Attorney Docket No. ACGI-43252.601 AMPLIFICATION OF MACROMOLECULES SEQUENCE LISTING The text of the computer readable sequence listing filed herewith, titled “43252- 601_SEQUENCE_LISTING”, created May 28, 2025, having a file size of 1,933 bytes, is hereby incorporated by reference in its entirety. FIELD Provided herein are methods, compositions, systems and kits to amplify DNA, RNA and protein macromolecules. In particular, provided herein are compositions and methods to amplify macromolecules for molecular analysis including low abundance template, single- cell and single nucleus applications. BACKGROUND Detection and identification of DNA, RNA and protein macromolecules from very low abundance templates, even DNA, RNA and protein macromolecules from a single cell and a single nucleus is critical is a multiplicity of research and health care applications including, for example, diagnosis of rare diseases with severely constrained tissue access, identification of rare circulating and biopsy cells, diagnosis of disorders expressed in the embryo, and management of cancer in apparent remission after therapy. SUMMARY Provided herein are methods, compositions, systems and kits to amplify DNA, RNA and protein macromolecules. In particular, provided herein are compositions and methods to amplify macromolecules for molecular analysis including low abundance template, single- cell and single nucleus applications. In some embodiments, the present invention provides a composition for amplifying a nucleic acid molecule, comprising: a nucleic acid template: a PolB enzyme that functions at equal to or greater than 43o C; a reducing agent that is not DTT; one or more primers comprising a phosphorothioate; a buffer; and a pyrophosphatase; wherein said composition does not comprise a nucleic acid chain terminator. In some embodiments, the PolB enzyme is comprises Phi29, Gp32, DPO1, DP02, Dp03, Equiphi29 -thermal stable phi29-(Thermo), Phi29-XT thermal stable (NEB), or Hotja Phi29. In some embodiments, the reducing agent is
Attorney Docket No. ACGI-43252.601 Tris (2-carboxyethyl) phosphine (TCEP). In some embodiments, the one or more primers is one or more 7mer random primers. In some embodiments, the buffer is [N- Morpholino]propane sulfonic acid (MOPS), 2-Hydroxy-3-morpholinopropanesulfonic acid (MOPSO), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2,2′-[(2- amino-2-oxoethyl)azanediyl]diacetic acid (ADA), Piperazine-1,4-bis(2- hydroxypropanesulfonic Acid (POPSO), or 2-Hydroxy-3-(4-(2-hydroxyethyl)piperazin-1- yl)propane-1-sulfonic acid(HEPSO. In some embodiments, the buffer is not Tris(hydroxymethyl)aminomethane (TRIS). In some embodiments, the pyrophosphatase is TheraPure™ GMP Pyrophosphatase, Thermostable Inorganic Pyrophosphatase (e.Coli), Thermostable Inorganic Pyrophosphatase (yeast), or NudC Pyrophosphatase. In some emboidments, the method comprises a polysorbate detergent wherein the concentration of the detergent controls the size of amplified sizes of said nucleic acids. In some embodiments, the polysorbate detergent is polyoxyethylene (20) sorbitan monolaurate and/or polyoxyethylene sorbitan monooleate. In some embodiments the one one or more primers are present at 50uM or less. In some embodiments, the method comprises Thermolabile Exo1. In some embodiments, the present invention provides a method, comprising: providing two or more different macromolecule templates selected from a group comprising a DNA macromolecule template; an RNA macromolecule template and a protein macromolecule template; providing a B-family DNA polymerase (PolB); and simultaneously co-amplifying the two or more different macromolecule templates with the PolB in an isothermal co-amplification reaction to generate a product comprising the amplified templates of the two or more different macromolecules. In some embodiments, the method comprises genomic DNA, whole genome genomic DNA, reverse transcriptase derived cDNA, or DNA synthesized from two or more oligonucleotides derived from the sequence of a protein macromolecule template. In some embodiments, the genomic DNA is converted to detect one or more methylated CpGs and/or one or more hydroxymethylated CpGs. In some embodiments, the genomic DNA is converted by bisulfite conversion or by enzymatic conversion. In some embodiments, transcription mediated amplification (TMA) is used to amplify the genomic DNA or said reverse transcriptase derived cDNA before said co- amplifying. In some embodiments, reverse transcription is followed by E.coli Pol 1, RNAse H and a ligase to generate a second strand of the first strand cDNA from the transcriptome, that is subsequenty amplified using a A (BST) or B (phi29) type polymerase I combination with the genome amplification. In some embodiments, the RNA macromolecule template comprises the whole transcriptome of an organism or a virus. In some embodiments, the
Attorney Docket No. ACGI-43252.601 whole transcriptome of an organism or virus is amplified by promoter-based isothermal transcription amplification before the co-amplifying. In some embodiments, the protein macromolecule template comprises one or more targeted proteins, a protein derived from the sequence of a DNA or RNA macromolecule template or a prion. In some embodiments, the two or more different macromolecule templates selected from a group comprising a DNA macromolecule template, an RNA macromolecule template, and a protein macromolecule template are derived from a single cell or a single nucleus. DESCRIPTION OF THE FIGURES Figure 1 shows time (minutes) on the x-axis vs. amplification abundance (Rn) on the y-axis using methods and compositions of the present invention over a range of initial template abundance. Line colors indicate template abundance i.e., pink 2.5 ng, purple 250 pg, blue 25 pg, aqua 2.5 pg, dark green 250 fg, light green 25 fg, yellow 2.5 fg, red no template control (NTC). Figure 2 shows amplification of DNA from single and multiple cells. Figure 3 shows amplified DNA product abundance in ng from one cell to 3 cell templates detected in less than 30 minutes and completed reactions in less than one hour. Figure 4 shows fluorescent intensity vs. amplified product DNA and NGS library lengths in base pairs (bp). NGS library sizes are depicted by a blue line, and amplified DNA sizes are depicted by a red line. Figure 5 shows analysis of a cancer cell line to determine the degree of chromosomal abnormality after DNA amplification using methods and compositions of the present invention. Single single-cell CNV data accurately detected CNV at low NGS depth in support of decreased NGS cost and time. Figure 6 shows precision and sensitivity of DNA amplification and detection with different amounts of target DNA. Figure 7 shows DNA amplification over a wide range of input amounts. Figure 8 shows real-time detection of single cell genomic DNA (gDNA) amplification. Figure 9 shows DNA yields from the amplification of a single cell genome. Figure 10 shows fragment sizes of amplified DNA products. Figure 11 shows optimized amplified fragment sizes. Figures 12A-B show effects of polysorbate detergent during the denaturation steps of the DNA amplification reaction (Fig.12A) with a 2-hour RT incubation (Fig.12B).
Attorney Docket No. ACGI-43252.601 Figure 13 shows a copy number analysis of single cell amplified DNA. Figure 14 shows single nucleotide variant detection with low sequencing depth compared to alternative chemistry platforms. Figure 15 shows reverse transcription (RT) efficiency. Figure 16 shows in vitro transcription (IVT) amplification yields at 4 hours. Figure 17 shows IVT amplification over a range of RNA concentrations. Figure 18 shows a RT reaction followed by isothermal amplification of the RT- produced cDNA. Figures 19A-C show RT optimization (SEQ ID NO.:1) for cDNA amplification. Figure 20 shows whole genome amplified (WGA) DNA yields from RT of total RNA. Figure 21 shows amplified DNA yields (ng) from RT of 1, 3 and 5 cells. Figure 22 shows amplified DNA yields from RT of 1, 3 and 5 cells with the RT reaction after an isothermal process to amplify the RT-produced cDNA. Figure 23 shows preparation-to-preparation variability in DNA yields. Figure 24 shows DNA yields in ng using WarmStart reverse transcriptase technology over a temperature range from 5oC to 60oC. DEFINITIONS Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention. For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control. As used herein, a “nucleic acid” or “nucleic acid molecule” generally refers to any ribonucleic acid or deoxyribonucleic acid, which may be unmodified or modified DNA or RNA. “Nucleic acids” include, without limitation, single- and double-stranded nucleic acids. As used herein, the term “nucleic acid” also includes DNA as described above that contains one or more modified bases. Thus, DNA with a backbone modified for stability or for other reasons is a “nucleic acid.” The term “nucleic acid” as it is used herein embraces such
Attorney Docket No. ACGI-43252.601 chemically, enzymatically, or metabolically modified forms of nucleic acids, as well as the chemical forms of DNA characteristic of viruses and cells, including for example, simple and complex cells. The terms “oligonucleotide” or “polynucleotide” or “nucleotide” or “nucleic acid” refer to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides for DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides for RNA are uracil, adenine, cytosine, and guanine. As used herein, the terms “locus” or “region” of a nucleic acid refer to a subregion of a nucleic acid, e.g., a gene on a chromosome, a single nucleotide, etc. The terms “complementary” and “complementarity” refer to nucleotides (e.g., 1 nucleotide) or polynucleotides (e.g., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence 5’-A-G-T-3’ is complementary to the sequence 3'-T-C-A-5'. Complementarity may be “partial,” in which only some of the nucleic acids’ bases are matched according to the base pairing rules. Or there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands effects the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions and in detection methods that depend upon binding between nucleic acids. The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA, or of a polypeptide or its precursor. A functional polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the polypeptide are retained. The term “portion” when used in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, “a nucleotide comprising at least a portion of a “gene” may comprise fragments of the gene or the entire gene. The term “gene” also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends, e.g., for a distance of about 1 kb on either end, such that the gene corresponds to the length of the full-
Attorney Docket No. ACGI-43252.601 length mRNA (e.g., comprising coding, regulatory, structural and other sequences). The sequences that are located 5' of the coding region, and that are present on the mRNA, are referred to as 5' non-translated or untranslated sequences. The sequences that are located 3' or downstream of the coding region and that are present on the mRNA are referred to as 3' non- translated or 3' untranslated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. In some organisms (e.g., eukaryotes), a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' ends of the sequences that are present on the RNA transcript. These sequences are referred to as “flanking” sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, posttranscriptional cleavage, and polyadenylation. The term “wild-type” when made in reference to a gene refers to a gene that has the characteristics of a gene isolated from a naturally occurring source. The term “wild-type” when made in reference to a gene product refers to a gene product that has the characteristics of a gene product isolated from a naturally occurring source. The term “wild-type” when made in reference to a protein refers to a protein that has the characteristics of a naturally occurring protein. The term “naturally-occurring” as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature, and which has not been intentionally modified by the hand of a person in the laboratory is naturally-occurring. A wild-type gene is often that gene or allele that is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. In contrast, the term “modified” or “mutant” when made in reference to a gene or to a gene product refers, respectively, to a gene or to a gene product that displays modifications in sequence and/or functional properties (e.g., altered characteristics) when
Attorney Docket No. ACGI-43252.601 compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. The term “allele” refers to a variation of a gene; the variations include but are not limited to single nucleotide or structural variants and mutants, polymorphic loci, and single nucleotide polymorphic loci, frameshift, and splice mutations. An allele may occur naturally in a population, or it might arise during the lifetime of any particular individual of the population. Thus, the terms “variant” and “mutant” when used in reference to a nucleotide sequence refer to a nucleic acid sequence that differs by one or more nucleotides from another, usually related, nucleotide acid sequence. A “variation” is a difference between two different nucleotide sequences; typically, one sequence is a reference sequence. As used herein, “integrated genome viewer (IGV)” is a computer program used to visualize sequence reads aligned to a reference genome. “IGV pileup” describes all sequence reads aligned to a specific location that create a stack. Allele fractions can be computed by examination at a specific genomic coordinate on the pileup, and reporting mutant alleles versus wild-type alleles (e.g., VAF = mut/(mut+wt) * 100). The precise allele fraction is a consequence of all wet lab and bio-informatic analysis. IGV pileup is an efficient method of visualizing and computing VAFs. A “false discovery rate” is computed as the VAF of mutant alleles when sequencing a known wild-type target. The term “primer” refers to an oligonucleotide, whether occurring naturally as, e.g., a nucleic acid fragment from a restriction digest, or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid template strand is induced, (e.g., in the presence of nucleotides and an inducing agent such as a DNA polymerase, and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer, and the use of the method. The term “probe” refers to an oligonucleotide (e.g., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically,
Attorney Docket No. ACGI-43252.601 recombinantly, or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification, and isolation of particular nucleic acid sequences (e.g., a “capture probe”). It is contemplated that any probe used in the embodiments of the present disclosure may, in some embodiments, be labeled with any “reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the various embodiments of the present disclosure be limited to any particular detection system or label. The term “target,” as used herein refers to a nucleic acid sought to be sorted out from other nucleic acids, e.g., by probe binding, amplification, isolation, capture, etc. For example, when used in reference to the polymerase chain reaction, “target” refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction, while when used in an assay in which target DNA is not amplified. Accordingly, as used herein, “non-target”, e.g., as it is used to describe a nucleic acid such as a DNA, refers to nucleic acid that may be present in a reaction, but that is not the subject of detection or characterization by the reaction. In some embodiments, non-target nucleic acid may refer to nucleic acid present in a sample that does not, e.g., contain a target sequence, while in some embodiments, non-target may refer to exogenous nucleic acid, i.e., nucleic acid that does not originate from a sample containing or suspected of containing a target nucleic acid, and that is added to a reaction, e.g., to normalize the activity of an enzyme (e.g., polymerase) to reduce variability in the performance of the enzyme in the reaction. As used herein, “multiplex” refers to the simultaneous amplification of different nucleic acid templates with different template-specific primer pairs in a shared vessel with shared amplification reagents (e.g., shared choice and concentrations of pH, buffers, ions, etc.) and shared amplification conditions (e.g., thermocycling conditions). The term “next generation sequencing” refers to highly parallelized methods of performing nucleic acid sequencing and comprises the sequencing-by-synthesis or sequencing-by-ligation platforms (e.g., employed by Illumina, Life Technologies, Pacific Biosciences and Roche etc.). Next generation sequencing methods may also include, but not be limited to, nanopore sequencing methods such as offered by Oxford Nanopore or electronic detection-based methods such as the Ion Torrent technology commercialized by Life Technologies.
Attorney Docket No. ACGI-43252.601 As used herein, the term “circulating tumor DNA” (or “ctDNA”) is tumor-derived DNA that is circulating in the peripheral blood of a patient. ctDNA is of tumor origin and originates directly from the tumor or from circulating tumor cells (CTCs), which are viable, intact tumor cells that shed from primary tumors and enter the bloodstream or lymphatic system. The term “cf-tDNA” refers to cell free tumor DNA in a circulating or non- circulating body fluid. As used herein, a “diagnostic” test application includes the detection or identification of a disease state or condition of a subject, determining the likelihood that a subject will contract a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to therapy, determining the prognosis of a subject with a disease or condition (or its likely progression or regression), and/or determining the effect of a treatment on a subject with a disease or condition. For example, a diagnostic test can be used for detecting the presence or likelihood of a subject contracting a neoplasm or the likelihood that such a subject will respond favorably to a compound (e.g., a pharmaceutical, e.g., a drug) or other treatment. The term “isolated” when used in relation to a nucleic acid, as in “an isolated oligonucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids, such as DNA and RNA, are found in the state they exist in nature. Examples of non-isolated nucleic acids include a given DNA sequence (e.g., a gene) found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, found in the cell as a mixture with numerous other mRNAs which encode a multitude of proteins. However, isolated nucleic acid encoding a particular protein includes, by way of example, such nucleic acid in cells ordinarily expressing the protein, where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid or oligonucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide may be single- stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide may be double-stranded). An isolated nucleic acid may, after isolation from its natural or typical environment, be combined with other nucleic acids or molecules. For example, an isolated
Attorney Docket No. ACGI-43252.601 nucleic acid may be present in a host cell into which it has been placed, e.g., for heterologous expression. The term “purified” refers to molecules, either nucleic acid or amino acid sequences that are removed from their natural environment, isolated, or separated. An “isolated nucleic acid sequence” may therefore be a purified nucleic acid sequence. “Substantially purified” molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated. As used herein, the terms “purified” or “to purify” also refer to the removal of contaminants from a sample. The removal of contaminating proteins results in an increase in the percent of polypeptide or nucleic acid of interest in the sample. In another example, recombinant polypeptides are expressed in plant, bacterial, yeast, or mammalian host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample. As used herein, the terms “patient” or “subject” refer to organisms to be subject to various tests described herein. The term “subject” includes animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human. Further with respect to diagnostic methods, a preferred subject is a vertebrate subject. A preferred vertebrate is warm-blooded; a preferred warm-blooded vertebrate is a mammal. A preferred mammal is most preferably a human. As used herein, the term “subject” includes both human and animal subjects. Thus, veterinary therapeutic uses are provided herein. As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials. As used herein, the term “fragmented kit” refers to delivery systems comprising two or more separate containers that each contain a sub-portion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides. The term “fragmented kit” is intended to encompass kits containing Analyte specific reagents (ASR’s) regulated under the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or
Attorney Docket No. ACGI-43252.601 more separate containers that each contains a sub-portion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits. As used herein, the term “information” refers to any collection of facts or data. In reference to information stored or processed using a computer system(s), including but not limited to internets, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.). As used herein, the term “information related to a subject” refers to facts or data pertaining to a subject (e.g., a human, plant, or animal). The term “genomic information” refers to information pertaining to a genome including, but not limited to, nucleic acid sequences, genes, percentage methylation, allele frequencies, RNA expression levels, protein expression, phenotypes correlating to genotypes, etc. As used herein, the terms “sample,” “test sample,” and “biological sample” refer to a sample containing or suspected of containing a biomarker of the present disclosure. The sample may be derived from any suitable source. In some cases, the sample may comprise a liquid, fluent particulate solid, or fluid suspension of solid particles. In some cases, the sample may be processed prior to the analysis described herein. For example, the sample may be separated or purified from its source prior to analysis. In a particular example, the source is a mammalian (e.g., human) bodily substance (e.g., bodily fluid, blood such as whole blood, buffy coat, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, lacrimal fluid, lymph fluid, amniotic fluid, interstitial fluid, cerebrospinal fluid, feces, tissue, organ, one or more dried blood spots, or the like). The sample may be a liquid sample or a liquid extract of a solid sample. In some embodiments, the source of the sample may be an organ or tissue, such as a biopsy sample and/or an endoscopic brushing sample (e.g., endoscopic esophageal brushing sample), which may be solubilized by tissue disintegration/cell lysis. Samples can be obtained by any number of methodologies. Cell free or substantially cell free samples can be obtained by subjecting the sample to various techniques including but are not limited to, centrifugation and filtration. In some embodiments, nucleic acid is isolated from a sample (e.g., a tissue sample, a blood sample, a plasma sample, a serum sample, a whole blood sample, a buffy coat sample, a secretion sample, an organ secretion sample, a cerebrospinal fluid (CSF) sample, a saliva sample, a urine sample, and/or a stool sample) using direct gene capture, e.g., as detailed in U.S. Pat. Nos.8,808,990 and 9,169,511, and in WO 2012/155072, or by a related method.
Attorney Docket No. ACGI-43252.601 As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of antiviral agents, such delivery systems include systems that allow for the storage, transport, or delivery of antiviral agents and/or supporting materials (e.g., written instructions for using the materials, etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant antiviral agents. As used herein, the term “fragmented kit” refers to delivery systems comprising two or more separate containers that each contain a sub-portion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain a composition comprising an antiviral composition for a particular use, while a second container contains a second agent (e.g., a second antiviral). Any delivery system comprising 2 or more separate containers that each contains a sub-portion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of an antiviral agent needed for a particular use in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list
Attorney Docket No. ACGI-43252.601 of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. The term “about” as used herein means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ± 20 %, preferably up to ± 10 %, more preferably up to ± 5 %, and more preferably still up to ± 1 % of a given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value. As used herein, a “system” refers to a plurality of components operating together for a common purpose. In some embodiments, a “system” is an integrated assemblage of hardware and/or software components. In some embodiments, each component of the system interacts with one or more other components and/or is related to one or more other components. In some embodiments, a system refers to a combination of components and software for controlling and directing methods. For example, a “system” or “subsystem” may comprise one or more of, or any combination of, the following: mechanical devices, hardware, components of hardware, circuits, circuitry, logic design, logical components, software, software modules, components of software or software modules, software procedures, software instructions, software routines, software objects, software functions, software classes, software programs, files containing software, etc., to perform a function of the system or subsystem. Thus, the systems or methods provided herein, or certain aspects or portions thereof, may take the form of program code (e.g., instructions) embodied in tangible media, such as floppy diskettes, CD-ROMs, hard drives, flash memory, or any other machine-readable storage medium wherein, when the program code is loaded into and executed by a machine, such as a computer, the machine becomes an apparatus for practicing the embodiments. In the case of program code execution on programmable computers, the computing device generally includes a processor, a storage medium readable by the processor (e.g., volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device. One or more programs may implement or utilize the processes
Attorney Docket No. ACGI-43252.601 described in connection with the embodiments, e.g., through use of an application programming interface (API), reusable controls, or the like. Such programs are preferably implemented in a high-level procedural or object-oriented programming language to communicate with a computer system. However, the program(s) can be implemented in assembly or machine language, if desired. In any case, the language may be a compiled or interpreted language, and combined with hardware implementations. DETAILED DESCRIPTION Provided herein are methods, compositions, systems and kits to amplify DNA, RNA and protein macromolecules. In particular, provided herein are compositions and methods to amplify macromolecules for molecular analysis including low abundance template input, single-cell and single nucleus applications. In some embodiments, the present invention provides methods, compositions, systems and kits for whole genome DNA amplification (WGA). In some embodiments, the WGA is compatible with both short and long read next generation sequencing (NGS). In some embodiments, the present invention provides amplification for whole genome sequencing (WGS) and whole epigenome sequencing (WES). In some embodiments, WGS and WES are configured for very low template input including single-cell and single-nucleus template input comprising, for example, less than 1 picogram, and 1 femtogram of template. In some embodiments, the amplification product is suitable for detection of CNVs, SNPs, indels and/or single nucleotide variants (SNVs). In some embodiments the amplification product is suitable for diverse library fragment sizes comprising, for example, repetitive regions, microsatellite regions, and somatic expansion or contraction of pathogenic trinucleotide repeats as seen, for example, in fragile X syndrome and Huntington’s disease. In some embodiments, the present invention provides improved genome coverage of G:C regions. In some embodiments, the present invention provides methods, compositions, systems and kits for enrichment of extranuclear nucleic acids including, for example exosomal DNA and RNA, mitochondrial DNA, cytoplasmic DNA and RNA, plasmid DNA, chloroplast DNA, and DNA and RNA from other organelles. In some embodiments, the present invention provides a 2-step WGA method comprising, for example, 2 hands-on steps (e.g., denaturation and amplification) and 10 or fewer pipetting steps in under 30 minutes with completion of WGA in one hour or less. In some embodiments, the present invention comprises isothermal reaction conditions. In some
Attorney Docket No. ACGI-43252.601 embodiments, the present invention provides warm start reactions (e.g., at 42oC) that preclude reaction initiation prior to thermal control. In some embodiments, the reaction set up takes place at room temperature. In some embodiments, elevated thermal control ameliorates NTC amplification. In some embodiments, a buffering system eliminates an error-prone neutralization step. In some embodiments, pipetting volume prevents sample dropouts in plates. In some embodiments, greater amounts of amplified DNA/reaction support multiple analyses. In some embodiments, the methods, compositions, systems and kits of the present invention provide up to 2 micrograms of amplified DNA product from as low as 2.5 femtograms of input (for example, a range of 2.5 ng-2.5fg of input human gDNA) template, and/or the DNA from a single cell or nucleus with limited variability introduced by operator error. (Figure 1) In some embodiments, single and multiple cells are amplified by the methods, compositions, systems and kits of the present invention with detection supported in less than 30 minutes and completion in one hour. In some embodiments, 97% of a WGA reaction may be detected from a single cell after flow activated cell sorting (FACS). (Figure 2 and Figure 3) In some embodiments, the amplified product has a size range of ~ 6 kB average (blue curve), and the library product has a size range of ~ 0.3 kB average (red curve). (Figure 4) In some embodiments, the present invention provides methods, compositions, systems and kits for WGA suitable for detection of copy number variation (CNV). In some embodiments, CNV analysis after WGA methods and compositions of the present invention supports detection of both amplification and deletion. In some embodiments, both whole chromosome and sub-chromosomal aneuploidy may be detected. In some embodiments, a range of NGS analysis depth (~0.25X) is supported that directly decreases cost per cell for both NGS and computation with close correlation between and within sample sets. (Figure 5) Figure 6 shows detection of SNVs after WGA using methods and compositions of the present invention. Using methods and compositions of the present invention DNA template abundance ranging from low 2.50 ng. and 2.5 fg. from multiple cells down to templates from 3 cell sand one cell are comparable in sensitivity and precision with high repeatability between and within sets of sample sources. In some embodiments, methods, compositions, systems and kits of the present invention provide DNA NGS-workflow comprising stepwise DNA isolation (including single cell DNA isolation), whole genome amplification, and NGS library construction in support of the detection and identification of one or more CNVs and SNVs from exomes and whole genome sequences in sequential and/or simultaneous formats on shared platforms at low cost, for example, at less than $1.00 per NGS cell. In
Attorney Docket No. ACGI-43252.601 In some embodiments, the present invention provides methods, compositions, systems and kits for RNA amplification. In some embodiments, the RNA amplification is whole transcriptome amplification. In some embodiments, the present invention provides methods, compositions, systems and kits for combined WGS and whole transcriptome amplification. In some embodiments, the combined amplification is sequential amplification. In some embodiments, the combined amplification is simultaneous amplification. In some embodiments, the present invention provides methods of amplifying RNA alone and in combination amplifying DNA including template switching, second strand synthesis by nick translation, second strand synthesis by incorporating RT, RNAse H, DNA Pol1 and DNA ligase into a single reaction, helicase mediated Phi 29 amplification after first and second strand synthesis, and BST (Type A Pol). In some embodiments, the present invention provides methods, compositions, systems and kits for protein amplification. In some embodiments, the present invention provides methods, compositions, systems and kits for combined WGS, WES, whole transcriptome and protein amplification, In some embodiments, the compositions, systems, kits and methods of the present invention find use in applications wherein accurate and precise measurement of nucleic acid allele frequencies in a sample are desired. In some embodiments, the allele frequency of a target polymorphism is 1% or less. In some embodiments, the allele frequency of a target polymorphism is 1% or greater. In some embodiments, the nucleic acid is DNA, for example, genomic DNA, complementary DNA, cell-free DNA (cfDNA), cell-free tumor DNA (cf-tDNA) circulating DNA, exosomal DNA, human DNA, eukaryote DNA, prokaryote DNA and pathogen DNA. In some embodiments, the nucleic acid is RNA, for example, mRNA, rRNA, tRNA, miRNA, siRNA piRNA, human RNA, eukaryote RNA, prokaryote RNA and pathogen RNA. In some embodiments, the sample is a fluid sample, a blood cell sample, a plasma sample, a serum sample, a urine sample, a saliva sample, a CSF sample, a healthy tissue sample, a tumor tissue sample, a biopsy sample and a biopsy margin sample. In some embodiments, the methods, compositions, systems and kits of the present invention are used for single cell genome measurements in oncology that are critical for appraisal of, for example, the genetic heterogeneity of cancers, the detection of rare mutations, and the guidance for individualized therapy. In some embodiments, cancer cells are isolated using, for example, Fluorescence-Activated Cell Sorting (FACS) and Laser Capture Microdissection (LCM). In some embodiments, the present invention supports
Attorney Docket No. ACGI-43252.601 analysis of single cells and/or ultra-low amounts of nucleic acid in support of, for example, minimal residual disease and circulating cancer cells. In some embodiments, methods and compositions of the present invention support high depth WGS, for example, ~450 million reads per cell for comprehensive analysis. In some embodiments, methods and compositions of the present invention support targeted sequencing, for example, exome and panel sequencing that focus on specific genes and gene regions of interest with less depth required. In some embodiments, the present invention supports high sensitivity and precision detection and identification of genetic variants including SNPs, indels, SNVs and CNVs. In some embodiments, the detection and identification is detection and identification or rare mutations at low frequency that are often missed in bulk sequencing albeit critical for management based on cancer heterogeneity and drug resistance. In some embodiments, methods and compositions of the present invention support analysis of genetic diversity within tumors, that track clonal evolution and metastatic potential. In some embodiments, methods and compositions of the present invention support individualized treatments based on the unique genetic profile of a patient's tumor, including the identification of actionable mutations. In some embodiments, methods and compositions of the present invention support detection of minimal residual disease comprising monitoring of residual cancer cells during and after treatment to detect relapse early. In some embodiments, methods and compositions of the present invention support analysis of circulating cancer cells for non-invasive cancer diagnosis and monitoring. In some embodiments, the methods, compositions, systems and kits of the present invention are used for single-cell genome measurements in archived DNA samples to detect genetic information from preserved specimens that are often essential for retrospective studies. In some embodiments, methods and compositions support DNA extraction from diverse sources including formalin-fixed paraffin-embedded (FFPE) tissues, frozen tissues, and blood samples. In some embodiments, methods and compositions support amplification of degraded or fragmented DNA often found in archived samples that further supports DNA sequencing. In some embodiments, the present invention provides methods and composition for conversion of amplified DNA into sequencing libraries compatible with both short-read and long-read sequencing platforms, and adaptable to diverse coverage depths based on the type of analysis. For example, WGS may require high depth for comprehensive analysis whereas target sequencing for analysis of specific regions of interest may require less depth.
Attorney Docket No. ACGI-43252.601 In some embodiments, methods, composition, systems and kits of the present invention support detection and characterization of DNA sequence variation in embryo biopsies. In some embodiments the present invention supports quantitative, genome-wide assessment of genome editing at the single cell level. In some embodiments, the present invention supports microbial genomics for the detection and identification of rare and difficult to challenge microorganisms. In some embodiments, input templates are prepared in a plate format, a tube format or a combination plate and tube format. In some embodiments, the methods, compositions, systems and kits comprise steps, reagents, components, hardware and software for cell lysis and nucleic acid isolation, purification, quantification, amplification library preparation and clean up, for example, bead-based clean up. In some embodiments, methods, composition, systems and kits of the present invention comprise an enzyme. In some embodiments, the enzyme is an A-Type or B-type polymerase (PolB) that requires RNA (or DNA) primed templates for DNA synthesis is found in all domains of life and many DNA viruses. Members of the Family B include Polymerase α, ε, ζ, and δ that function to proofread newly synthesized DNA in the 3′→5′ direction, and are capable of synthesizing DNA on both the leading and lagging strands. PolB enzymes are sufficiently accurate to correct mispairings that occur during DNA synthesis. In some embodiments, a PolB enzyme of the present invention is Phi29, Gp32, DPO1, DP02, Dp03, Equiphi29 -thermal stable phi29-(Thermo), Phi29-XT thermal stable (NEB) or HotJa Phi29. In some embodiments, methods, composition, systems and kits of the present invention comprise a buffer. In some embodiments the buffer is [N-Morpholino]propane sulfonic acid (MOPS), 2-Hydroxy-3-morpholinopropanesulfonic acid (MOPSO), 2-[4-(2- hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2,2′-[(2-amino-2- oxoethyl)azanediyl]diacetic acid (ADA), Piperazine-1,4-bis(2-hydroxypropanesulfonic Acid (POPSO), or 2-Hydroxy-3-(4-(2-hydroxyethyl)piperazin-1-yl)propane-1-sulfonic acid(HEPSO. In some embodiments, the buffer is not Tris(hydroxymethyl)aminomethane (TRIS). In some embodiments, methods, composition, systems and kits of the present invention comprise a pyrophosphatase. In some embodiments, the pyrophosphatase is TheraPure™ GMP Pyrophosphatase, Thermostable Inorganic Pyrophosphatase (e.Coli), Thermostable Inorganic Pyrophosphatase (yeast), or NudC Pyrophosphatase.
Attorney Docket No. ACGI-43252.601 In some embodiments, methods, composition, systems and kits of the present invention comprise a reducing agent. In some embodiments, the reducing agent is Tris (2- carboxyethyl) phosphine (TCEP). In some embodiments, the reducing agent is not dithiothreitol (DTT). In some embodiments, methods, compositions, kits and systems of the present invention support amplification of diverse macromolecules alone or simultaneously in a single vessel or multiple vessels. In some embodiments, the present invention provides reagents in 1, 2 or 3 or more vessels, In some embodiments, the present invention comprises a kit comprising 3 vessels wherein a reducing agent that is stable in a basic solution (pH 13) is in a first vessel, one or more primers are present in a second vessel comprising a detergent, and amplification components are provided as a mastermix in a third vessel. EXPERIMENTAL EXAMPLES Example 1 – DNA amplification form multiple cell and single cell templates The nucleic acid amplification methods, compositions, systems and kits of the present invention have demonstrated robust DNA amplification from both multiple cell and single cell templates with amounts of DNA templates that vary over orders of magnitude. (Figure 1, Figure 2, Figure 3) Real-time analysis on a thermal cycler showed rapid and robust amplification with minimal non-template control amplification. (Figure 1, Figure 2) Over 95% of single-cell reactions generated reproducible amplification curves. (Figure 3) The amplified DNA fragments ranged from ~0.5-10 kb, while library fragments were around 350 bp. (Figure 4) Libraries generated from samples, including cells known to have no aneuploidy (HG0001), and samples with chromosomal deletions or amplifications, were sequenced at a depth of 1 million reads per cell ($1.00 USD per cell). Example 2 – Next generation sequencing (NGS) of single cell DNA Whole Genome Sequencing (WGS) requires 450 million reads ($150 USD per cell) for ~10-20X coverage, Exome sequencing requires 20-40 million reads ($10-15 USD per cell), and panel NGS costs vary based on panel size ($0.5-5.0 USD per cell). Figure 5 shows analysis of a cancer cell line to determine the degree of chromosomal abnormality after DNA amplification using methods and compositions of the present invention. Single single-cell CNV data accurately detected CNV at low NGS depth in support of decreased NGS cost and time. High-depth NGS analysis achieved 92% sensitivity and over 99% precision for single-
Attorney Docket No. ACGI-43252.601 cell samples. (Figure 6). Multi-cell samples showed even higher sensitivity (>95%) with similar precision. Example 3 - DNA amplification over a wide range of input DNA amounts DNA reactions were assembled including a DNA binding dye (EvaGreen) to detect the amplification of genomic DNA over a range of inputs (2.5ng – 2.5fg) including a non- template reaction (NTC). (Figure 7) Reactions were incubated at 45oC for 90 minutes, and imaged on a 3 minute interval to observe an increase in amplified DNA. The highest concentrations were detectable within 6 minutes (2.5ng), while low input reactions (e.g., 2.5fg), were detectable after 54 minutes. Detection of the NTC did not occur until 66 minutes thereby supporting amplification and detection DNA amounts over7 logs of input dynamic range. Reactions are terminated by increasing the temperature to 65 degrees for 10 min. Example 4 - Real-time detection of single cell genomic DNA (gDNA) amplification Reactions were assembled including a DNA binding dye (EvaGreen) to detect amplification of genomic DNA within single cells. Reactions were incubated at 45oC for 60 minutes and imaged on a 3-minute interval to observe an increase in amplified DNA. Single cell templates amplified in a reproducible manner becoming detectable within 12 minutes. (Figure 8) Reactions were terminated by increasing the temperature to 65 degrees for 10 min. Example 5 - DNA yields from the amplification of a single cell genome Ten microliter reactions were assembled to detect amplification of genomic DNA within single cells. Reactions were incubated at 45oC for 60 minutes and terminated by increasing the temperature to 65 degrees for 10 min. The amplified samples were diluted 1:20, and 2 ul of the dilution was subsequently analyzed by Qubit to determine the yield of amplified DNA from single cell samples and three cell samples. Single cells amplified in a reproducible manner yielding an average of ~ 2100 ng of DNA (range 1800-2740 ng). (Figure 9) Sample with 3 cells generated ~ 4500ng of amplified DNA in 60 minutes of isothermal amplification. Example 6 - Fragment sizes of amplified DNA products Ten microliter reactions were assembled to detect the amplification of genomic DNA within single cells. Reactions were incubated at 45oC for 60 minutes and terminated by
Attorney Docket No. ACGI-43252.601 increasing the temperature to 65 degrees for 10 min. The amplified samples were diluted to ~ 1ng/ul and subsequently analyzed by Agilent BioAnalyzer high sensitivity DNA chip. Single cell DNA was amplified to produce a pool of large fragments that were approximately 8-10 kilobases in length. (Figure 10, blue line) To determine if the amplified products could be converted into a next generation sequencing (NGS) library, a kit including fragmentation, end repair, and ligation was used to generate a NGS library (Agilent BioAnalyzer 2100 HS DNA chip). The fragment size created by this process (redline) generated an amplicon of ~ 350 base pairs suitable for short read next generation sequencing analysis. Example 7 - Optimized amplified fragment sizes Ten microliter reactions were assembled to detect the extent of fragment size modification that occurred due to the inclusion of a polysorbate detergent during the denaturation steps of the DNA amplification reaction. Reactions were incubated at 45oC for 60 minutes and terminated by increasing the temperature to 65 degrees for 10 min. The amplified samples were diluted to ~ 1ng/ul and subsequently analyzed by Agilent BioAnalyzer high sensitivity DNA chip. Decreasing the amount of the polysorbate in the reaction had a pronounced effect of the size of the amplified DNA fragment length. (Figure 11) Reactions that included 0.4% Tween generated fragments approximately 2500 bp in length. Decreasing from 0.4 to 0.3 % polysorbate in the reaction increased the fragment size approximately 2000 bp. By reducing the polysorbate concentration further to 0.1% the size fraction again increased to ~ 7000 bp. The largest product fragments were generated with elimination of the polysorbate detergent with an increase in fragment size to nearly 4-fold. Example 8 - Effects of polysorbate detergent during denaturation steps of the DNA amplification reaction Ten microliter reactions were assembled to detect the DNA yield modification that occurred due to the inclusion of a polysorbate detergent (0.3%) during the denaturation steps of the DNA amplification reaction. Reactions were incubated at 45oC for 60 minutes and terminated by increasing the temperature to 65 degrees for 10 min. The amplified samples diluted 1:20, and 2 ul of the dilution was subsequently analyzed by Qubit to determine the yield of amplified DNA. Different concentrations of detergent had a pronounced effect on the yield of the DNA amplification reaction. (Figure 12A.) Comparing 7 logs of DNA concentration differences, DNA products were generated from all reactions that omitted the polysorbate detergent, while reactions that included the polysorbate had less comparable
Attorney Docket No. ACGI-43252.601 yield, and were unable to detect the lowest 3 concentrations of DNA (250fg, 25fg, and 2.5fg), resulting in just 4 log of detection range, compared to 7 logs of the polysorbate exclusive DNA amplification reactions. Inhibition of yield was also more pronounced with time as a factor. (Figure 12B.) When reactions were allowed to stand for 2 hours, the polysorbate inclusive samples failed to generate any product, resulting in no dynamic range or detectable DNA amplification compared to those reactions that omitted the polysorbate. Reactions comprising polysorbate detergent generated fragment truncation and yield loss and was thus omitted from consequent DNA amplification chemistry. Example 9 - Copy number analysis of single cell amplified DNA Ten microliter reactions were assembled to detect the extent of copy number variation (CNV) from single cell samples. Reactions were incubated at 45oC for 60 minutes and terminated by increasing the temperature to 65 degrees for 10 min. Approximately 300 ng of the amplified DNA was used to create a NGS library for analysis. Libraries were sequenced at a read depth of 1 million reads /cell. Data was processed using a customized version of Ginkgo to determine the extent of CNV present in the reference samples. (Garvin T. et al. Interactive analysis and assessment of single-cell copy-number variations. Nat Methods.2015 Nov;12(11):1058-60.) Single cell copy CNVs were prominent in NGS displays (Figure 13, square highlights). Example 10 - Single nucleotide variant detection with low sequencing depth compared to alternative chemistry platforms Ten microliter reactions were assembled to detect the extent of genome coverage from single cell samples. Reactions were incubated at 45oC for 60 minutes and terminated by increasing the temperature to 65 degrees for 10 min. Approximately 300 ng of the amplified DNA was used to create a NGS library for analysis. Libraries were sequenced at a read depth of 2 million reads (~ 1x)/cell. Data was processed using a custom bioinformatics platform compared to HG38 human reference genome to determine the degree of genome coverage present in the reference samples. Even with low depth of sequencing, genome coverage was estimated to cover over 95% of genome using low pass estimation tools and compared equivalently or better to a diversity of conventional chemistry platforms. (Figure 14) (Lianti, Chen C. et al. Single-cell whole-genome analyses by Linear Amplification via Transposon Insertion (LIANTI). Science.2017 Apr 14;356(6334):189-194.) (Malbac, Yikon Genomics, Jingsu, China), (PTA, BioSkryb Genomics, Durham, NC), DOP PCR, ReproSeq,
Attorney Docket No. ACGI-43252.601 Thermofisher, Waltham, MA), (PicoPlex, Takara, San Jose, CA), BE/Cytiva, MDA, Genomiphi, Milipore Sigma, St. Lousi, MO), Repligen Qiagen MDA, Hilden, German) Example 11 - Reverse transcription (RT) efficiency Ten microliter reactions were assembled to detect the ability of an embodiment of the present invention to reverse transcribe (RT) total RNA over a range of concentrations. Reactions were incubated at 45oC for 90 minutes and terminated by increasing the temperature to 75 degrees for 10 min. First strand cDNA products were then analyzed using real-time qPCR. The RT reaction converted mRNA amongst the total RNA samples containing from 200ng input to as low as 2 picograms (~ 20pgs in a single cell) (Figure 15) with no detection of cDNA from the sample containing 0.2 pg (0.0002 ng) and the no- template control (NTC). Example 12 – In vitro transcription (IVT) amplification yields at 4 hours 10 microliter reactions were assembled to detect the ability of an embodiment of the present invention to amplify transcripts from a total RNA sample over a range of concentrations. Reactions containing cDNA from the total RNA were incubated at 42oC for 240 minutes and terminated by increasing the temperature to 72 degrees for 10 min. By analyzing the outputted single -stranded RNA, it was found that the dynamic range was a slightly reduced compared to the qPCR results of amplifying the first strand cDNA, reaching 10pg/sample as a detection limit. (Figure 16) No RNA was detected in the lowest 3 concentration samples (1pg, 0.1pg and NTC). Example 13 - IVT amplification over a range of RNA concentrations Ten microliter reactions were assembled to detect the ability of an embodiment of the present invention to amplify transcripts from a total RNA sample over a range of concentrations. Reactions containing the cDNA from the total RNA were incubated at 42oC for 240 minutes and terminated by increasing the temperature to 72 degrees for 10 min. By analyzing the outputted single -stranded RNA, it was found found that the size range of the RNA transcribed created a range of transcripts up to 20kb. (Figure 17) While electrophoretic analysis demonstrated a preponderance of small species (likely < 100 bases), evidence of long transcripts, of 1000+ kb in size are also present. Because the samples were not purified to remove small RNA species prior to analysis, it’s clear that the IVT reaction is functioning and creates long transcripts.
Attorney Docket No. ACGI-43252.601 Example 14 - RT reaction followed by isothermal amplification of the RT-produced cDNA To test an isothermal process to perform reverse transcription (RT) and cDNA amplification, the RT reaction was followed by an isothermal process to amplify resultant RT- generated cDNA. (Figure 18) First total RNA was exposed to a combination of random primers as well as an oligo dT primer. The reaction was carried out at 42oC for 90 minutes and was immediately followed by addition of reagents required to amplify long cDNAs generated. The reaction was then carried out 45oC for one hour. Total RNA templates in a range of concentrations were added to a microcentrifuge tube. A reaction buffer comprising primer (1uM), dNTP (1mM), and detergent (Triton x-100, (0.1%)), NH4SO4 (5mM), MgAoC (22mM), TRIS (50mM), pH 8.2 including a thermo-stable reverse transcriptase was added. The total reaction volume was 10ul. The sample was incubated at 42oC for 90 min, after which a solution (20ul) of high concentration KOH base (475mM) was added to denature the sample along with EDTA (2.5mM), and allowed to stand at room temperature for 15 min. The reaction mix containing primer (2uM), dNTP (1mM), and Triton x-100 detergent (0.1%), NH4SO4 (5mM), MgAoC (22mM), MOPS (50mM) including a thermal stable B-type polymerase were added (40ul) to the denatured sample and incubated at 45oC for one hour, including an enzyme deactivation step of 75oC for ten minutes. Example 15 - RT optimization for cDNA amplification Ten microliter reactions were assembled to detect reverse transcription of total RNA. Reactions containing a range of RNA inputs were incubated at 42oC for 90 minutes and terminated by increasing the temperature to 75 degrees for 10 min. cDNA products were then analyzed by qPCR to identify the optimal buffer for the reverse transcription, as well as ensuring that the RT step may be integrated with the isothermal DNA amplification step. While all buffer components generated cDNA across the RNA dilution range, the TRIS buffer (pH 8.2) performed optimally. (Figures 19A-C) By adding 2X the volume of denaturing buffer the denaturation pH exceeded 12.0, a requirement to reach optimal DNA denaturation. The reaction buffer buffered the b-type polymerase reaction to ~ 7.5pH, that is optimal for the cDNA amplification. In Figure 19A (MOPS – pH 6.9), Figure 19B (HEPES pH 7.4) and Figure 19C (Tris-HCL pH 8.2) show that the reactions are RT reactions that are optimal in Tris-HCL (pH 8.2), with detection of 20.0 ng ( red line), 20ng (yellow line), 200pg (light green line), 20pg (dark green line), 2 pg (light blue line), 200fg (pink-line), and 20 fg
Attorney Docket No. ACGI-43252.601 (dark blue line), and NTC (not detected purple line) in linear regression. In contrast, the MOPS and HEPES buffers did not perform with the comparable linearity. Example 16 - Whole genome amplified (WGA) DNA yields from RT of total RNA To test an isothermal process to perform reverse transcription (RT) and cDNA amplification in an embodiment of the present invention, the RT reaction was followed by an isothermal process to amplify the resultant RT-produced cDNA. The reaction was carried out at 42oC for 90 minutes, which was immediately followed by adding reagents required to amplify long cDNAs that are generated. The reaction was then carried out 45oC for one hour. The amplified samples were diluted 1:10, and 2 ul of the dilution was analyzed by Qubit to determine the yield of amplified DNA from single cell samples and from 3 cell samples. Total RNA was both reverse transcribed and amplified by the 2-step system in a reproducible manner, yielding ~ 3500 ng of DNA from the 40ng input. (Figure 20) At lower concentrations, cDNA from as low as 40 fg was also amplified, generating 242 ng compared to the no-template control that showed no amplification. These data indicate that amplification of the reverse transcribed RNA was concentration dependent and generated abundant material to prepare libraries for downstream analysis processes including NGS and qPCR. Of note, even with single-cell concentrations of total RNA (~ 4-40pg) the combined process generated from 400-500 ng of amplified cDNA, compared to NTC. Example 17 - Amplified DNA yields (ng) from RT of 1, 3 and 5 cells To test an isothermal process to perform reverse transcription (RT) and cDNA amplification in one embodiment of the present invention, the RT reaction was followed by an isothermal process to amplify the result RT produced cDNA. The reaction was carried out at 42oC for 90 minutes, which was immediately followed by adding the reagents required to amplify the long cDNAs that are generated. This reaction was then carried out 45oC for one hour. The amplified samples were diluted 1:10, and 2 ul of the dilution was subsequently analyzed by Qubit to determine the yield of amplified DNA from single cell and three cell samples. RNA from 1,3, and 5 cells was both reverse transcribed and amplified by the 2-step system in a reproducible manner, yielding ~ 1800 ng of DNA from the samples containing 1 and 3 cells, while 5 cell samples generated roughly 3000ng of amplified cDNA. (Figure 21) The cell samples were incubated with thermal labile DNAse 1 prior to the RT and cDNA amplification steps to ensure that the amplified DNA was from the reverse transcribed cellular RNA, and not from genomic DNA within the cell.
Attorney Docket No. ACGI-43252.601 Example 18 - Amplified DNA yields from RT of 1, 3 and 5 cells with the RT reaction after an isothermal process to amplify the RT-produced cDNA To test an isothermal process to perform reverse transcription (RT) and cDNA amplification in an embodiment of the present invention, the RT reaction was followed by an isothermal process to amplify the resultant RT-generated cDNA. The reaction was carried out at 42oC for 90 minutes, which was immediately followed by adding the reagents required to amplify the long cDNAs that are generated. This reaction was then carried out 45oC for one hour. The amplified samples were diluted 1:100, and 1 ul of the dilution (~ 1ng) was subsequently analyzed by Bioanalyzer to determine the size range of amplified cDNA from single cell samples and from 3 cell samples. RNA from 1,3, and 5 cells was both reverse transcribed and amplified by the 2-step system in a reproducible manner, generating fragments in excess of 2000 bases, with a range from ~ 500 bases to nearly 20,000 bases. (Figure 22) Size range was not affected by the input of cells (1,3 or 5) with similar size ranges, Example 19 – Preparation to preparation variability in DNA yields To test lot-to-lot and preparation-to-preparation variability, we tested DNA yields in 9 independent component combinations. Samples containing genomic DNA were added as a dilution series to an 8-strip well, including 7 logs of DNA concentration and non-template control (0 or NTC). Reproducibility of the compositions, method steps and systems was high with deviation observed in the preparation of control standards rather than the chemistry of an embodiment of the present invention. (Figure 23) No evidence of significant NTC contamination or amplification was observed. Surprisingly, a single hour of amplification carried out 45oC for a single hour yields over 7 logs dynamic range of template concentrations generated > 100 ng of DNA from 2.5fg of input with the highest 2.5ng inputs generating up to 6000 ng of amplified DNA. No evidence of reproducible NTC amplification was observed with only one sample of 9 with amplification of the NTC at less than 150ng of DNA output. Example 20 WarmStart RTX reverse transcriptase (New England BioLabs, Ipswich, MA) supports multi-omic applications with room temperature reaction setup with inactive enzyme, and restoration of enzyme activity by heating. Figure 14 shows amplified DNA yields in ng over
Attorney Docket No. ACGI-43252.601 a temperature range from 5oC to 60oC with varying amounts of template DNA. These data show that the methods, compositions, systems and kits of the present invention are compatible with WarmStart protocols. INCORPORATION BY REFERENCE All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.