons from a tumor sample and amplicons from a non-diseased sample. Each pair can be aligned, to detect and/or identify mutations present in the target nucleic acid molecules.
In some embodiments, methods for generating an amplicon library can include:
-
- amplifying RNA expression or cDNA targets using exon-specific or target-specific primers to generate amplicons; purifying the amplicons from the input RNA or cDNA and primers;
phosphorylating the amplicons; ligating adapters to the phosphorylated amplicons; purifying the ligated amplicons; nick-translating the amplified amplicons; and purifying the nick-translated amplicons to generate the amplicon library. In some embodiments, additional amplicon library manipulations can be conducted following the step of amplification of RNA expression targets to generate the amplicons. In some embodiments, any combination of additional reactions can be conducted in any order, and can include: purifying; phosphorylating; ligating adapters; nick-translating; amplification and/or sequencing. In some embodiments, any of these reactions can be omitted or can be repeated. It will be readily apparent to one of skill in the art that the method can repeat or omit any one or more of the above steps. It will also be apparent to one of skill in the art that the order and combination of steps may be modified to generate the required amplicon library, and is not therefore limited to the exemplary methods provided.
A phosphorylated amplicon can be joined to an adapter to conduct a nick translation reaction, subsequent downstream amplification (e.g., template preparation), or for attachment to particles (e.g., beads), or both. For example, an adapter that is joined to a phosphorylated amplicon can anneal to an oligonucleotide capture primer which is attached to a particle, and a primer extension reaction can be conducted to generate a complementary copy of the amplicon attached to the particle or surface, thereby attaching an amplicon to a surface or particle. Adapters can have one or more amplification primer hybridization sites, sequencing primer hybridization sites, barcode sequences, and combinations thereof. In some embodiments, amplicons prepared by the methods disclosed herein can be joined to one or more Ion Torrent™ compatible adapters to construct an amplicon library. Amplicons generated by such methods can be joined to one or more adapters for library construction to be compatible with a next generation sequencing platform. For example, the amplicons produced by the teachings of the present disclosure can be attached to adapters provided in the Ion Fragment Library Kit (Life Technologies, Catalog No. 4466464).
In some embodiments, amplification of expressed RNA targets or FFPE samples can be conducted using a 2× AmpliSeq Hi Fi Master Mix. In some embodiments, the AmpliSeq Hi Fi Master Mix can include glycerol, dNTPs, and a DNA polymerase, such as Platinum® Taq DNA polymerase High Fidelity. In some embodiments, the 2× AmpliSeq Hi Fi Master Mix can further include at least one of the following: a preservative, magnesium sulfate, tris-sulfate and/or ammonium sulfate.
In some embodiments, amplification of expressed RNA targets or FFPE samples can be conducted using a 5× Ion AmpliSeq Hi Fi Master Mix. In some embodiments, the 5× Ion AmpliSeq Hi Fi Master Mix can include glycerol, dNTPs, and a DNA polymerase such as Platinum® Taq DNA polymerase High Fidelity. In some embodiments, the 5× Ion AmpliSeq Hi Fi Master Mix can further include at least one of the following: a preservative, magnesium chloride, magnesium sulfate, tris-sulfate and/or ammonium sulfate.
In some embodiments, phosphorylation of the amplicons can be conducted using a FuPa reagent. In some embodiments, the FuPa reagent can include a DNA polymerase, a DNA ligase, at least one uracil cleaving or modifying enzyme, and/or a storage buffer. In some embodiments, the FuP reagent can further include at least one of the following: a preservative and/or a detergent.
In some embodiments, phosphorylation of the amplicons can be conducted using a FuPa reagent. In some embodiments, the FuPa reagent can include a DNA polymerase, at least one uracil cleaving or modifying enzyme, an antibody and/or a storage buffer. In some embodiments, the FuPa reagent can further include at least one of the following: a preservative and/or a detergent. In some embodiments, the antibody is provided to inhibit the DNA polymerase and 3′-5′ exonuclease activities at ambient temperature.
In some embodiments, the amplicon library produced by the teachings of the present disclosure are sufficient in yield to be used in a variety of downstream applications including the Ion Xpress™ Template Kit using an Ion Torrent™ PGM system (e.g., PCR-mediated addition of the nucleic acid fragment library onto Ion Sphere™ Particles)(Life Technologies, Part No. 4467389). For example, instructions to prepare a template library from the amplicon library can be found in the Ion Xpress Template Kit User Guide (Thermo Fisher Scientific). Instructions for loading the subsequent template library onto the Ion Torrent™ Chip for nucleic acid sequencing are described in the Ion Sequencing User Guide (Thermo Fisher Scientific). In some embodiments, the amplicon library produced by the teachings of the present disclosure can be used in paired end sequencing (e.g., paired-end sequencing on the Ion Torrent™ PGM system (Thermo Fisher Scientific).
It will be apparent to one of ordinary skill in the art that numerous other techniques, platforms or methods for clonal amplification such as wildfire PCR and bridge amplification can be used in conjunction with the amplified target sequences of the present disclosure. It is also envisaged that one of ordinary skill in art upon further refinement or optimization of the conditions provided herein can proceed directly to nucleic acid sequencing (for example using the Ion Torrent PGM™ or Proton™ sequencers, Life Technologies) without performing a clonal amplification step.
In some embodiments, at least one of the amplified targets sequences to be clonally amplified can be attached to a support or particle. The support can be comprised of any suitable material and have any suitable shape, including, for example, planar, spheroid or particulate. In some embodiments, the support is a scaffolded polymer particle as described in U.S. Published App. No. 20100304982, hereby incorporated by reference in its entirety.
In some embodiments, nucleic acid sequencing of the amplified target sequences produced by the teachings of this disclosure include de novo sequencing or targeted resequencing. In some embodiments, nucleic acid sequencing further includes comparing the nucleic acid sequencing results of the amplified target sequences against a reference nucleic acid sample. In some embodiments, the reference sample can be normal tissue or well documented tumor sample. In some embodiments, nucleic acid sequencing of the amplified target sequences further includes determining the presence or absence of a mutation within the nucleic acid sequence. In some embodiments, the method further includes correlating the presence of a mutation with drug susceptibly, prognosis of treatment and/or organ rejection. In some embodiments, nucleic acid sequencing includes the identification of genetic markers associated with cancer. In some embodiments, nucleic acid sequencing includes the identification of copy number variation in a sample under investigation.
In some embodiments, a kit is provided for amplifying multiple immune response target expression sequences from a population of nucleic acid molecules in a single reaction. In some embodiments, the kit includes a plurality of target-specific primer pairs containing one or more cleavable groups, one or more DNA polymerases, a mixture of dNTPs and at least one cleaving reagent. In one embodiment, the cleavable group can be 8-oxo-deoxyguanosine, deoxyuridine or bromodeoxyuridine. In some embodiments, the at least one cleaving reagent includes RNaseH, uracil DNA glycosylase, Fpg or alkali. In one embodiment, the cleaving reagent can be uracil DNA glycosylase. In some embodiments, the kit is provided to perform multiplex PCR in a single reaction chamber or vessel. In some embodiments, the kit includes at least one DNA polymerase, which can be a thermostable DNA polymerase. In some embodiments, the concentration of the one or more DNA polymerases is present in a 3-fold excess as compared to a single PCR reaction. In some embodiments, the final concentration of each target-specific primer pair is present at about 25 nM to about 50 nM. In one embodiment, the final concentration of each target-specific primer pair can be present at a concentration that is 50% lower than conventional single-plex PCR reactions. In some embodiments, the kit provides amplification of at least 100, 150, 200, 250, 300, 350, 398, or more, target immune response expression sequences from a population of nucleic acid molecules in a single reaction chamber. In particular embodiments, a provided kit of the invention is a test kit.
In some embodiments, the kit further comprises one or more adapters, barcodes, and/or antibodies.
A common concern in study of RNA expression analyses is determination of how gene expression changes between different samples. Current methodology for RNA sequencing typically requires an amplification step followed by sequence analysis of select amplified products (e.g., transcripts) to a fixed number of reads. In particular, with the provided methods of the invention, expression of each gene can be inferred by amplifying an amplicon that represents a transcript (e.g., an amplicon crosses a selected exon/exon junction). Due to the selected amplification, there is a dependency between the number of reads associated with a particular gene transcript and the number of reads produced by all other genes. Such interdependency means an apparent identical fold-change can be induced by two distinct biological processes: an increase in the ration of expression of a particular gene, or a decrease in the expression ration of all other genes measured. To accurately measure which of the two scenarios takes place, number of reads should be correctly normalized within each sample before cross comparison of samples takes place. Normalization has been studies for full transcriptome RNA sequencing analyses. Among such studies, log RPM (read counts per million) has been widely accepted to provide an interpretable measure of gene expression that robustly reflects the difference in expression between samples. The accuracy of this measure, however, depends on the underlying assumption that the majority of the genes measured are not differentially expressed. This assumption tends to hold true for transcriptomic studies, where more than 20,000 genes of the same tissues are typically profiled. However, such an assumption is unlikely to always hold for smaller gene panels, for panels comprising very low expression genes, or for panels that focus on similar biological processes, such as, for example, the methods and compositions provided herein. As a simple illustration, consider a targeted panel of 20 genes made up of 2 functional categories, one with 16 and the other with 4 genes, where each gene of a function is perfectly correlated to the others. In this example, the actual number of RNA molecules in sample 1 and sample 2 is known, and can sequence an exact of proportion of reads. When a standard RPM computation is applied to measure the expression of each gene and calculate the fold change using our estimated expression, there is 2-fold increase in the smaller category, the fold change estimate maintains a fairly accurate estimate of true fold change (
Thus, in conjunction with the compositions and methods of the invention, provided compositions include primer pairs and methods for determination of expression of a set of housekeeping genes for use of one or more as endogenous controls and an effective means for normalization of expression levels and determination of expression changes across samples.
The following description of various exemplary embodiments is exemplary and explanatory only and is not to be construed as limiting or restrictive in any way. Other embodiments, features, objects, and advantages of the present teachings will be apparent from the description and accompanying drawings, and from the claims.
Although the present description described in detail certain exemplary embodiments, other embodiments are also possible and within the scope of the present invention. Variations and modifications will be apparent to those skilled in the art from consideration of the specification and figures and practice of the teachings described in the specification and figures, and the claims.
EXEMPLIFICATIONMaterials and Methods
Provided immune response assay compositions include reagents directed to 395 immune response markers designed for library preparation technology of expressed target sequences. Generally, RNAs extracted from samples (e.g., tumor samples, e.g., fresh, frozen, FFPE, of various types, e.g., lung tumor, NSCLC) were reverse transcribed; libraries were generated, template prepared, e.g., using Ion Chef™ or Ion OneTouch™ 2 System, then prepared samples were sequenced using next generation sequencing technology, e.g., an Ion S5™, an Ion PGM™ System.
Total RNA was extracted from samples, 10 ng of total RNA was first reverse transcribed to and used as template to amplify targets (e.g., target expression sequences in Table 1) with the immune response assay comprising primer pairs in Table 2. Manual library prep was performed with the Ion AmpliSeg™ Library Kit 2.0 which was then templated with either the Ion OneTouch™ 2 System or Ion Chef™ instrument. Automated library prep and templating was performed with the Ion AmpliSeg™ Kit for Chef DL8 and Ion Chef™ Instrument. Sequencing was performed on either the Ion S5™ System or the Ion PGM™ System. Gene expression was quantified with the Ion Torrent Suite™ software.
HL-60 and human normal lung (AM7968) were purchased from ATCC and Ambion, respectively. De-identified melanoma and NSCLC FFPE samples were procured from Asterand and Research Dx respectively.
RNA and cDNA Preparation
Total RNA was extracted from samples with the RecoverAll™ Total Nucleic Acid Isolation Kit (Ambion, Inc.), according to the manufacturer instructions, and quantified, e.g., using the Qubit™ RNA HS Assay Kit (Thermo Fisher) for quantifying RNA. A total of 10 ng of total RNA was first reverse transcribed to cDNA with SuperScript® VILO™ cDNA Synthesis Kit (Thermo Fisher, Cat. No. 11754050) according to manufacturer instructions. Prepared template was used to amplify targets with the immune response assay comprising primer pairs in Table 2.
PCR Amplify RNA Targets
A multiplex polymerase chain reaction was performed to amplify 398 individual amplicons across a RNA expression sample. A list of genes and the resulting amplicon sequences generated are provided in Table 1. Primer pairs are presented in Table 2. To a single well of a 96-well PCR plate was added 4 microliters of immune response primer pool containing 398 primer pairs at a concentration of 15 μM in TE, 10 microliters prepared cDNA and 4 microliters of an amplification reaction mixture (5× AmpliSeq HiFi Master Mix) that can include glycerol, dNTPs, and Platinum® Taq High Fidelity DNA Polymerase (Invitrogen, Catalog No. 11304) to a final volume of 20 microliters with DNase/RNase Free Water (Life Technologies, CA, Part No. 600004).
The PCR plate was sealed and loaded into a thermal cycler (GeneAmp® PCR system Dual 96-well thermal cycler (Life Technologies, CA, Part No. N8050200 and 4314445)) and run on the following temperate profile to generate the preamplified amplicon library.
An initial holding stage was performed at 99° C. for 2 minutes, followed by 19 cycles of denaturing at 99° C. for 15 seconds and an annealing and extending stage at 60° C. for 4 minutes. After cycling, the preamplified amplicon library was held at 10° C. until proceeding.
Partially Digest Amplicons
The sample was briefly centrifuged to collect contents before proceeding. To the preamplified amplicon library (˜20 microliters), 2 microliters of FuPa reagent was added. The reaction mixture was sealed, mixed thoroughly to ensure uniformity and incubated at 50° C. for 10 minutes, 55° C. for 10 minutes, 60° C. for 20 minutes, then held at 10° C. for up to 1 hour. The sample was briefly centrifuged to collect contents before proceeding.
Ligate Adapters to Amplicons and Purify
After incubation, the reaction mixture proceeded directly to a ligation step. Here, the reaction mixture now containing the phosphorylated amplicon library was combined with 2 microliters of A/P1 Adapters (sold as a component of the Ion Fragment Library Kit, Thermo Fisher), microliters of Switch Solution (sold as a component of the Fragment Library Kit, Thermo Fisher) and 2 microliters of DNA ligase, added last (sold as a component of the Ion Fragment Library Kit, Thermo Fisher), then incubated at the following: 22° C. for 30 minutes, 72° C. for 10 minutes, then held at 10° C. for up to 1 hour. The sample was briefly centrifuged to collect contents before proceeding.
After the incubation step, 45 microliters (1.5× sample volume) of room temperature AgenCourt® AMPure® Reagent (Beckman Coulter, Calif.) was added to ligated DNA. The mixture was pipetted thoroughly to mix the bead suspension with the ligated DNA. The mixture was pulse-spin and incubated at room temperature for 5 minutes. Samples underwent another pulse-spin and were placed on a magnetic rack such as a DynaMag™-2 spin magnet (Life Technologies, CA, Part No. 123-21D) for two minutes. After the solution had cleared, the supernatant was discarded. Without removing the tube from the magnetic rack, 150 microliters of freshly prepared 70% ethanol was introduced into the sample, and incubated while gently rotating the tube on the magnetic rack. After the solution cleared, the supernatant was discarded without disturbing the pellet. A second ethanol wash was performed and the supernatant discarded. Any remaining ethanol was removed by pulse-spinning the tube and carefully removing residual ethanol while not disturbing the pellet. The pellet was air-dried for about 5 minutes at room temperature.
The pellet was resuspended in 20 microliters of DNase/RNase Free Water (Life Technologies, CA, Part No. 600004) and vortexed to ensure the sample was mixed thoroughly. The sample was pulse-spin and placed on the magnetic rack for two minutes. After the solution cleared, the supernatant containing the ligated DNA was transferred to a new Eppendorf LoBind™ tube for long-term storage. After quantification, determine the dilution factor that results in a concentration of ˜100 μM, which is suitable for template preparation using an Ion template kit.
Quantify the Library
Ligated library was quantified, using a Qubit™ 2.0 or 3.0 Fluorometer and/or by qPCR using the Ion Library TaqMan® Quantitation Kit (Cat. No. 4468802), according to manufacturer instructions.
Amplify and Purify the Library
The ligated preamplified library (˜20 microliters) was combined with 50 microliters of Platinum® PCR SuperMix High Fidelity (Thermo Fisher, sold as a component of the Ion Fragment Library Kit) and 2 microliters of Library Amplification Primer Mix (sold as a component of the Ion Fragment Library Kit). The solution was applied to a single well of a 96-well PCR plate and sealed. The plate was loaded into a thermal cycler (GeneAmp® PCR system 9700 Dual 96-well thermal cycler (Life Technologies, CA, Part No. N8050200 and 4314445)) and run on the following temperate profile to generate the final amplicon library: hold at 98° C. for 2 minutes, followed by 5 cycles of denaturing at 98° C. for 15 seconds and an annealing and extending stage at 64° C. for 1 minute. After cycling, the final amplicon library was held at 4° C. until proceeding to the final purification step outlined below.
A two-round purification of the final library was carried out. 25 μL (0.5× sample volume) of Agencourt™ AMPure™ XP Reagent was added to each plate well containing ˜50 μL of sample. The bead suspension was pipetted up and down to thoroughly mix the bead suspension with the final amplicon library. The sample was then pulse-spun and incubated for 5 minutes at room temperature. The plate containing the final amplicon library was placed on a magnetic rack such as a DynaMag™-side magnet (Thermo Fisher) for 5 minutes to capture the beads. Once the solution cleared, the supernatant was carefully transferred without disturbing the bead pellet. A second round of purification was carried out, adding 60 microliters (1.2× sample volume) of Agencourt™ AMPure™ XP Reagent was added to each plate well containing sample. The bead suspension was pipetted up and down to thoroughly mix the bead suspension and incubated for 5 minutes at room temperature. The plate containing the final amplicon library was placed on a magnetic rack for 3 minutes to capture the beads. Without removing the plate from the magnetic rack, 150 microliters of freshly prepared 70% ethanol was introduced into the beads containing sample. The sample was incubated for 30 seconds while gently rotating the tube on the magnetic rack. After the solution cleared, the supernatant was discarded without disturbing the pellet. A second ethanol wash was performed and the supernatant discarded. Any remaining ethanol was removed by pulse-spinning the tube and carefully removing residual ethanol while not disturbing the pellet. The pellet was air-dried for about 5 minutes at room temperature.
Once the tube was dry, the tube was removed from the magnetic rack and 50 microliters of Low TE was added (Thermo Fisher), pipetted and vortexed to ensure the sample was mixed thoroughly. The sample was pulse-spin and placed on the magnetic rack for two minutes. After the solution cleared, the supernatant containing the final amplicon library was analyzed using Qubit™ Fluorometer and Qubit™ dsDNA HS Assay Kit according to manufacturer instructions to quantify the library and calculate the dilution factor for template preparation and sequencing. Library was diluted to ˜50 pM for use in template preparation or stored in 1.5-mL Eppendorf LoBind™ tube for long-term storage.
Template Preparation and Sequencing
An aliquot of the final library was used in template preparation with either Ion OneTouch™ 2 System or Ion Chef™ instrument according to the manufacturer's instructions.
Sequencing was performed on either the Ion S5™ System or the Ion PGM™ System. According to manufacturer instructions, and gene expression was quantified with the Ion Torrent Suite™ software subjected to analysis and sequencing according to manufacturer instructions.
Automated Processes
Alternatively, automated library prep and templating can be performed using cDNA prepared as described above in conjunction with the Ion AmpliSeg™ Kit for Chef DL8 and Ion Chef™ Instrument according to manufacturer instructions and described in the Ion AmpliSeq Library Preparation on the Ion Chef System (Thermo Fisher Scientific publication number MAN0013432), hereby incorporated by reference.
Multiple Library Preparation
When sequencing multiple libraries, a different barcode will be ligated to each library. DNA and RNA libraries from the same sample also require different barcodes. IonCode Adapters are provided at the appropriate concentration and include forward and reverse adapters in a single well. For each barcode selected, prepare a mix of Ion P1 adapter and IonXpress Barcode X (Thermo Fisher) at a final dilution of 1:4 for each adapter. Substitute 2 uL of this barcode adapter mix for the Ion AmpliSeq Adapters in ligation reactions above; and scale volumes as necessary. Multiple barcoded libraries can be sequenced on a single chip by combining equal volumes of each library before template preparation. The manufacturer recommends up to eight libraries for the following chips: Ion 318 Chip v2 (up to 4), Ion 520 Chip (up to 4), Ion 530 Chip (up to 8). A combined library is prepared by diluting all individual libraries to 50 pM concentration; combining 10 uL of each library in a single tube (preferably Eppendorf LoBind tube). After combining the last library, mix thoroughly by pipetting up and down 5 times, then centrifuge briefly to collect in the bottom of the tube. Proceed to templating and sequencing as desired.
Data Analysis
Gene expression level quantification is performed using the Torrent Suite™ RNA plugin that produces gene transcript quantification from sequence read data. The plugin utilizes target region .bed file and an associated reference library .fasta file, and optionally accepts a secont .bed file that specifies a subset of target genes allowing sample clustering. Resulting report includes gene expression counts (number of aligned reads to a given target sequence), data analysis summary, and QC plots. Normalized gene level count data can be determined using the Affymetrix Transcriptome Analysis Console (TAC) 3.1 software (Thermo Fisher Scientific). CHP files generated from Torrent Suite™ plugin can be used directly with Transcriptome Analysis Console (TAC3 software for differential gene expression analysis. Affymetrix® Transcriptome Analysis Console (TAC) Software is freely available, http://www.affymetrix.com/
Normalization of Expression
To observe how expression profiles among the selected target immune response genes vary within a large cohort of samples, we downloaded and processed more than 9,600 patient samples across 18 cancer studies from the ICGC database [Zhang et al., 2011]. To ensure high-quality RNA profiles, we excluded all samples with a library size less than 20 million reads mapped to the transcriptome and ended up with 9,148 full transcriptome datasets. For this study, we used the log 2 CPM full transcriptome (FT) expression of each gene as the ‘true’ expression value and the total number of reads mapping to genes as the ‘true’ normalizing constant for each sample in the cohort.
Results
Total RNA extracted from, samples e.g., NSCLC FFPE samples, were reverse transcribed. Libraries were generated, templated on Ion Chef™ or Ion OneTouch™ 2 System and sequenced on the Ion 55™ or Ion PGM™ System. With an input requirement as low as 10 ng of total RNA, we were able to measure robust expression, including for low expressing transcripts (e.g., IFN, IL2, IL21, IL10, IL23) from samples, e.g., NSCLC FFPE samples, CRC samples. Results show high sensitivity and specificity, with broad dynamic range and great reproducibility. Technical replicates were highly reproducible (see
Analysis report files include, for example, a sample summary report (table listing each sample, total reads, aligned reads, percent aligned); absolute read count data (absolute reads for genes of each sample), RPM data (normalized by total read count for each sample), mean housekeeping scaled log 2 RPM data (for each sample), CHP files normalized by RPM, CHP files normalized by mean housekeeping genes, and expression data for genomic DNA and water samples. See
Thus, we verified a linear and unbiased estimate of fold change in our assays across mixing concentrations of a cell-line titration experiment.
Using endogenous controls accurately estimates fold change. When most genes do not change between the samples, the fold change estimate has negligible error. When most genes do change, the fold change estimate is wildly inaccurate. See
To first understand how assaying only genes from a common function can affect estimate of expression, an assay was made comprising only the 5 genes associated with the Leukocyte Migration category (ITGAL, ITGAM, ITGB7, SELL, VCAM1). Assaying only those genes does not provide an accurate expression estimate. Expanding the assay by 2 Leukocyte Inhibition and 19 TCR co-expression genes (CCR7, CD247, CD3D, CD3E, CD3G, CD6, CD8A, CD8B, CRTAM, GPR18, GRAP2, IKZF3, IL2RB, IL7R, ITK, LAMP3, LCK, PTPRCAP, TIGIT) does not increase the correlation. However, assaying 27 genes from the unrelated Tumor Marker category (AKT1, BRCA1, BRCA2, CDKN2A, EFNA4, EGFR, EGR3, IRS1, KRT5, KRT7, MAPK1, MMP2, MMP9, MYC, NOTCH3, PGF, PTGS2, PTK7, RB1, RPS6, SNAIL SNAI2, TCF7, TP63, TRIM29, TWIST1, ZEB1) or 11 housekeeping (HK) genes (ABCF1, G6PD, GUSB, HMBS, LMNA, LRP1, POLR2A, SDHA, TBP, TFRC, TUBB) improves the correlation with true expression values. See
Thus, we also found that normalization using endogenous controls (housekeeping genes) provides accurate fold change estimates for small panel assays with highly-correlated genes. Uncorrelated gene categories can have the same effect as house-keeping genes on normalization, however, close cases where uncorrelated gene categories are differentially expressed between samples will bias expression estimates when not using housekeeping genes. The method of housekeeping gene normalization provided herein can improve correlation with true values of expression assays. In particular, housekeeping gene normalization may improve assessment of expression in sub-panels of the provided immune response assay, and may be useful in connection with the relatively large immune response assay provided herein.
From the previous analysis, it is clear that normalization using housekeeping genes in conjunction with compositions and methods of the invention are necessary for accurately estimating fold change expression. It is also clear that using only the housekeeping genes to calculated a normalizing constant is a preferred way to estimate gene expression for accurate fold change estimation. An important factor to using housekeeping genes for calculating normalizing constant is to determine which is metric of the gene counts provides the most accurate fold change estimates. We thus compared 6 metrics across 18 cancer studies; for each study we calculate the log 10 of the total counts and determine the correlation to the following metrics: log 10 of the sum of housekeeping counts (analogous to RPM normalization), log 10 of the geometric mean of the housekeeping counts, the median of the log 10 of housekeeping counts, the 75th quantile of the log 10 of housekeeping counts, and the trimmed mean (15% and 25% of the log 10 of HK counts). Interestingly, the sum of housekeeping counts does not provide an optimal correlation value, instead the geometric mean provides the highest average correlation and number of cancers where the R-squared is greater than 0.5 (data not shown). Of the more robust measures, the 15% trimmed mean provides the highest average R-squared. To further estimate which metric is most robust, we simulate a dropout of a random housekeeping genes for every sample by setting a random housekeeping gene of each sample to have 0 reads. We then reproduce the same analysis to compare the various metrics using dropout samples. Interestingly, the geometric mean still provides the highest correlation with the full transcriptome normalizing constant even in the dropout samples, while the 15% trimmed mean also shows high performances (data not shown). This suggests that the geometric mean is an optimal metric due to its high correlation with the true normalizing constant values and its robustness to dropouts. Additionally, we carried out analysis of normalizing constant, correlations and drop out correlation among various cancer types of interest and confirmed even across various sample types the housekeeping set of genes provided in the instant compositions and methods performed consistently across populations of interest.
Housekeeping (HK) constant:
-
- Let x be a vector of counts for every gene on the immune response assay and let HKgenes be the set of housekeeping genes; add a pseudocount 1 to every entry in the vector x, creating a new vector y.
- Find the geometric mean of the counts of housekeeping genes, calculated as 10{circumflex over ( )}mean (log 10(y_HKgenes)), where y_HKgenes is a subset of the y vector to only include the counts associated with the housekeeping genes. This geometric mean value will be denoted asHKnorm.
- Multiply each entry of y by the (10{circumflex over ( )}6/HKnorm), creating a new vector z.
- Finally take the log 2 value of each entry in z and add, creating the final housekeeping gene normalized expression estimate.
Once established, an HKnorm constant was calculated by taking the geometric mean of our 11 housekeeping genes and comparing our normalizing strategy to a standard RNA-seq RPM measurement using the 398 genes of the provided immune response assay. Since this assay is made up of a relatively large number of genes that are on average uncorrelated to each other, one could make a valid argument that housekeeping genes are unnecessary to the assay and do not provide additional information. To address this concern, we compared the HKnorm constant against the standard RPM constant and the RPM constant when housekeeping genes are removed from the assay. We first evaluated how these normalizing strategies compare to the full transcriptome constant across all samples, where we found that removing housekeeping genes led to decrease in the correlation of the RPM constant compared to a standard RPM calculation, however, even the standard RPM calculation under-performs when compared to HKnorm. (data not shown). We then examined which normalizing strategy would be optimal for each cancer study that we evaluated, and found that HKnorm largely outperforms the RPM strategies across all studies except for LIHC-US, where no normalization strategy does particularly well and all the strategies produce equivalent results. When looking at the performance each strategy across target genes on the immune response assay of the invention, we see that HKnorm outperforms the RPM strategies in all genes, as expected. Interestingly, genes with smaller variance have a smaller correlation to their FT values than those with higher variance. See
- Schalper K A, et al. Objective measurement and clinical significance of TILs in non-small cell lung cancer. J Natl Cancer Inst. 2015 Feb. 3; 107(3).
- Padmanee S, et al. Immune Checkpoint Targeting in Cancer Therapy: Toward Combination Strategies with Curative Potential. Cell 2015 April 161(2):205-214.
- Love M, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 2014.
- Law C, Chen Y, Shi W, Smyth G. voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biology, 2014.
- Hansen K, Irizarry R. Removing technical variability in RNA-seq data using conditional quantile normalization. Biostatistics, 2011.
- Risso D, Schwartz K, Sherlock G and Dudoit S. GC-Content Normalization for RNA-Seq Data. BMC Bioinformatics, 2011.
- Zhang, J., Baran, J., Cros, A., Guberman, J. M., Haider, S., Hsu, J. Kasprzyk, A. (2011). International Cancer Genome Consortium Data Portal—a one-stop shop for cancer genomics data. Database: The Journal of Biological Databases and Curation, 2011.
Claims
1-20. (canceled)
21. A method for determining immune response activity in a biological sample, comprising:
- multiplex amplification of a plurality of target sequences from the biological sample, wherein the amplification comprises contacting at least a portion of the sample with a plurality of sets of primer pair reagents and a polymerase under amplification conditions, wherein the plurality of target sequences are selected from the group consisting of the following functions: checkpoint pathways, T cell related signaling pathways, markers of tumor infiltrating lymphocytes (TILs), and tumor markers, and housekeeping genes, and wherein the plurality of primer pair reagents are selected from the group consisting of SEQ ID NOs: 399-1194, thereby producing amplified immune response target sequences; and
- detecting a level of expression of each of the plurality of immune response target sequences, wherein a change in the level of expression of one or of the immune response target sequences as compared with a control sample determines a change in immune response activity in the sample.
22. The method of claim 21, wherein the immune response target sequences are from target genes selected from the group consisting of immune checkpoint pathways and targets; T and B cell signaling genes, markers of lymphocyte subsets, interferon signaling genes, cytokine signaling genes; tumor markers, tumor antigens, proliferation markers; and housekeeping genes.
23. The method of claim 22, wherein the target genes are selected from the genes of CD63, CD69, CXCL1, KLRD1, HLA-DOB, CXCR5, IL12B, PTK7, CEACAM1, CXCL9, IL13, NT5E, VEGFA, ABCF1, D38, JAML, S100A8, MYC, IRF1, CCL22, CXCR2, IFIT1, IFIT2, CD68, M6PR, SH2D1A, ISG20, GBP1, TBP, STAT6, ID3, CX3CL1, KLRB1, TNFSF4, CD52, IL10RA, HLA-DOA, IFNB1, CCR5, IKZF3, STAT1, CD6, BRCA1, CORO1A, TBX21, KLRK1, CXCR6, PTEN, PMEL, DMBT1, IFI44L, LAPTM5, CD226, TNFSF13B, ICOS, CD160, TRIM29, LST1, ZBTB46, VTCN1, KREMEN1, PDCD1LG2, TUBB, CLEC4C, CD86, HAVCR2, GZMH, NFATC1, CD8B, BCL2, GADD45GIP1, CBLB, ITGA1, CD8A, IL2RA, EIF2AK2, MADCAM1, PTPN6, LRG1, ADGRE5, SH2D1B, ITGB2, HLA-DPA1, DGAT2, IGF1R TAGAP, LMNA, NCAM1, TIGIT, IL17F, HLA-F-AS1, CD247, CD79B, IDO2, IL4, TYROBP, BTLA, AKT1, IL2RG, POLR2A, ITGAX, IL1B, CSF2RB, DDX58, KIAA0101, CD274, LAMPS, TNFAIP8, FOXP3, IL12A, SAMHD1, SIT1, CD3E, ICOSLG, HGF, MELK, IGSF6, GNLY, TDO2, KRT7, HLA-E, HLADMA, LAMP1, NTN3, CD28, TARP, EGFR, CCR4, MAGEA3, BATF, KLRG1, IRS1, CSF1R, CTLA4, TNFSF18, POU2AF1, GZMA, PIK3CA, ITK, IFI27, EOMES, LCN2, CD80, CD83, CXCL13, MTOR, FCER1G, TFRC, RORC, MMP9, BST2, PIK3CD, FCGR2B, TNFRSF14, OAS3, GRAP2, CCNB2, MLANA, MAGEA12, VCAM1, CDKN3, NCR1, FAS, GZMB, IRF9, IFITM2, TNFSF14, HLA-B, SDHA, NRP1, EBB, EFNA4, PVR BUB1, SKAP2, PRF1, CCL20, TNFRSF18, CTSS, NKG7, ISG15, PDCD1, SNAI1, CXCL11, CIITA, IFI35, TNFSF9, TNFSF10, MMP2, EGR3, MAGEA1, CD163, IL6, KLRF1, B3GAT1, C1QA, OAS1, IKZF2, TLR9, KLF2, GUSB, NFKBIA, IL23A, HERC6, SLAMF8, IL15, TLR7, OAS2, HLA-DRA, CRTAM, MAGEC2, ICAM1, CD4, MAPK14, C1QB, NOTCH3, NCR3, STAT3, TLR8, CYBB, IKZF4, IFIH1, LCK, BCL2L11, ITGAM, ITGB7, JCHAIN, CD209, SLAMF7, IL10, IL1A, FCGR3A, IFNA17, EGR2, TOP2A, C10orf54, FOXM1, AXL, MS4A1, IFI6, CD3D, GPR18, CD3G, ZAP70, HMBS, IL7, IFIT3, RBI, PTGS2, TGFB1, NCF1, TWIST1, CA4, SELL, LILRB1, CD14, ALOX15B, PECAM1, NOS2, FASLG, CD44, ENTPD1, CMKLR1, CD53, TNF, CXCL8, CD40LG, HLA-F, GATA3, LYZ, ARG1, IL2RB, NECTIN2, MPO, CCR2, BRCA2, ADORA2A, G6PD, TAP1, MXI, HLA-DQB2, CD27, CD276, STAT4, PTPN7, PTPRC, PSMB9, CD244, CXCR4, MAPK1, TP63, IRF4, CCL3, CCL18, IL7R, HLA-DRB1, CEACAM8, CXCL10, CCL2, SRGN, CD19, ITGB1, IFITM1, CCL21, MRC1, PGF, ITGAL, ID2, CD22, CCL17, ITGAE, IL3RA, CCR7, CD1C, MAD2L1, PYGL, CD40, LY9, HLA-G, TLR3, CD48, STAT5A, FCRLA, BCL6, ZEB1, CCL5, IDOL IL18, TNFRSF9, HIF1A, HLA-DPB1, FOXO1, CD33, S100A9, HLA-DMB, HLAA, SNAI2, TNFRSF17, LRP1, MAGEA4, HLA-DQA1, CD1D, RPS6, MKI67, GZMK, CD79A, CD37, FUT4, AIF1, CCR1, PRDM1, CD47, CD74, LAGS, TNFRSF4, CD2, CCL4, BAGE, LEXM, CCR6, CD70, CDK1, CTAG1B, CTAG2, CX3CR1, GAGE1, GAGE121, GAGE12F, GAGE12J, GAGE2C, GAGE2A, GAGE2E, GAGE10, GAGE13, IKZF1, IL17A, IL2, IL21, IL22, KIR2DL2, KIR2DL3, MAGEA10, MIF, PTPRCAP, SSX2, TCF7, XAGE1B, CXCR3, FCGR1A, FCGR3B, FYB, HLA-C, HLA-DQA2, IFNG, KIR2DL1, KRT5, and PTPN11.
24. The method of claim 23, wherein the target genes comprise the genes of CD63, CD69, CXCL1, KLRD1, HLA-DOB, CXCR5, IL12B, PTK7, CEACAM1, CXCL9, IL13, NT5E, VEGFA, ABCF1, D38, JAML, S100A8, MYC, IRF1, CCL22, CXCR2, IFIT1, IFIT2, CD68, M6PR, SH2D1A, ISG20, GBP1, TBP, STAT6, ID3, CX3CL1, KLRB1, TNFSF4, CD52, IL10RA, HLA-DOA, IFNB1, CCR5, IKZF3, STAT1, CD6, BRCA1, CORO1A, TBX21, KLRK1, CXCR6, PTEN, PMEL, DMBT1, IFI44L, LAPTM5, CD226, TNFSF13B, ICOS, CD160, TRIM29, LST1, ZBTB46, VTCN1, KREMEN1, PDCD1LG2, TUBB, CLEC4C, CD86, HAVCR2, GZMH, NFATC1, CD8B, BCL2, GADD45GIP1, CBLB, ITGA1, CD8A, IL2RA, EIF2AK2, MADCAM1, PTPN6, LRG1, ADGRE5, SH2D1B, ITGB2, HLA-DPA1, DGAT2, IGF1R TAGAP, LMNA, NCAM1, TIGIT, IL17F, HLA-F-AS1, CD247, CD79B, IDO2, IL4, TYROBP, BTLA, AKT1, IL2RG, POLR2A, ITGAX, IL1B, CSF2RB, DDX58, KIAA0101, CD274, LAMPS, TNFAIP8, FOXP3, IL12A, SAMHD1, SIT1, CD3E, ICOSLG, HGF, MELK, IGSF6, GNLY, TDO2, KRT7, HLA-E, HLADMA, LAMP1, NTN3, CD28, TARP, EGFR, CCR4, MAGEA3, BATF, KLRG1, IRS1, CSF1R, CTLA4, TNFSF18, POU2AF1, GZMA, PIK3CA, ITK, IFI27, EOMES, LCN2, CD80, CD83, CXCL13, MTOR, FCER1G, TFRC, RORC, MMP9, BST2, PIK3CD, FCGR2B, TNFRSF14, OAS3, GRAP2, CCNB2, MLANA, MAGEA12, VCAM1, CDKN3, NCR1, FAS, GZMB, IRF9, IFITM2, TNFSF14, HLA-B, SDHA, NRP1, EBB, EFNA4, PVR BUB1, SKAP2, PRF1, CCL20, TNFRSF18, CTSS, NKG7, ISG15, PDCD1, SNAI1, CXCL11, CIITA, IFI35, TNFSF9, TNFSF10, MMP2, EGR3, MAGEA1, CD163, IL6, KLRF1, B3GAT1, C1QA, OAS1, IKZF2, TLR9, KLF2, GUSB, NFKBIA, IL23A, HERC6, SLAMF8, IL15, TLR7, OAS2, HLA-DRA, CRTAM, MAGEC2, ICAM1, CD4, MAPK14, C1QB, NOTCH3, NCR3, STAT3, TLR8, CYBB, IKZF4, IFIH1, LCK, BCL2L11, ITGAM, ITGB7, JCHAIN, CD209, SLAMF7, IL10, IL1A, FCGR3A, IFNA17, EGR2, TOP2A, C10orf54, FOXM1, AXL, MS4A1, IFI6, CD3D, GPR18, CD3G, ZAP70, HMBS, IL7, IFIT3, RBI, PTGS2, TGFB1, NCF1, TWIST1, CA4, SELL, LILRB1, CD14, ALOX15B, PECAM1, NOS2, FASLG, CD44, ENTPD1, CMKLR1, CD53, TNF, CXCL8, CD40LG, HLA-F, GATA3, LYZ, ARG1, IL2RB, NECTIN2, MPO, CCR2, BRCA2, ADORA2A, G6PD, TAP1, MXI, HLA-DQB2, CD27, CD276, STAT4, PTPN7, PTPRC, PSMB9, CD244, CXCR4, MAPK1, TP63, IRF4, CCL3, CCL18, IL7R, HLA-DRB1, CEACAM8, CXCL10, CCL2, SRGN, CD19, ITGB1, IFITM1, CCL21, MRC1, PGF, ITGAL, ID2, CD22, CCL17, ITGAE, IL3RA, CCR7, CD1C, MAD2L1, PYGL, CD40, LY9, HLA-G, TLR3, CD48, STAT5A, FCRLA, BCL6, ZEB1, CCL5, IDOL IL18, TNFRSF9, HIF1A, HLA-DPB1, FOXO1, CD33, S100A9, HLA-DMB, HLAA, SNAI2, TNFRSF17, LRP1, MAGEA4, HLA-DQA1, CD1D, RPS6, MKI67, GZMK, CD79A, CD37, FUT4, AIF1, CCR1, PRDM1, CD47, CD74, LAGS, TNFRSF4, CD2, CCL4, BAGE, LEXM, CCR6, CD70, CDK1, CTAG1B, CTAG2, CX3CR1, GAGE1, GAGE121, GAGE12F, GAGE12J, GAGE2C, GAGE2A, GAGE2E, GAGE10, GAGE13, IKZF1, IL17A, IL2, IL21, IL22, KIR2DL2, KIR2DL3, MAGEA10, MIF, PTPRCAP, SSX2, TCF7, XAGE1B, CXCR3, FCGR1A, FCGR3B, FYB, HLA-C, HLA-DQA2, IFNG, KIR2DL1, KRT5, and PTPN11.
25. The method of claim 21, wherein the plurality of immune response target sequences comprise a plurality of amplicon sequences selected from SEQ ID NOs: 1-398.
26. The method of claim 25, wherein the plurality of target sequences comprises each of the amplicon sequences of SEQ ID NOs: 1-398.
27. The method of claim 26, wherein the plurality of target sequences consists of each of the amplicon sequences of SEQ ID NOs: 1-398.
28. The method of claim 21, wherein the plurality of primer pair reagents comprises each of the primer pairs of SEQ ID NOs: 399-1194.
29. The method of claim 28, wherein the plurality of primer pair reagents consists of each of the primer pairs of SEQ ID NOs: 399-1194.
30. The method of claim 21, wherein the one or more housekeeping genes are selected from ABCF1, G6PD, GUSB, HMBS, LMNA, LRP1, POLR2A, SDHA, TBP, TFRC, and TUBB.
31. The method of claim 21, wherein the biological sample comprises a tumor, tissue surrounding a tumor, or both.
32. The method of claim 21, wherein the biological sample and the control sample are from the same individual.
Type: Application
Filed: Jul 29, 2022
Publication Date: Mar 23, 2023
Applicant: Life Technologies Corporation (Carlsbad, CA)
Inventors: Ann Mongan (San Francisco, CA), Alex Atkins (San Francisco, CA)
Application Number: 17/816,214
