Single cell analysis

Abstract

Provided herein are compositions and methods for accurate and scalable primary template directed amplification (PTA) nucleic acid amplification and sequencing methods, and their applications for mutation analysis in research, diagnosis, and therapy. Further provided herein are multi-omics methods for parallel analysis of DNA, RNA, and / or protein from single cells. Provided herein are multi-omics single-cell analysis methods, comprising: (a) isolating a single cell from a cell population; (b) sequencing a cDNA library comprising polynucleotides amplified from mRNA transcripts from the single cell; (c) sequencing the genome of the single cell.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 881,183, filed on July 31, 2019, the contents of which are incorporated herein by reference in their entirety. Background Art

[0003] Research methods using nucleic acid amplification, such as next-generation sequencing, provide a wealth of information about complex samples, genomes, and other nucleic acid sources. In some cases, these samples are obtained in small quantities from single cells. For research, diagnosis, and treatment involving small samples, highly accurate, scalable, and efficient nucleic acid amplification and sequencing methods are needed, especially for the simultaneous analysis of RNA, DNA, and protein. Summary of the invention

[0004] Provided herein are methods for multi-omics single-cell analysis, the methods comprising: (a) isolating a single cell from a cell population; (b) sequencing a cDNA library comprising polynucleotides amplified from mRNA transcripts from the single cell; and (c) sequencing the genome of the single cell, wherein sequencing the genome comprises: (i) contacting the genome with at least one amplification primer, at least one nucleic acid polymerase, and a nucleotide mixture, wherein the nucleotide mixture comprises at least one terminator nucleotide, the at least one terminator nucleotide terminating nucleic acid replication of the polymerase; and (ii) amplifying at least some of the genome to produce a plurality of terminated amplification products, wherein the replication is performed by strand displacement replication; (iii) connecting the molecule obtained in step (ii) to an adapter, thereby producing a genomic DNA library; and (iv) sequencing the genomic DNA library. Also provided herein are methods, wherein the mRNA transcripts comprise polyadenylated mRNA transcripts. Also provided herein are methods, wherein the mRNA transcripts do not comprise polyadenylated mRNA transcripts. Also provided herein are methods, wherein sequencing the cDNA library comprises amplifying mRNA transcripts with template switching primers. Also provided herein is a method, wherein at least some of the polynucleotides in the cDNA library comprise a barcode. Also provided herein is a method, wherein the barcode comprises a cell barcode or a sample barcode. Also provided herein is a method, wherein the cDNA library and the genomic DNA library are combined prior to sequencing. Also provided herein is a method, wherein the single cell is a primary cell. Also provided herein is a method, wherein the single cell is derived from liver, skin, kidney, blood or lung. Also provided herein is a method, wherein the single cell is isolated by flow cytometry. Also provided herein is a method, wherein the method further comprises removing at least one terminator nucleotide from the terminated amplification product. Also provided herein is a method, wherein the length of the multiple terminated amplification products comprises an average of 1000-2000 bases. Also provided herein is a method, wherein the length of the multiple terminated amplification products is 250-1500 bases. Also provided herein is a method, wherein the multiple terminated amplification products comprise at least 97% of the genome of the single cell. Also provided herein is a method, wherein at least some of the amplification products comprise a cell barcode or a sample barcode. Also provided herein is a method, wherein sequencing the cDNA library comprises cytosolic lysis of the single cell, and reverse transcription. Also provided herein is a method, wherein the mRNA transcript is amplified via template switching reverse transcription. Also provided herein is a method, wherein the cDNA library comprises at least 10,000 genes. Also provided herein is a method, wherein sequencing the genome of the single cell also comprises nuclear lysis of the single cell. Also provided herein is a method, wherein the method further comprises an additional amplification step using PCR.Also provided herein are methods, wherein at least one mutation is identified in the genome of the cell, wherein the mutation is different from the corresponding position in the reference sequence. Also provided herein are methods, wherein the at least one mutation occurs in less than 1% of the cell population. Also provided herein are methods, wherein the at least one mutation occurs in no more than 0.1% of the cell population. Also provided herein are methods, wherein the at least one mutation occurs in no more than 0.001% of the cell population. Also provided herein are methods, wherein the at least one mutation occurs in no more than 1% of the amplified product sequence. Also provided herein are methods, wherein the at least one mutation occurs in no more than 0.1% of the amplified product sequence. Also provided herein are methods, wherein the at least one mutation occurs in no more than 0.001% of the amplified product sequence.

[0005] Provided herein is a method for multi-omics single-cell analysis, the method comprising: (a) isolating a single cell from a cell population; (b) identifying at least one protein on the surface of the single cell; and (c) sequencing the genome of the single cell, wherein sequencing the genome comprises: (i) contacting the genome with at least one amplification primer, at least one nucleic acid polymerase, and a nucleotide mixture, wherein the nucleotide mixture comprises at least one terminator nucleotide, the at least one terminator nucleotide terminating nucleic acid replication of the polymerase; (ii) amplifying at least some of the genome to produce a plurality of terminated amplification products, wherein the replication is performed by strand displacement replication; (iii) connecting the molecule obtained in step (ii) to an adapter, thereby producing a genomic DNA library; and (iv) sequencing the genomic DNA library. Also provided herein is a method, wherein identifying at least one protein on the surface of the cell comprises contacting the cell with a labeled antibody that binds to the at least one protein. Also provided herein is a method, wherein the labeled antibody comprises at least one fluorescent label or mass tag. Also provided herein is a method, wherein the labeled antibody comprises at least one nucleic acid barcode.

[0006] Provided herein is a method for multi-omics single-cell analysis, the method comprising: (a) isolating a single cell from a cell population; (b) sequencing the genome of the single cell, wherein sequencing the genome of the cell comprises: (i) digesting the genome with a methylation-sensitive restriction enzyme to produce genomic fragments; (ii) contacting at least some of the genomic fragments with at least one amplification primer, at least one nucleic acid polymerase, and a nucleotide mixture, wherein the nucleotide mixture comprises at least one terminator nucleotide, and the at least one terminator nucleotide terminates nucleic acid replication of the polymerase; (iii) amplifying at least some of the genome to produce multiple terminated amplification products, wherein the replication is performed by strand displacement replication; (iv) amplifying at least some of the genomic fragments using methylation-specific PCR; (v) ligating the molecules obtained in steps (iii and iv) to adapters to produce a genomic DNA library and a methylome DNA library; and (vi) sequencing the genomic DNA library and the methylome library.

[0007] Incorporation by reference

[0008] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and accompanying drawings which set forth illustrative embodiments in which the principles of the invention are utilized, in which:

[0010] Figure 1A An overview of the overall workflow for isolating and analyzing protein, DNA, and RNA from single cells is presented.

[0011] Figure 1B A workflow is demonstrated for isolating and analyzing protein, DNA, and RNA from single cells using sample splitting to minimize cross-contamination.

[0012] Figure 1C Demonstrated workflow for isolating and analyzing protein, DNA, and RNA from single cells using single-tube preamplification.

[0013] Figure 1D A workflow is demonstrated for isolating and analyzing protein, DNA, and RNA from single cells using a single-tube preamplification using terminators to reduce amplicon size.

[0014] Figure 1EA workflow for isolating and analyzing protein, DNA, and RNA from single cells using co-amplification is demonstrated.

[0015] Figure 1F An informatics workflow for combining data from the protein / DNA / RNA single-cell experiments described herein is presented.

[0016] Figure 1G A comparison of MDA and PTA-irreversible terminator methods is shown when it comes to mutation propagation. The PTA method results in a direct copy number increase of the original DNA template.

[0017] Figure 2A The process steps performed after amplification are shown, including removal of terminators, repair of ends, and A-tailing prior to adapter ligation. Hybridization-mediated enrichment of all exons or other specific regions of interest can then be performed in the pooled cell library prior to sequencing. The source cell of each read is identified by a cell barcode (shown as green and blue sequences).

[0018] Figure 2B (GC) shows the comparison of GC content of sequenced bases from MDA and PTA experiments.

[0019] Figure 2C Shown are the mapping quality scores (e) (mapQ) of single cells mapped to the human genome (p_mapped) after PTA or MDA.

[0020] Figure 2D Percentage of reads mapping to the human genome (p_mapped) after single cells were subjected to PTA or MDA.

[0021] Figure 2E (PCR) shows a comparison of the percentage of reads that are PCR replicates among 20 million subsampled reads after single cells were subjected to MDA and PTA.

[0022] Figure 2F A workflow for RT amplification of single cells for use with PTA is demonstrated.

[0023] Figure 2G The generation of a library from cDNA obtained by RT is shown.

[0024] Figure 3A Shown are the mapping quality scores (c) (mapQ2) of single cells mapped to the human genome (p_map2) after reversible or irreversible terminator PTA.

[0025] Figure 3B Shown are the percentages of reads that mapped to the human genome (p_mapped2) after single cells were subjected to reversible or irreversible terminator PTA.

[0026] Figure 3C A series of box plots are shown using various methods, depicting the average percentage of reads that overlap with Alu elements for aligned reads. PTA has the maximum number of reads aligned to the genome.

[0027] Figure 3D Shown is a series of box plots obtained using various methods, depicting the average percentage of reads overlapping Alu elements across PCR replicates.

[0028] Figure 3E Shown is a series of box plots using various methods, depicting the GC content of reads for the average percentage of reads overlapping with Alu elements.

[0029] Figure 3F Shown is a series of box plots using various methods, depicting the mapping quality of the average percentage of reads overlapping Alu elements. PTA had the highest mapping quality of the methods tested.

[0030] Figure 3G Shown is a comparison of the SC mitochondrial genome coverage breadth using different WGA methods at a fixed 7.5X sequencing depth.

[0031] Figure 4A The average coverage depth for a 10 kilobase window on chromosome 1 is shown after selecting high-quality MDA cells (representing approximately 50% of cells) compared to cells amplified with random primers PTA after downsampling to 40 million paired reads per cell. The figure shows that MDA is less uniform, with more windows having a coverage depth that is two-fold greater (box A) or less (box C) than the average coverage depth. There is no coverage at the centromere for both MDA and PTA (box B) due to the high GC content and low mapping quality of the repetitive region.

[0032] Figure 4B The plots showing the relationship between sequencing coverage and genomic position for MDA and PTA methods (top). The lower box plots show the allele frequencies for MDA and PTA methods compared to bulk samples.

[0033] Figure 5A A plot of genome coverage score versus number of genome reads is shown to evaluate the coverage of various methods at increasing sequencing depth. The PTA method approached both bulk samples at each depth, which is an improvement over the other methods tested.

[0034] Figure 5B The coefficient of variation of genome coverage versus the number of reads is shown to assess coverage uniformity. The PTA method was found to have the highest uniformity among the methods tested.

[0035] Figure 5C Lorenz plots of the cumulative fraction of total reads versus the cumulative fraction of the genome are shown. The PTA method was found to have the highest uniformity among the tested methods.

[0036] Figure 5D A series of box plots of the calculated Gini index for each method tested are shown to estimate the variance of each amplification reaction in terms of complete uniformity. The PTA method was found to have reproducibly higher uniformity than the other methods tested.

[0037] Figure 5E A plot of the fraction of called bulk variants versus the number of reads is shown. The variant call rate of each method was compared to the corresponding bulk sample at increasing sequencing depth. To estimate sensitivity, the percentage of variants found in each cell that were called in the corresponding bulk sample downsampled to 650 million reads was calculated at each sequencing depth ( Figure 5E The improved coverage and uniformity of PTA allowed the detection of 30% more variants compared to the second most sensitive method, Q-MDA.

[0038] Fig. 5F A series of box plots showing the average percentage of reads overlapping with Alu elements. The PTA method significantly reduced the allelic skew at these heterozygous sites. The PTA method amplified both alleles in the same cell more evenly relative to the other methods tested.

[0039] Figure 5G A plot of the specificity of variant calls versus the number of reads is shown to assess the specificity of mutation calls. Variants found using various methods that were not found in a large number of samples were considered false positives. Among the methods tested, the PTA method had the lowest false positive calls (highest specificity).

[0040] Figure 5H The proportion of false positive base changes for each type of base change is shown for various methods. Without being bound by theory, this pattern may be polymerase dependent.

[0041] Fig.5I A series of box plots showing the average percentage of reads overlapping Alu elements for false positive variant calls are shown. The PTA method produced the lowest allele frequency for false positive variant calls.

[0042] Figure 6(Part A) shows beads with oligonucleotides attached with cleavable linkers, unique cell barcodes and random primers. Part B shows that a single cell and beads are encapsulated in the same droplet, the cell is then lysed and the primers are cut. The droplet can then be fused with another droplet comprising a PTA amplification mixture. Part C shows that the droplet breaks after amplification and the amplicons from all cells are merged. The protocol according to the present disclosure is then used to remove terminators, end repair and add A tails, followed by linkers. The merged cell library is then subjected to hybridization-mediated enrichment for the exons of interest before sequencing. The cell barcode is then used to identify the source cell of each read.

[0043] Fig. 7A A workflow for analyzing single cells using PTA multiomic (or polyomic) is shown. Step A: Contact cells with antibodies containing fluorescent markers and oligonucleotide barcode tags. Step B: Sort cells based on fluorescent markers. Step C: Coat tubes with antibodies that bind to nuclei; lyse cells; cytoplasmic mRNA undergoes reverse transcription, while intact nuclei are bound to the tube wall.

[0044] Figure 7B Shown for upper connection Fig. 7A Step C of , workflow for multi-omics analysis of single cells using PTA. Step D: After reverse transcription, the RT fraction is taken out for sequencing analysis. Step E: Nuclei are lysed and the PTA method is performed on genomic DNA. Step F: PTA results in a short fragment cDNA pool with approximately 1000-fold amplification.

[0045] Fig. 8A Primers for reverse transcription and pre-amplification in a multi-omics DNA / RNA single-cell analysis workflow are presented.

[0046] Figure 8B The reverse transcription and pre-amplification workflow for multi-omics DNA / RNA single-cell analysis is demonstrated. Fig. 8A Primers.

[0047] Fig.9A A graph showing the growth rate of a parental cell line treated with 2 nM Quizartinib (2 nM) for a three-week period to generate an AML cell line that grows robustly in the presence of a FLT3 inhibitor. Resistant and parental single cells (FACS enriched) were then analyzed by RNA sequencing and low-throughput DNA sequencing analysis.

[0048] Fig. 9BRNA expression from both parental and resistant cultures was shown demonstrating the ability to generate cDNA pools (C) using one-pot RNAseq chemistry, and the genes expressed in these cells produced distinct patterns that enabled visualization of cell populations by gene expression on an average of approximately 10K genes detected per cell. In a separate workflow, single-cell genomes were amplified using the PTA method.

[0049] Fig. 9C Normalized gene expression profiles for an RNAseq-only control experiment are shown.

[0050] Fig.9D A graph showing the amount of amplified DNA produced by PTA relative to different protocols. Transcripts produced during the RT step (R) were not efficiently amplified by the PTA reaction compared to DNA, and DNA in single cells was efficiently amplified using the combined protocols (SC1-SC8) compared to standard PTA amplified genomes (D, RD) from single cells. NTC = no template control; R = RT step; D = PTA DNA step; RD = dual RT / PTA.

[0051] Fig. 10A Shown are mitochondrial chromosome amounts (%) for two different protocols (dual RNAseq / PTA, standard RNAseq) using a low-throughput sequencing protocol (~5 million reads / cell).

[0052] And the estimated genome size is greater than 3 billion bases.

[0053] Fig. 10B The replicate percentages are shown for two different protocols (dual RNAseq / PTA, standard RNAseq) using a low-throughput sequencing protocol (~5 million reads / cell).

[0054] Fig. 10C Shown are the estimated genome sizes for two different protocols (dual RNAseq / PTA, standard RNAseq) using a low-throughput sequencing protocol (~5 million reads / cell).

[0055] Fig. 10D Feature assignments for three scRNAseq datasets from molm13 cells using the dual RNAseq / PTA protocol are shown.

[0056] Fig.10E A graph showing the normalized expression profile of the Sum159 cell line obtained using a standard RNAseq protocol. P = parental cells. R = resistant cells.

[0057] Fig.10FA graph showing the normalized expression profile of the Sum159 cell line obtained using the dual RNAseq / PTA protocol. P = parental cells. R = resistant cells.

[0058] Fig.11A Results of deep sequencing of 7 parental and 5 resistant molm13 cells to an approximate depth (K) of 25x are shown. Reads were aligned to Hg38 using bwa mem. Quality control and SNV determination were performed using GATK4 optimal operation. SNVs were considered only if they were limited to at least 2 resistant cells, alternative alleles were not determined in any parental cell, and at least 6 parental cells were genotyped. All cells covered at least 96% of the genome at 1x coverage and at least 76% at 10x. The illustration shows the detection of known Flt3 indels in molm13 cells in all cells (4 are shown for clarity).

[0059] Fig. 11B A heat map of gene expression profiles including the overexpressed gene GAS6, a known mechanism of resistance to quizartinib, is shown. Gas6 is a ligand for AXL, a clinically relevant resistance mechanism in relapsed patients who have failed quizartinib therapy.

[0060] Fig. 12A A plot showing the proportion of exons covered in the bulk sample relative to the single-cell sample.

[0061] Fig. 12B A graph showing the proportion of exons that are not covered in the bulk sample relative to the single-cell sample.

[0062] Fig. 12C Plot showing the percentage of bases selected in the bulk sample relative to the single-cell sample.

[0063] Fig.12D A graph showing the proportion of bases covered at 20X in the bulk sample relative to the single cell sample.

[0064] Fig.13A A diagram showing the positions of mapped read bases in the genome stratified by treatment and shaded by sample type.

[0065] Fig. 13B A plot of sample intensity versus trapped insert size is shown.

[0066] Fig.14A Shown is a graph of the percentage of replicates versus the percentage of selected bases for a 12-plex experiment.

[0067] Fig. 14B A plot of the number of target bases versus coverage level is shown. DETAILED DESCRIPTION

[0068] It is necessary to develop new scalable, accurate and effective nucleic acid amplification (including single cell and multi-cell genome amplification) and sequencing methods, which will overcome the limitations of current methods by increasing sequence presentation, uniformity and accuracy in a reproducible manner. Provided herein are compositions and methods for providing accurate and scalable primary template directional amplification (PTA) and sequencing. Also provided herein are methods for multi-omics analysis, including combining analysis with PTA of proteins, DNA and RNA from single cells, and corresponding post-transcriptional or post-translational modifications. These methods and compositions promote high-precision amplification of target (or "template") nucleic acids, which increases the accuracy and sensitivity of downstream applications (such as next-generation sequencing).

[0069] definition

[0070] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which these inventions belong.

[0071] Throughout this disclosure, numerical features are represented in range format. It should be understood that the description of range format is only for convenience and simplicity, and should not be interpreted as a strict limitation of the scope of any embodiment. Therefore, unless the context clearly indicates otherwise, it should be considered that the description of the range has specifically disclosed all possible sub-ranges and a single numerical value up to one-tenth of the lower limit unit within the range. For example, the description of the range such as from 1 to 6 should be considered as specifically disclosing a sub-range, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and a single value within the range, for example, 1.1, 2, 2.3, 5 and 5.9. Regardless of the width of the range, this applies. The upper and lower limits of these intermediate ranges can be independently included in a smaller range, and are also included in the present invention, subject to any explicit exclusion of restrictions within the specified range. When the range includes one or two limits, unless the context clearly indicates otherwise, the scope that does not include one or two of the included limits is also included in the present invention.

[0072] The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit any embodiments. As used herein, the singular forms "a", "an", and "the" are also intended to include the plural forms, unless the context clearly indicates otherwise. It will also be understood that when the terms "comprises" and / or "comprising" are used in this specification, they specify the presence of the features, wholes, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0073] Unless otherwise specified or apparent from the context, as used herein, the term "about" when referring to a numerical value or a range of numerical values ​​should be understood to mean the stated numerical value + / - 10% thereof, or, for values ​​listed in a range, from 10% below the listed lower limit to 10% above the listed upper limit.

[0074] As used herein, the term "subject" or "patient" or "individual" refers to animals, including mammals, such as humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and disease experimental animal models (e.g., mice, rats). According to the present invention, conventional molecular biology, microbiology and recombinant DNA techniques can be used within the skill of the art. These techniques are fully explained in the literature. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (hereinafter "Sambrook et al., 1989"); DNA Cloning: A practical Approach, Volumes I and II (DN Glover, 1985); Oligonucleotide Synthesis (MJ. Gait, 1984); Nucleic Acid Hybridization (BD Hames and SJ Higgins, (1985)); Transcription and Translation (BD Hames and SJ Higgins, (1984)); Animal Cell Culture (RI Freshney, (1986)); Immobilized Cells and Enzymes (IRL Press (1986)); B. Perbal, A practical Guide To Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (hereinafter "Sambrook et al., 1989"); DNA Cloning: A practical Approach, Volumes I and II (DN Glover, 1985); Oligonucleotide Synthesis (MJ. Gait, 1984); Nucleic Acid Hybridization (BD Hames and SJ Higgins, (1985)); Transcription and Translation (BD Hames and SJ Higgins, (1984)); Animal Cell Culture (RI Freshney, (1986)); Immobilized Cells and Enzymes (IRL Press (1986)); B. Perbal, A practical Guide To Molecular Cloning (1984); FM Ausubel et al. (authors), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994); etc.

[0075] The term "nucleic acid" encompasses multi-stranded and single-stranded molecules. In double-stranded or triple-stranded nucleic acids, the nucleic acid chains do not need to be co-extended (i.e., double-stranded nucleic acids do not need to be double-stranded along the entire length of the two chains). Nucleic acid templates as described herein can have any size according to the sample (from small cell-free DNA fragments to the entire genome), including but not limited to a length of 50-300 bases, 100-2000 bases, 100-750 bases, 170-500 bases, 100-5000 bases, 50-10,000 bases, or 50-2000 bases. In some cases, the length of the template is at least 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000 or more than 1,000,000 bases. The methods described herein provide amplification of nucleic acids such as nucleic acid templates. The methods described herein additionally provide the generation of isolated and at least partially purified nucleic acids and nucleic acid libraries. In some cases, the methods described herein provide extracted nucleic acids (e.g., extracted from tissues, cells or culture media). Nucleic acids include, but are not limited to, those including the following: DNA, RNA, circular RNA, mtDNA (mitochondrial DNA), cfDNA (cell-free DNA), cfRNA (cell-free RNA), siRNA (small interfering RNA), cffDNA (cell-free fetal DNA), mRNA, tRNA, rRNA, miRNA (microRNA), synthetic polynucleotides, polynucleotide analogs, any other nucleic acid that meets this specification, or any combination thereof. When provided, the length of a polynucleotide is described in bases and abbreviated as nt (nucleotides), bp (bases), kb (kilobases), or Gb (gigabases).

[0076] As used herein, the term "droplet" refers to a volume of liquid on a droplet actuator. In some cases, for example, the droplet is aqueous or non-aqueous, or can be a mixture or emulsion including aqueous and non-aqueous components. For non-limiting examples of droplet fluids that can be subjected to droplet operations, see, for example, International Patent Application Publication No. WO2007 / 120241. In the embodiments presented herein, any system suitable for forming and manipulating droplets can be used. For example, in some cases, a droplet actuator is used. For non-limiting examples of droplet actuators that can be used, see, e.g., U.S. Patent Nos. 6,911,132, 6,977,033, 6,773,566, 6,565,727, 7,163,612, 7,052,244, 7,328,979, 7,547,380, 7,641,779, U.S. Patent Application Publication Nos. US20060194331, US20030205632, US 20060164490, US20070023292, US20060039823, US20080124252, US20090283407, US20090192044, US20050179746, US20090321262, US20100096266, US20110048951, International Patent Application Publication No. WO2007 / 120241. In some cases, the bead is provided in a droplet, in a droplet operations gap, or on a droplet operations surface. In some cases, the bead is provided in a reservoir located outside the droplet operations gap or separate from the droplet operations surface, and the reservoir can be associated with a flow path that allows a droplet including the bead to enter the droplet operations gap or contact the droplet operations surface. Non-limiting examples of droplet actuator technology for immobilizing magnetically responsive beads and / or non-magnetically responsive beads and / or using beads for droplet manipulation protocols are described in U.S. Patent Application Publication No. US20080053205, International Patent Application Publication No. WO2008 / 098236, WO2008 / 134153, WO2008 / 116221, WO2007 / 120241. Bead properties can be employed in multiplexing embodiments of the methods described herein. Examples of beads with properties suitable for multiplexing, and methods for detecting and analyzing signals emitted from such beads, can be found in U.S. Patent Application Publication Nos. US20080305481, US20080151240, US20070207513, US20070064990, US20060159962, US20050277197, US20050118574.

[0077] Primers and / or template switching oligonucleotides may also be attached to a solid substrate to facilitate reverse transcription and template switching of mRNA polynucleotides. In this arrangement, a portion of the RT or template switching reaction occurs in the bulk solution of the device, wherein the second step of the reaction is carried out close to the surface. In other arrangements, the primers of the template switching oligonucleotides are released from the solid substrate to allow the entire reaction to proceed above the surface in the solution. In multi-omics methods, in some cases, primers for multi-stage reactions are attached to a solid substrate or combined with beads to achieve a combination of multi-stage primers.

[0078] Some microfluidic devices also support multi-omics methods. For example, devices made of PDMS usually have continuous chambers for each reaction step. Such multi-chambered devices are usually isolated using microvalve structures, which can be controlled by pressure with air or fluids such as water or inert hydrocarbons (i.e., fluorinert). In multi-omics methods, each level of the reaction can be isolated and allowed to proceed discretely. When a specific level is completed, the valve between adjacent chambers can be released, and the substrate for subsequent reactions can be added in series. The result is that it is possible to use individual cells as input template materials to simulate the sequence group of reactions, such as the multi-omics (protein / RNA / DNA / epigenomics) group of reactions. Various microfluidic platforms can be used to analyze single cells. In some cases, cells are manipulated by fluid dynamics (droplet microfluidics, inertial microfluidics, vortex, microvalve, microstructure (e.g., micropore, microtrap)), electrical methods (electrophoresis (DEP), electroosmosis), optical methods (optical tweezers, optically induced dielectrophoresis (ODEP), optical thermocapillary), acoustic methods or magnetic methods. In some cases, the microfluidic platform comprises microwells. In some cases, the microfluidic platform comprises a PDMS (polydimethylsiloxane) based device. Non-limiting examples of single-cell analysis platforms compatible with the methods described herein are: ddSEQ single-cell isolator (Bio-Rad, Hercules, CA, USA and Illumina, San Diego, CA, USA); Chromium (10x Genomics, Pleasanton, CA, USA); Rhapsody single-cell analysis system (BD, Franklin Lakes, NJ, USA); Tapestri platform (MissionBio, San Francisco, CA, USA); Nadia Innovate (Dolomite Bio, Royston, UK); C1 and Polaris (Fluidigm, South San Francisco, CA, USA); ICELL8 single-cell system (Takara); MSND (Wafergen); Puncher platform (Vycap); CellRaft AIR system (CellMicrosystems); DEPArray NxT and DEPArray systems (Menarini Silicon Biosystems); AVISO CellCelector (ALS); and InDrop system (1CellBio) and TrapTx (Celldom).

[0079] As used herein, the term "unique molecular identifier (UMI)" refers to a unique nucleic acid sequence attached to each of a plurality of nucleic acid molecules. When incorporated into a nucleic acid molecule, in some cases, the UMI is used to correct for subsequent amplification bias by directly counting the UMIs sequenced after amplification. The design, incorporation, and application of UMIs are described, for example, in International Patent Application Publication No. WO 2012 / 142213; Islam et al. Nat. Methods (2014) 11: 163-166; Kivioja, T. et al. Nat. Methods (2012) 9: 72-74; Brenner et al. (2000) PNAS 97(4), 1665; and Hollas and Schuler, (2003) Conference: 3rd International Workshop on Algorithms in Bioinformatics, Vol. 2812.

[0080] As used herein, the term "barcode" refers to a nucleic acid tag that can be used to identify a sample or source of a nucleic acid material. Therefore, in the case where the nucleic acid sample is from multiple sources, in some cases, the nucleic acid in each nucleic acid sample is labeled with different nucleic acid tags, so that the source of the sample can be identified. Barcodes, also commonly referred to as indexes, labels, etc., are well known to those skilled in the art. Any suitable barcode or barcode set can be used. See, for example, the non-limiting examples provided in U.S. Patent No. 8,053,192 and International Patent Application Publication No. WO2005 / 068656. The barcoding of a single cell can be performed, for example, as described in U.S. Patent Application Publication No. 2013 / 0274117.

[0081] The terms "solid surface," "solid support," and other grammatical equivalents herein refer to any material that is suitable or can be modified to be suitable for attachment of primers, barcodes, and sequences described herein. Exemplary substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylic, polystyrene, and copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon, and polyvinyl alcohol). TM The solid support may be a polymer, such as polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials (e.g., silicon or modified silicon), carbon, metals, inorganic glass, plastics, fiber optic bundles, and various other polymers. In some embodiments, the solid support comprises a patterned surface suitable for immobilizing primers, barcodes, and sequences in an ordered pattern.

[0082] As used herein, the term "biological sample" includes, but is not limited to, tissues, cells, biological fluids and isolates thereof. In some cases, the cells or other samples used in the methods described herein are isolated from human patients, animals, plants, soil or other samples including microorganisms such as bacteria, fungi, protozoa. In some cases, the biological sample is derived from humans. In some cases, the biological sample is not derived from non-humans. In some cases, cells undergo PTA methods and sequencing as described herein. Variants detected in the entire genome or a specific position can be compared with all other cells isolated from the object to track the history of cell lineages for research or diagnostic purposes. In some cases, variants are confirmed by other analytical methods such as direct PCR sequencing.

[0083] Single cell analysis

[0084] Methods and compositions for analyzing single cells are described herein. Analysis of a large number of cells provides general information about the cell population, but low-frequency mutants are often not detected above background. Such mutants may include important properties, such as drug resistance or mutations associated with cancer. In some cases, DNA, RNA, and / or proteins from the same single cell are analyzed in parallel. The analysis may include identification of epigenetic post-translational modifications (e.g., glycosylation, phosphorylation, acetylation, ubiquitination, histone modifications) and / or post-transcriptional modifications (e.g., methylation, hydroxymethylation). Such methods may include "primary template directed amplification" (PTA) to obtain a nucleic acid library for sequencing. In some cases, PTA is combined with additional steps or methods, such as RT-PCR or proteomic / protein quantification techniques (e.g., mass spectrometry, antibody staining, etc.). In some cases, during a separate analysis step, the various components of the cell are physically or spatially separated from each other. For example, in some cases the workflow includes Figure 1AThe overall steps in. First, the protein is labeled with an antibody. In some cases, at least some antibodies include a label or marker (e.g., a nucleic acid / oligonucleotide label, a mass label or a fluorescent label). In some cases, a part of the antibody includes an oligonucleotide label. In some cases, a part of the antibody includes a fluorescent marker. In some cases, the antibody is labeled with two or more labels or markers. In some cases, a part of the antibody is sorted based on a fluorescent marker. After RT-PCR, the first strand mRNA product is produced, and then taken out for analysis. Then a library is produced by the RT-PCR product and the barcode present on the protein-specific antibody, and then sequenced. At the same time, PTA is performed to the genomic DNA from the same cell, a library is produced, and sequenced. In some cases, the sequencing results from the genome, proteome and transcriptome are merged using bioinformatics methods. In some cases, the methods described herein include any combination of labeling, cell sorting, affinity separation / purification, cracking of specific cell components (e.g., outer membrane, core, etc.), RNA amplification, DNA amplification (e.g., PTA) or other steps related to protein, RNA or DNA separation or analysis. In some cases, the methods described herein include one or more enrichment steps, such as exome enrichment.

[0085] Described herein is the first method for single cell analysis, which comprises analyzing RNA and DNA from single cells. Figure 1B ). In some cases, the method includes isolating a single cell, lysing a single cell and reverse transcription (RT). In some cases, reverse transcription is performed with a template switching oligonucleotide (TSO). In some cases, TSOS comprises a molecular tag such as biotin, thereby allowing the cDNA RT product to be subsequently pulled down, and the RT product is amplified by PCR to produce a cDNA library. Alternatively or in combination, centrifugation is used to separate the RNA in the supernatant from the cDNA in the cell pellet. In some cases, the remaining cDNA is fragmented and removed with UDG (uracil DNA glycosylase), and alkaline lysis is used to degrade RNA and denature the genome. After neutralization, addition of primers and PTA, in some cases, the amplified product is purified on SPRI (solid phase reversible fixation) beads and connected to an adapter to produce a gDNA library.

[0086] A second method of single cell analysis is described herein, which comprises analyzing RNA and DNA from single cells. Figure 1C). In some cases, the method includes isolating a single cell, lysing a single cell and reverse transcription (RT). In some cases, reverse transcription is performed with a template switching oligonucleotide (TSO). In some cases, TSOS comprises a molecular tag such as biotin, thereby allowing the cDNA RT product to be subsequently pulled down, and the RT product is amplified by PCR to produce a cDNA library. In some cases, alkaline lysis is then used to degrade RNA and denature the genome. After neutralization, addition of random primers and PTA, in some cases, the amplified product is purified on SPRI (solid phase reversible fixation) beads and connected to an adapter to produce a gDNA library. In some cases, the RT product is separated by pulling down, such as pulling down with streptavidin beads.

[0087] A third method of single cell analysis is described herein, comprising analyzing RNA and DNA from a single cell ( Figure 1D ). In some cases, the method includes isolating a single cell, lysing a single cell and reverse transcription (RT). In some cases, reverse transcription is performed with a template switching oligonucleotide (TSO) in the presence of a terminator nucleotide. In some cases, TSOS comprises a molecular tag such as biotin, thereby allowing the cDNA RT product to be subsequently pulled down, and the RT product is amplified by PCR to produce a cDNA library. In some cases, alkaline lysis is then used to degrade RNA and denature the genome. After neutralization, addition of random primers and PTA, in some cases, the amplified product is purified on SPRI (solid phase reversible fixation) beads and connected to an adapter to produce a DNA library. In some cases, the RT product is separated by pulling down, such as pulling down with streptavidin beads.

[0088] A fourth method of single cell analysis is described herein, comprising analyzing RNA and DNA from a single cell. Figure 1E ). In some cases, the method includes separating single cells, lysing single cells and reverse transcription (RT). In some cases, reverse transcription is performed with template switching oligonucleotides (TSO). In some cases, TSOS include molecular tags such as biotin, thereby allowing subsequent pull-down of cDNA RT products, and PCR amplification of RT products to produce cDNA libraries. In some cases, alkaline lysis is then used to degrade RNA and denature the genome. After neutralization, adding random primers and PTA, in some cases, the amplified product is subjected to RNase and cDNA amplification using closed and labeled primers. The gDNA is purified on SPRI (solid phase reversible fixation) beads and connected to adapters to produce gDNA libraries. In some cases, the RT product is separated by pull-down, such as pull-down with streptavidin beads.

[0089] A fifth method of single cell analysis is described herein, comprising analyzing RNA and DNA from a single cell. Fig. 7A and Figure 7B). The cell colony is contacted with an antibody library, wherein the antibody is labeled. In some cases, the antibody is labeled with a fluorescent marker, a nucleic acid barcode, or both. The labeled antibody is bound to at least one cell in the colony, and such cells are sorted, and one cell is placed in each container (e.g., a tube, a vial, a microwell, etc.). In some cases, the container includes a solvent. In some cases, the region on the surface of the container is coated with a capture portion. In some cases, the capture portion is a small molecule, antibody, protein, or other agent that can bind to one or more cells, organelles, or other cell components. In some cases, at least one cell, a single cell, or a component thereof is bound to the region on the surface of the container. In some cases, the core is bound to the region of the container. In some cases, the outer membrane of the cell is lysed, thereby releasing mRNA into the solution in the container. In some cases, the nucleus of the cell containing genomic DNA is bound to the region on the surface of the container. Next, RT is usually performed using the mRNA in the solution as a template to produce cDNA. In some cases, the template switching primer includes a TSS region (transcription start site), an anchor region, an RNA BC region, and a poly-dT tail from 5' to 3'. In some cases, the poly-dT tail is combined with the poly-A tail of one or more mRNAs. In some cases, the template switching primer comprises a TSS region, an anchor region and a poly-G region from 3' to 5'. In some cases, the poly-G region includes ribose G. In some cases, the poly-G region is combined with the poly-C region on the mRNA transcript. In some cases, ribose G is added to the mRNA transcript by terminal transferase. After taking out the RT PCR product for subsequent sequencing, any remaining RNA in the cell is removed by UNG. Then the nucleus is lysed, and the PTA method is carried out to the released genomic DNA using random primers and isothermal polymerase. In some cases, the length of the primer is 6-9 bases. In some cases, PTA produces a genomic amplicon with a length of 100-5000, 200-5000, 500-2000, 500-2500, 1000-3000 or 300-3000 bases. In some cases, PTA produces a genomic amplicon with an average length of 100-5000, 200-5000, 500-2000, 500-2500, 1000-3000 or 300-3000 bases. In some cases, PTA produces a genomic amplicon with a length of 250-1500 bases. In some cases, the method described herein produces a short fragment cDNA pool with about 500, about 750, about 1000, about 5000 or about 10,000 times of amplification. In some cases, the method described herein produces a short fragment cDNA pool with 500-5000, 750-1500 or 250-10,000 times of amplification. Optionally, the PTA product is subjected to additional amplification and is sequenced.

[0090] Sample preparation and isolation of single cells

[0091] Methods described herein may need to separate single cells for analysis. Any single cell separation method can be used together with PTA, such as oral pipetting, micro-pipetting, flow cytometry / FACS, microfluidics, methods for sorting nuclear (tetraploid or other) or manual dilution. Such methods are assisted by other reagents and steps, such as antibody-based enrichment (e.g., circulating tumor cells), enrichment methods or fluorescent labels based on other small molecules or proteins. In some cases, multi-omics analysis methods described herein include mechanical or enzymatic dissociation of cells from larger tissues.

[0092] Preparation and analysis of cell fractions

[0093] The multi-omics analysis method including PTA as described herein can include one or more methods for processing cell components such as DNA, RNA and / or protein. In some cases, the core (comprising genomic DNA) is physically separated from the cytosol (comprising mRNA), followed by a membrane selective lysis buffer to dissolve the membrane, but keep the core intact. The cytosol is then separated from the core using a method including micro-pipetting, centrifugation or antibody-conjugated magnetic microbeads. In another case, the coated magnetic beads of oligodT primers are combined with polyadenylated mRNA to separate from DNA. In another case, preamplify DNA and RNA simultaneously, and then separate to analyze. In another case, the single cell is split into two equal parts, wherein mRNA is processed from one half and genomic DNA is processed from the other half.

[0094] Multi-omics

[0095] The methods described herein (e.g., PTA) can be used as an alternative to any number of other known methods in the art for single cell sequencing (multi-omics, etc.). PTA can replace genomic DNA sequencing methods such as MDA, PicoPlex, DOP-PCR, MALBAC, or target-specific amplification. In some cases, PTA replaces standard genomic DNA sequencing methods in multi-omics methods, including DR-seq (Dey et al., 2015), G&T seq (MacAulay et al., 2015), scMT-seq (Hu et al., 2016), sc-GEM (Cheow et al., 2016), scTrio-seq (Hou et al., 2016), RNA and protein simultaneous multiplexed measurement (Darmanis et al., 2016), scCOOL-seq (Guo et al., 2017), CITE-seq (Stoeckius et al., 2017), REAP-seq (Peterson et al., 2017), scNMT-seq (Clark et al., 2018) or SIDR-seq (Han et al., 2018). In some cases, the methods described herein include methods of PTA and polyadenylated mRNA transcripts. In some cases, the methods described herein include methods of PTA and non-polyadenylated mRNA transcripts. In some cases, the methods described herein include methods of PTA and total (polyadenylated and non-polyadenylated) mRNA transcripts.

[0096] In some cases, PTA is combined with standard RNA sequencing methods to obtain genomic and transcriptome data. In some cases, the multi-omics methods described herein include PTA and one of the following: Drop-seq (Macosko et al. 2015), mRNA-seq (Tang et al., 2009), InDrop (Klein et al., 2015), MARS-seq (Jaitin et al., 2014), Smart-seq2 (Hashimshony et al., 2012; Fish et al., 2016), CEL-seq (Jaitin et al., 2014), STRT -seq (Islam et al., 2011), Quartz-seq (Sasagawa et al., 2013), CEL-seq2 (Hashimshony et al., 2016), cytoSeq (Fan et al., 2015), SuPeR-seq (Fan et al., 2011), RamDA-seq (Hayashi et al., 2018), MATQ-seq (Sheng et al., 2017), or SMARTer (Verboom et al., 2019).

[0097] Various reaction conditions and mixtures can be used to generate cDNA libraries for transcriptome analysis. In some cases, RT reaction mixtures are used to generate cDNA libraries. In some cases, RT reaction mixtures include crowding reagents, at least one primer, template switching oligonucleotide (TSO), reverse transcriptase and dNTP mixtures. In some cases, RT reaction mixtures include RNAse inhibitors. In some cases, RT reaction mixtures include one or more surfactants. In some cases, RT reaction mixtures include Tween-20 and / or Triton-X. In some cases, RT reaction mixtures include betaine. In some cases, RT reaction mixtures include one or more salts. In some cases, RT reaction mixtures include magnesium salts (e.g., magnesium chloride) and / or tetramethylammonium chloride. In some cases, RT reaction mixtures include gelatin. In some cases, RT reaction mixtures include PEG (PEG1000, PEG2000, PEG4000, PEG6000, PEG8000 or other lengths of PEG).

[0098] The multi-omics methods described herein can provide both genomic information and RNA transcript information from a single cell (e.g., a combination or dual scheme). In some cases, the genomic information from a single cell is obtained from a PTA method, and the RNA transcript information is obtained from reverse transcription for generating a cDNA library. In some cases, a full transcript method is used to obtain a cDNA library. In some cases, a cDNA library is obtained using 3' or 5' end counting. In some cases, a cDNA library is not obtained using UMI. In some cases, the multi-omics method provides RNA transcript information about at least 500, 1000, 2000, 5000, 8000, 10,000, 12,000 or at least 15,000 genes from a single cell. In some cases, the multi-omics method provides RNA transcript information about about 500, 1000, 2000, 5000, 8000, 10,000, 12,000 or about 15,000 genes from a single cell. In some cases, the multi-omics method provides RNA transcript information about 100-12,000, 1000-10,000, 2000-15,000, 5000-15,000, 10,000-20,000, 8000-15,000, or 10,000-15,000 genes from a single cell. In some cases, the multi-omics method provides at least 80%, 90%, 92%, 95%, 97%, 98%, or at least 99% of the genome sequence information about the genome of a single cell. In some cases, the multi-omics method provides about 80%, 90%, 92%, 95%, 97%, 98%, or about 99% of the genome sequence information about the genome of a single cell.

[0099] Multi-omics methods can include analysis of single cells from a cell population. In some cases, at least 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000 or at least 8000 cells are analyzed. In some cases, about 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000 or about 8000 cells are analyzed. In some cases, 5-100, 10-100, 50-500, 100-500, 100-1000, 50-5000, 100-5000, 500-1000, 500-10000, 1000-10000 or 5000-20,000 cells are analyzed.

[0100] Multi-omics methods can produce the yield of genomic DNA from PTA reaction based on the type of single cell. In some cases, the amount of DNA produced from a single cell is about 0.1, 1, 1.5, 2, 3, 5 or about 10 micrograms. In some cases, the amount of DNA produced from a single cell is about 0.1, 1, 1.5, 2, 3, 5 or about 10 femtograms. In some cases, the amount of DNA produced from a single cell is at least 0.1, 1, 1.5, 2, 3, 5 or at least 10 micrograms. In some cases, the amount of DNA produced from a single cell is at least 0.1, 1, 1.5, 2, 3, 5 or at least 10 femtograms. In some cases, the amount of DNA produced from a single cell is about 0.1-10, 1-10, 1.5-10, 2-20, 2-50, 1-3 or 0.5-3.5 micrograms. In some cases, the amount of DNA produced from a single cell is about 0.1-10, 1-10, 1.5-10, 2-20, 2-4, 1-3, or 0.5-4 femtograms.

[0101] Methylome analysis

[0102] This paper describes a method including PTA, wherein the site of methylated DNA in a single cell is determined using the PTA method. In some cases, these methods also include parallel analysis of the transcriptome and / or proteome of the same cell. The method for detecting methylated genomic bases includes selective restriction with a methylation-sensitive endonuclease, and then processing with the PTA method. Determine the site cut by this type of enzyme according to sequencing, and identify the methylated base. In another case, the bisulfite treatment of the genomic DNA library converts unmethylated cytosine into uracil. Then in some cases, the library is amplified with a methylation-specific primer that selectively anneals to the methylated sequence. Alternatively, non-methylation-specific PCR is performed, and then one or more methods for distinguishing the base of the bisulfite reaction are performed, including direct pyrophosphate sequencing, MS-SnuPE, HRM, COBRA, MS-SSCA or base-specific cutting / MALDI-TOF. In some cases, the genomic DNA sample is split for parallel analysis of genome (or its enriched part) and methylation group analysis. In some cases, analysis of the genome and methylome includes enrichment of genomic fragments (e.g., exome or other targets) or whole genome sequencing.

[0103] Bioinformatics

[0104] The data obtained from the single cell analysis method utilizing PTA as described herein can be compiled into a database. The method and system of bioinformatics data integration are described herein. In some cases, the data from proteome, genome, transcriptome, methylation group or other data are combined / integrated into a database and analyzed. In some cases, the bioinformatics data integration method and system include one or more of protein detection (FACS and / or NGS), mRNA detection and / or genome variation detection. In some cases, the data are related to disease state or disease. In some cases, the data from multiple single cells are compiled to describe the characteristics of a larger cell population (such as cells from a specific sample, region, organism or tissue). In some cases, protein data is obtained from fluorescently labeled antibodies selectively combined with proteins on cells. In some cases, the method of protein detection includes grouping cells based on fluorescent markers and reporting sample positions after sorting. In some cases, the method of protein detection includes detecting sample barcodes, detecting protein barcodes, comparing with the designed sequence and grouping cells based on barcodes and copy numbers. In some cases, protein data is obtained from barcoded antibodies selectively combined with proteins on cells. In some cases, transcriptome data is obtained from sample and RNA specific barcode. In some cases, the method for mRNA detection includes detection sample and RNA specific barcode, comparison with genome, comparison with RefSeq / Encode, reporting exon / intron / intergenic sequence, analyzing exon-exon junction, cell grouping and cluster analysis variation and top variable gene based on barcode and expression variation. In some cases, genome data is obtained from sample and DNA specific barcode. In some cases, the method for genomic variation detection includes detection sample and DNA specific barcode, comparison with genome, determination of genome recovery rate and SNV mapping rate, filtering reads on exon-exon junction, generation of variant determination file (VCF) and cluster analysis variation and top variable mutation.

[0105] mutation

[0106] In some cases, the method described herein (for example, multi-omics PTA) leads to higher detection sensitivity and / or lower false positive rate of mutation detection.In some cases, mutation is the difference between analysis sequence (for example, using method described herein) and reference sequence.In some cases, reference sequence is obtained from other individuals, organism colonies or other regions of the same genome of other organisms, identical or similar species.In some cases, mutation is identified on plasmid or chromosome.In some cases, mutation is SNV (single nucleotide variation), SNP (single nucleotide polymorphism) or CNV (copy number variation or CNA / copy number abnormality).In some cases, mutation is base substitution, insertion or deletion.In some cases, mutation is conversion, transversion, nonsense mutation, silent mutation, synonymous or non-synonymous mutation, non-pathogenic mutation, missense mutation or frameshift mutation (deletion or insertion). In some cases, PTA results in higher detection sensitivity and / or lower false positive rate for mutation detection when compared to methods such as prediction via computer simulation, ChIP-seq, GUIDE-seq, circular-seq, HTGTS (high-throughput whole-genome translocation sequencing), IDLV (integration-defective lentivirus), Digenome-seq, FISH (fluorescence in situ hybridization), or DISCOVER-seq.

[0107] Directed amplification of primary template

[0108] Nucleic acid amplification methods, such as "primary template directed amplification (PTA)", are described herein. In some cases, PTA is combined with other analytical workflows for multi-omics analysis. For example, one embodiment of the PTA method described herein is schematically represented in Figure 1GIn the PTA method, polymerase (e.g., strand displacement polymerase) is used to preferentially generate amplicons from primary templates ("direct copies"). Therefore, compared with MDA, in the subsequent amplification process, errors are propagated from subamplicons at a lower rate. The result is a method that is easy to perform, which is different from the existing WGA scheme, and the method can amplify low input DNA (including the genome of a single cell) in an accurate and reproducible manner, and has high coverage and uniformity. In addition, the terminated amplification product can be directed connected after removing the terminator, allowing the cell barcode to be attached to the amplification primer, so that the products from all cells can be merged after performing parallel amplification reactions. In some cases, the template nucleic acid is not bound to a solid support. In some cases, a direct copy of the template nucleic acid is not bound to a solid support. In some cases, one or more primers are not bound to a solid support. In some cases, no primer is not bound to a solid support. In some cases, a primer is attached to a first solid support, and the template nucleic acid is attached to a second solid support, wherein the first solid support and the second solid support are not identical. In some cases, PTA is used to analyze single cells from a larger cell population. In some cases, PTA is used to analyze more than one cell from a larger cell body, or an entire cell population.

[0109] This article describes a method for amplification using a nucleic acid polymerase with strand displacement activity. In some cases, this polymerase has strand displacement activity and a low error rate. In some cases, this polymerase has strand displacement activity and proofreading exonuclease activity, such as 3'->5' proofreading activity. In some cases, nucleic acid polymerase is used in combination with other components, such as reversible or irreversible terminators, or other strand displacement factors. In some cases, polymerase has strand displacement activity, but does not have exonuclease proofreading activity. For example, in some cases, these polymerases include bacteriophage phi29 (Φ29) polymerase, which also has a very low error rate, which is the result of 3'->5' proofreading exonuclease activity (see, e.g., U.S. Patent Nos. 5,198,543 and 5,001,050). In some cases, non-limiting examples of strand-displacing nucleic acid polymerases include, for example, genetically modified phi29 (Φ29) DNA polymerase, Klenow fragment of DNA polymerase I (Jacobsen et al., Eur. J. Biochem. 45:623-627 (1974)), bacteriophage M2 DNA polymerase (Matsumoto et al., Gene 84:247 (1989)), bacteriophage phiPRD1 DNA polymerase (Jung et al., Proc. Natl. Acad. Sci. USA 84:8287 (1987); Zhu and Ito, Biochim. Biophys. Acta. 1219:267-276 (1994)), Bst DNA polymerases (e.g., Bst large fragment DNA polymerase (Exo(-)Bst; Aliotta et al., Genet. Anal. (Netherlands) 12:185-195 (1996)), exo(-)Bca DNA polymerase (Walker and Linn, Clinical Chemistry 42:1604-1608 (1996)), Bsu DNA polymerase, including Vent R (exo-)DNA polymerase Vent RDNA polymerase (Kong et al., J. Biol. Chem. 268: 1965-1975 (1993)), Deep Vent DNA polymerase including Deep Vent (exo-) DNA polymerase, IsoPol DNA polymerase, DNA polymerase I, Therminator DNA polymerase, T5 DNA polymerase (Chatterjee et al., Gene 97: 13-19 (1991)), Sequenase (US. Biochemicals), T7 DNA polymerase, T7-sequenase, T7 gp5 DNA polymerase, PRDI DNA polymerase, T4 DNA polymerase (Kaboord and Benkovic, Curr. Biol. 5: 149-157 (1995)). Additional strand displacement nucleic acid polymerases are also compatible with the methods described herein. The ability of a given polymerase to perform strand displacement replication can be determined, for example, by using the polymerase in a strand displacement replication assay (e.g., as disclosed in U.S. Pat. No. 6,977,148). In some cases, these assays are performed at a temperature suitable for the optimal activity of the enzyme used, for example, 32°C for phi29 DNA polymerase, 46°C to 64°C for exo(-)Bst DNA polymerase, or about 60°C to 70°C for enzymes from hyperthermogenic organisms. Another useful assay for selecting polymerases is the primer blocking assay described in Kong et al., J.Biol.Chem.268:1965-1975 (1993). The assay includes a primer extension assay using an M13 ssDNA template in the presence or absence of an oligonucleotide that hybridizes upstream of the extension primer to block its progress. In this assay, other enzymes that can displace the blocking primer are useful for the disclosed method in some cases. In some cases, the polymerase incorporates dNTPs and terminators at approximately equal ratios. In some cases, the polymerases described herein incorporate dNTPs and terminators at a ratio of about 1: 1, about 1.5: 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 10: 1, about 20: 1, about 50: 1, about 100: 1, about 200: 1, about 500: 1, or about 1000: 1. In some cases, the polymerases described herein incorporate dNTPs and terminators at a ratio of 1: 1 to 1000: 1, 2: 1 to 500: 1, 5: 1 to 100: 1, 10: 1 to 1000: 1, 100: 1 to 1000: 1, 500: 1 to 2000: 1, 50: 1 to 1500: 1, or 25: 1 to 1000: 1.

[0110] Amplification methods are described herein, wherein chain displacement can be promoted by using a chain displacement factor such as a helicase. In some cases, these factors are used in combination with other amplification components, such as polymerases, terminators or other components. In some cases, the chain displacement factor is used together with a polymerase that does not have chain displacement activity. In some cases, the chain displacement factor is used together with a polymerase with chain displacement activity. Not subject to theoretical constraints, the chain displacement factor can increase the speed at which less double-stranded amplicon is triggered. In some cases, any DNA polymerase that can carry out chain displacement replication in the presence of a chain displacement factor is suitable for the PTA method, even if the DNA polymerase cannot carry out chain displacement replication in the absence of this factor. In some cases, strand displacement factors that can be used for strand displacement replication include, but are not limited to, BMRF1 polymerase auxiliary subunit (Tsurumi et al., J. Virology 67(12):7648-7653 (1993)), adenovirus DNA binding protein (Zijderveld and van der Vliet, J. Virology 68(2):1158-1164 (1994)), herpes simplex virus protein ICP8 (Boehmer and Lehman, J. Virology 67(2):711-715 (1993); Skaliter and Lehman, Proc. Natl. Acad. Sci. USA 91(22):10665-10669 (1994)); single-stranded DNA binding protein (SSB; Rigler and Romano, J. Biol. Chem. 270:8910-8919 (1995)); bacteriophage T4 gene 32 protein (Villemain and Giedroc, Biochemistry 35:14395-14404 (1996); T7 helicase-primase; T7gp2.5 SSB protein; Tte-UvrD (from Thermoanaerobacter tengcongensis), calf thymus helicase (Siegel et al., J.Biol.Chem.267:13629-13635 (1992)); bacterial SSB (e.g., E. coli SSB), replication protein A (RPA) in eukaryotes, human mitochondrial SSB (mtSSB) and recombinases (e.g., recombinase A (RecA) family proteins, T4 UvsX, T4UvsY, Sak4 of bacteriophage HK620, Rad51, Dmc1 or Radb). Combinations of factors that promote strand displacement and initiation are also consistent with the methods described herein. For example, a helicase is used in combination with a polymerase.In some cases, the PTA method includes the use of a single-stranded DNA binding protein (SSB, T4gp32 or other single-stranded DNA binding protein), a helicase and a polymerase (e.g., SauDNA polymerase, Bsu polymerase, Bst2.0, GspM, GspM2.0, GspSSD or other suitable polymerases). In some cases, a reverse transcriptase is used in combination with a strand displacement factor as described herein. In some cases, a reverse transcriptase is used in combination with a strand displacement factor as described herein. In some cases, a polymerase and a nickase (e.g., "NEAR") (such as those described in US 9,617,586) are used for amplification. In some cases, the nicking enzyme is Nt.BspQI, Nb.BbvCi, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.BpulOI, or Nt.BpulOI.

[0111] Amplification methods are described herein, which include the use of terminator nucleotides, polymerases, and other factors or conditions. For example, in some cases, these factors are used to fragment one or more nucleic acid templates or amplicons during amplification. In some cases, these factors include endonucleases. In some cases, factors include transposases. In some cases, mechanical shearing is used to fragment nucleic acids during amplification. In some cases, nucleotides are added during amplification, and fragmentation can be achieved by adding other proteins or conditions. For example, uracil is incorporated into the amplicon; nucleic acids are fragmented at uracil-containing positions by treatment with uracil D-glycosylase. In some cases, other systems of selective nucleic acid fragmentation are also used, for example, engineered DNA glycosylases that cut modified cytosine-pyrene base pairs (Kwon, et al. ChemBiol. 2003, 10 (4), 351).

[0112] Amplification methods including the use of terminator nucleotides are described herein, which terminate nucleic acid replication, thereby reducing the size of the amplified product. In some cases, these terminators are used in combination with polymerases, strand displacement factors or other amplification components as described herein. In some cases, terminator nucleotides reduce or reduce the efficiency of nucleic acid replication. In some cases, these terminators reduce the elongation rate by at least 99.9%, 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70% or at least 65%. In some cases, these terminators reduce the elongation rate by 50%-90%, 60%-80%, 65%-90%, 70%-85%, 60%-90%, 70%-99%, 80%-99% or 50%-80%. In some cases, terminators reduce the average amplicon product length by at least 99.9%, 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70% or at least 65%. In some cases, the terminator reduces the average amplicon length by 50%-90%, 60%-80%, 65%-90%, 70%-85%, 60%-90%, 70%-99%, 80%-99%, or 50%-80%. In some cases, amplicons including terminator nucleotides form loops or hairpins, thereby reducing the ability of polymerases to use these amplicons as templates. In some cases, the use of terminators slows the rate of amplification at the initial amplification site by incorporating terminator nucleotides (e.g., dideoxynucleotides that have been modified to make them resistant to exonucleases and thereby terminate DNA extension), thereby producing smaller amplification products. By producing smaller amplification products than currently used methods (e.g., the average length of the PTA method is 50-2000 nucleotides, while the average product length of the MDA method is >10,000 nucleotides), the PTA amplification products can in some cases be directly ligated to adapters without fragmentation, thereby allowing efficient incorporation of cellular barcodes and unique molecular identifiers (UMIs) (see Figure 2A ).

[0113] Terminator nucleotides are present in various concentrations, depending on factors such as polymerase, template or other factors. For example, in some cases, in the methods described herein, the amount of terminator nucleotides is expressed as the ratio of non-terminator nucleotides to terminator nucleotides. In some cases, these concentrations allow the length of the amplicon to be controlled. In some cases, the ratio of terminator nucleotides to non-terminator nucleotides is changed for the size of the template amount or template present. In some cases, the ratio of terminator nucleotides to non-terminator nucleotides is reduced for smaller sample sizes (e.g., femtogram to picogram range). In some cases, the ratio of non-terminator nucleotides to terminator nucleotides is about 2:1, 5:1, 7:1, 10:1, 20:1, 50:1, 100:1, 200:1, 500:1, 1000:1, 2000:1 or 5000:1. In some cases, the ratio of non-terminator nucleotides to terminator nucleotides is 2: 1-10: 1, 5: 1-20: 1, 10: 1-100: 1, 20: 1-200: 1, 50: 1-1000: 1, 50: 1-500: 1, 75: 1-150: 1 or 100: 1-500: 1. In some cases, at least one nucleotide present during amplification using the methods described herein is a terminator nucleotide. Each terminator does not have to be present at about the same concentration; in some cases, the ratio of various terminators present in the methods described herein can be optimized for a particular set of reaction conditions, sample types or polymerases. Without being bound by theory, each terminator can be incorporated into the growing polynucleotide chain of the amplicon in response to pairing with the corresponding nucleotide on the template strand. For example, in some cases, the concentration of terminators paired with cytosine is about 3%, 5%, 10%, 15%, 20%, 25% or 50% higher than the average terminator concentration. In some cases, the concentration of the terminator paired with thymine is about 3%, 5%, 10%, 15%, 20%, 25% or 50% higher than the average terminator concentration. In some cases, the concentration of the terminator paired with guanine is about 3%, 5%, 10%, 15%, 20%, 25% or 50% higher than the average terminator concentration. In some cases, the concentration of the terminator paired with adenine is about 3%, 5%, 10%, 15%, 20%, 25% or 50% higher than the average terminator concentration. In some cases, the concentration of the terminator paired with uracil is about 3%, 5%, 10%, 15%, 20%, 25% or 50% higher than the average terminator concentration. In some cases, any nucleotide that can terminate the nucleic acid extension by nucleic acid polymerase is used as a terminator nucleotide in the method described herein. In some cases, reversible terminators are used to terminate nucleic acid replication. In some cases, irreversible terminators are used to terminate nucleic acid replication.In some cases, the non-limiting examples of terminators include reversible and irreversible nucleic acids and nucleic acid analogs, for example, including 3' sealed reversible terminators of nucleotides, including 3' unsealed reversible terminators of nucleotides, including 2' modified terminators of deoxynucleotides, including terminators of the modification of the nitrogenous bases of deoxynucleotides or any combination thereof. In one embodiment, terminator nucleotides are dideoxynucleotides. Nucleic acid replication is terminated and other nucleotide modifications applicable to the implementation of the present invention include but are not limited to any modification of the r group of the 3' carbon of deoxyribose, such as reverse dideoxynucleotides, 3' biotinylated nucleotides, 3' aminonucleotides, 3'-phosphorylated nucleotides, 3'-O-methyl nucleotides, including 3'C3 spacer nucleotides, 3'C18 nucleotides, 3' hexanediol spacer nucleotides 3' carbon spacer nucleotides, acyclic nucleotides, and combinations thereof. In some cases, terminators are polynucleotides with a length of 1, 2, 3, 4 or more bases. In some cases, terminators do not include detectable parts or labels (for example, mass labels, fluorescent labels, dyes, radioactive atoms or other detectable parts). In some cases, the terminator does not include a chemical moiety that allows a detectable portion or tag to be attached (e.g., "click" azide / alkyne, conjugate addition agent, or other chemical treatment for tag attachment). In some cases, all terminator nucleotides include the same modification that reduces amplification at a certain region of the nucleotide (e.g., sugar moiety, base moiety, or phosphate moiety). In some cases, at least one terminator has a different modification that reduces amplification. In some cases, all terminators have substantially similar fluorescence excitation or emission wavelengths. In some cases, terminators of unmodified phosphate groups are used with polymerases that do not have exonuclease proofreading activity. When terminators are used with polymerases (e.g., phi29) that have 3'->5' proofreading exonuclease activity that can remove terminator nucleotides, in some cases, further modification is required to make them resistant to exonucleases. For example, dideoxynucleotides are modified by α-sulfur groups to produce phosphorothioate bonds, making these nucleotides resistant to the 3'->5' proofreading exonuclease activity of nucleic acid polymerases. In some cases, the modification reduces the exonuclease proofreading activity of the polymerase by at least 99.5%, 99%, 98%, 95%, 90%, or at least 85%.In some cases, non-limiting examples of other terminator nucleotide modifications that provide resistance to 3'->5' exonuclease activity include: nucleotides with modifications to the alpha group, such as alpha-thiodideoxynucleotides that produce phosphorothioate bonds, C3 spacer nucleotides, locked nucleic acids (LNA), inverted nucleic acids, 2'fluoro bases, 3' phosphorylation, 2'-O-methyl modifications (or other 2'-O-alkyl modifications), propyne-modified bases (e.g., deoxycytosine, deoxyuridine), L-DNA nucleotides, L-RNA nucleotides, nucleotides with inverted linkages (e.g., 5'-5' or 3'-3'), 5' inverted bases (e.g., 5' inverted 2',3'-dideoxy dT), methylphosphonate backbones, and trans nucleic acids. In some cases, nucleotides with modifications include base-modified nucleic acids with free 3'OH groups (e.g., 2-nitrobenzyl alkylated HOMedU triphosphates, modified bases containing large chemical groups (e.g., solid supports or other larger moieties)). In some cases, a polymerase having strand displacement activity but not 3'->5' exonuclease proofreading activity is used with a terminator nucleotide that has or has not undergone modification that renders it resistant to exonucleases. These nucleic acid polymerases include, but are not limited to, Bst DNA polymerase, Bsu DNA polymerase, Deep Vent (exo-) DNA polymerase, Klenow fragment (exo-) DNA polymerase, Therminator DNA polymerase, and Vent. R (exo-).

[0114] Primers and Amplicon Libraries

[0115] Amplicon libraries produced by the amplification of at least one target nucleic acid molecule are described herein. In some cases, these libraries are generated using methods described herein, such as libraries using terminators. These methods include using strand displacement polymerases or factors, terminator nucleotides (reversible or irreversible) or other features and embodiments described herein. In some cases, the amplicon libraries produced using terminators described herein are further amplified in subsequent amplification reactions (e.g., PCR). In some cases, subsequent amplification reactions do not include terminators. In some cases, the amplicon libraries include polynucleotides, wherein at least 50%, 60%, 70%, 80%, 90%, 95% or at least 98% of the polynucleotides include at least one terminator nucleotide. In some cases, the amplicon libraries include target nucleic acid molecules derived from the amplicon libraries. The amplicon libraries include a variety of polynucleotides, wherein at least some of the polynucleotides are direct copies (e.g., directly from target nucleic acid molecules such as genomic DNA, RNA or other target nucleic acid copies). For example, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more than 95% of the amplicon polynucleotides are direct copies of at least one target nucleic acid molecule. In some cases, at least 5% of the amplicon polynucleotides are direct copies of at least one target nucleic acid molecule. In some cases, at least 10% of the amplicon polynucleotides are direct copies of at least one target nucleic acid molecule. In some cases, at least 15% of the amplicon polynucleotides are direct copies of at least one target nucleic acid molecule. In some cases, at least 20% of the amplicon polynucleotides are direct copies of at least one target nucleic acid molecule. In some cases, at least 50% of the amplicon polynucleotides are direct copies of at least one target nucleic acid molecule. In some cases, 3%-5%, 3-10%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 5%-30%, 10%-50% or 15%-75% of the amplicon polynucleotides are direct copies of at least one target nucleic acid molecule. In some cases, at least some polynucleotides are direct copies or progeny (first copy of target nucleic acid) of target nucleic acid molecules. For example, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more than 95% of the amplicon polynucleotides are direct copies or progeny of at least one target nucleic acid molecule. In some cases, at least 5% of the amplicon polynucleotides are direct copies or progeny of at least one target nucleic acid molecule. In some cases, at least 10% of the amplicon polynucleotides are direct copies or progeny of at least one target nucleic acid molecule. In some cases, at least 20% of the amplicon polynucleotides are direct copies or progeny of at least one target nucleic acid molecule. In some cases, at least 30% of the amplicon polynucleotides are direct copies or progeny of at least one target nucleic acid molecule.In some cases, 3%-5%, 3%-10%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 5%-30%, 10%-50% or 15%-75% of the amplicon polynucleotides are direct copies or progeny of at least one target nucleic acid molecule. In some cases, the length of the direct copy of the target nucleic acid is 50-2500, 75-2000, 50-2000, 25-1000, 50-1000, 500-2000 or 50-2000 bases. In some cases, the length of the progeny is 1000-5000, 2000-5000, 1000-10,000, 2000-5000, 1500-5000, 3000-7000 or 2000-7000 bases. In some cases, the average length of the PTA amplified product is 25-3000 nucleotides, 50-2500, 75-2000, 50-2000, 25-1000, 50-1000, 500-2000 or 50-2000 bases. In some cases, the length of the amplicon produced from PTA is no more than 5000, 4000, 3000, 2000, 1700, 1500, 1200, 1000, 700, 500 or no more than 300 bases. In some cases, the length of the amplicon produced from PTA is 1000-5000, 1000-3000, 200-2000, 200-4000, 500-2000, 750-2500 or 1000-2000 bases. In some cases, the amplicon library produced using the methods described herein includes at least 1000, 2000, 5000, 10,000, 100,000, 200,000, 500,000 or more than 500,000 amplicons including unique sequences. In some cases, the library includes at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 2500, 3000 or at least 3500 amplicons. In some cases, at least 5%, 10%, 15%, 20%, 25%, 30% or more than 30% of the amplicon polynucleotides less than 1000 bases in length are direct copies of at least one target nucleic acid molecule. In some cases, at least 5%, 10%, 15%, 20%, 25%, 30%, or more than 30% of the amplicon polynucleotides of no more than 2000 bases in length are direct copies of at least one target nucleic acid molecule. In some cases, at least 5%, 10%, 15%, 20%, 25%, 30%, or more than 30% of the amplicon polynucleotides of 3000-5000 bases in length are direct copies of at least one target nucleic acid molecule.In some cases, the ratio of direct copy amplicon to target nucleic acid molecule is at least 10: 1, 100: 1, 1000: 1, 10,000: 1, 100,000: 1, 1,000,000: 1, 10,000,000: 1, or greater than 10,000,000: 1. In some cases, the ratio of direct copy amplicon to target nucleic acid molecule is at least 10: 1, 100: 1, 1000: 1, 10,000: 1, 100,000: 1, 1,000,000: 1, 10,000,000: 1, or greater than 10,000,000: 1, wherein the direct copy amplicon is no more than 700-1200 bases in length. In some cases, the ratio of direct-copy amplicon and subamplifier to target nucleic acid molecule is at least 10: 1, 100: 1, 1000: 1, 10,000: 1, 100,000: 1, 1,000,000: 1, 10,000,000: 1, or greater than 10,000,000: 1. In some cases, the ratio of direct-copy amplicon and subamplifier to target nucleic acid molecule is at least 10: 1, 100: 1, 1000: 1, 10,000: 1, 100,000: 1, 1,000,000: 1, 10,000,000: 1, or greater than 10,000,000: 1, wherein the direct-copy amplicon is 700-1200 bases in length and the subamplifier is 2500-6000 bases in length. In some cases, the library includes about 50-10,000, about 50-5,000, about 50-2500, about 50-1000, about 150-2000, about 250-3000, about 50-2000, about 500-2000 or about 500-1500 amplicon, which are direct copies of the target nucleic acid molecule. In some cases, the library includes about 50-10,000, about 50-5,000, about 50-2500, about 50-1000, about 150-2000, about 250-3000, about 50-2000, about 500-2000 or about 500-1500 amplicon, which are direct copies or subamplicons of the target nucleic acid molecule. In some cases, the direct copy number can be controlled by the pcr amplification cycle number. In some cases, no more than 30, 25, 20, 15, 13, 11, 10, 9, 8, 7, 6, 5, 4, or 3 PCR cycles are used to generate copies of the target nucleic acid molecule. In some cases, about 30, 25, 20, 15, 13, 11, 10, 9, 8, 7, 6, 5, 4, or about 3 PCR cycles are used to generate copies of the target nucleic acid molecule. In some cases, 3, 4, 5, 6, 7, or 8 PCR cycles are used to generate copies of the target nucleic acid molecule.In some cases, 2-4, 2-5, 2-7, 2-8, 2-10, 2-15, 3-5, 3-10, 3-15, 4-10, 4-15, 5-10 or 5-15 PCR cycles are used to generate copies of the target nucleic acid molecule. In some cases, the amplicon library generated using the methods described herein is subjected to additional steps, such as adapter ligation and further PCR amplification. In some cases, these additional steps are prior to the sequencing step.

[0116] Methods described herein may additionally include one or more enrichment or purification steps. In some cases, one or more polynucleotides (such as cDNA, PTA amplicon or other polynucleotides) are enriched during the method described herein. In some cases, one or more polynucleotides are captured using polynucleotide probes. In some cases, probes are configured to capture one or more genomic exons. In some cases, the probe library includes at least 1000, 2000, 5000, 10,000, 50,000, 100,000, 200,000, 500,000 or more than 1 million different sequences. In some cases, the probe library includes sequences that can be combined with at least 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000 or more than 10,000 genes. In some cases, the probe includes a part for the capture of a solid support, such as biotin. In some cases, the enrichment step is carried out after the PTA step. In some cases, the enrichment step is performed before the PTA step. In some cases, the probe is configured to bind to a genomic DNA library. In some cases, the probe is configured to bind to a cDNA library.

[0117] In some cases, the amplicon library of polynucleotides produced by the PTA methods and compositions described herein (terminators, polymerases, etc.) has increased uniformity. In some cases, uniformity is measured using a Lorentz curve (e.g., Figure 5C) or other similar methods to describe. In some cases, this increase makes the sequencing reads required for covering the desired target nucleic acid molecule (for example, genomic DNA, RNA or other target nucleic acid molecules) less. For example, no more than 50% of the cumulative score of the polynucleotide includes at least 80% of the sequence of the cumulative score of the sequence of the target nucleic acid molecule. In some cases, no more than 50% of the cumulative score of the polynucleotide includes at least 60% of the sequence of the cumulative score of the sequence of the target nucleic acid molecule. In some cases, no more than 50% of the cumulative score of the polynucleotide includes at least 70% of the sequence of the cumulative score of the sequence of the target nucleic acid molecule. In some cases, no more than 50% of the cumulative score of the polynucleotide includes at least 90% of the sequence of the cumulative score of the sequence of the target nucleic acid molecule. In some cases, uniformity is described using the Gini index (wherein index 0 represents the complete equality of the library, and index 1 represents completely unequal). In some cases, the Gini index of the amplicon library described herein is no more than 0.55, 0.50, 0.45, 0.40 or 0.30. In some cases, the Gini index of the amplicon library described herein does not exceed 0.50. In some cases, the Gini index of the amplicon library described herein does not exceed 0.40. In some cases, this uniformity metric depends on the number of reads obtained. For example, the reads obtained do not exceed 100 million, 200 million, 300 million, 400 million or do not exceed 500 million. In some cases, the length of the read is about 50, 75, 100, 125, 150, 175, 200, 225 or about 250 bases. In some cases, the uniformity metric depends on the coverage depth of the target nucleic acid. For example, the average coverage depth is about 10X, 15X, 20X, 25X or about 30X. In some cases, the average coverage depth is 10-30X, 20-50X, 5-40X, 20-60X, 5-20X or 10-20X. In some cases, the Gini index of the amplicon library described herein does not exceed 0.55, wherein about 300 million reads are obtained. In some cases, the Gini index of the amplicon library described herein does not exceed 0.50, wherein about 300 million reads are obtained. In some cases, the Gini index of the amplicon library described herein does not exceed 0.45, wherein about 300 million reads are obtained. In some cases, the Gini index of the amplicon library described herein does not exceed 0.55, wherein no more than 300 million reads are obtained. In some cases, the Gini index of the amplicon library described herein does not exceed 0.50, wherein no more than 300 million reads are obtained. In some cases, the Gini index of the amplicon library described herein does not exceed 0.45, wherein no more than 300 million reads are obtained. In some cases, the Gini index of the amplicon library described herein does not exceed 0.55, wherein the average depth of sequencing coverage is about 15X. In some cases, the Gini index of the amplicon library described herein does not exceed 0.50, wherein the average depth of sequencing coverage is about 15X.In some cases, the Gini index of the amplicon library described herein is no more than 0.45, and the average depth of the sequencing coverage is about 15X. In some cases, the Gini index of the amplicon library described herein is no more than 0.55, and the average depth of the sequencing coverage is at least 15X. In some cases, the Gini index of the amplicon library described herein is no more than 0.50, and the average depth of the sequencing coverage is at least 15X. In some cases, the Gini index of the amplicon library described herein is no more than 0.45, and the average depth of the sequencing coverage is at least 15X. In some cases, the Gini index of the amplicon library described herein is no more than 0.55, and the average depth of the sequencing coverage is no more than 15X. In some cases, the Gini index of the amplicon library described herein is no more than 0.50, and the average depth of the sequencing coverage is no more than 15X. In some cases, the Gini index of the amplicon library described herein is no more than 0.45, and the average depth of the sequencing coverage is no more than 15X. In some cases, the uniform amplicon library generated using the method described herein needs to undergo additional steps, such as adapter connection and further PCR amplification. In some cases, these additional steps precede the sequencing step.

[0118] Primers include nucleic acids for initiating amplification reactions as described herein. In some cases, these primers include, but are not limited to, random deoxynucleotides of any length with or without modification that makes it resistant to exonucleases, random ribonucleotides of any length with or without modification that makes it resistant to exonucleases, modified nucleic acids, such as locked nucleic acids, DNA or RNA primers targeting specific genomic regions, and reactions initiated by enzymes such as initiators. In the case of whole genome PTA, a group of primers with random or partially random nucleotide sequences are preferably used. In very complex nucleic acid samples, it is not necessary to know the specific nucleic acid sequence present in the sample, and it is not necessary to design primers to be complementary to any particular sequence. On the contrary, the complexity of nucleic acid samples results in a large number of different hybridization target sequences in the sample, which will be complementary to various primers of random or partially random sequences. In some cases, the complementary portion of the primer used for PTA is completely random, including only random portions, or selectively random. In some cases, for example, the number of random base positions in the complementary portion of the primer is 20% to 100% of the total number of nucleotides in the complementary portion of the primer. In some cases, the number of random base positions in the complementary part of primers is 10% to 90%, 15-95%, 20%-100%, 30%-100%, 50%-100%, 75-100% or 90-95% of the total number of nucleotides in the complementary part of primers. In some cases, the number of random base positions in the complementary part of primers is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or at least 90% of the total number of nucleotides in the complementary part of primers. In some cases, the primer sets with random or partially random sequences are synthesized using standard techniques, which is by allowing any nucleotide to be randomly added at each position. In some cases, primer sets are composed of primers with similar length and / or hybridization properties. In some cases, the term "random primer" refers to a primer that can show four times of degeneracy at each position. In some cases, the term "random primer" refers to a primer that can show three times of degeneracy at each position. In some cases, the random primers used in the methods described herein include random sequences of 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bases in length. In some cases, primers include random sequences of 3-20, 5-15, 5-20, 6-12 or 4-10 bases in length. Primers can also include non-extendable elements that limit the subsequent amplification of the amplicon produced. For example, in some cases, primers with non-extendable elements include terminators. In some cases, primers include terminator nucleotides, such as 1, 2, 3, 4, 5, 10 or more than 10 kinds of terminator nucleotides. Primers do not need to be limited to the components added to the amplification reaction from the outside. In some cases, primers are produced in situ by adding nucleotides and proteins that promote initiation.For example, in some cases, the priming enzyme combined with nucleotides is used to generate random primers of methods described herein. In some cases, the priming enzyme is a member of the DnaG or AEP enzyme superfamily. In some cases, the priming enzyme is TthPrimPol. In some cases, the priming enzyme is T7 gp4 helicase-priming enzyme. In some cases, these priming enzymes are used together with polymerases or strand displacement factors as described herein. In some cases, the priming enzyme starts priming with deoxyribonucleotides. In some cases, the priming enzyme starts priming with ribonucleotides.

[0119] After PTA amplification, a specific subset of amplicon can be selected. In some cases, this selection depends on size, affinity, activity, hybridization with probe or other known selection factors in the art. In some cases, selection is performed before or after other steps as described herein such as adapter connection and / or library amplification. In some cases, selection is based on the size (length) of amplicon. In some cases, select a smaller amplicon, which is unlikely to experience exponential amplification, thereby enriching the product derived from the primary template, while further converting amplification from an exponential form into a quasi-linear amplification process ( Figure 1A). In some cases, the amplicon of 50-2000, 25-5000, 40-3000, 50-1000, 200-1000, 300-1000, 400-1000, 400-600, 600-2000 or 800-1000 bases in length is selected. In some cases, size selection is performed by using a scheme, for example, solid phase reversible immobilization (SPRI) is used on carboxylated paramagnetic beads to enrich for nucleic acid fragments of a specific size, or other schemes known to those skilled in the art. Optionally or in combination, selection is performed by preferentially connecting and amplifying smaller fragments during PCR while preparing a sequencing library, and also as a result of preferentially forming clusters by smaller sequencing library fragments during sequencing (for example, sequencing by synthesis, nanopore sequencing or other sequencing methods). Other strategies for selecting smaller fragments also conform to the methods described herein and include but are not limited to separating nucleic acid fragments of a specific size after gel electrophoresis, using a silica gel column in conjunction with nucleic acid fragments of a specific size, and using other PCR strategies that can more strongly enrich smaller fragments. The library preparation scheme of any number can be used together with PTA method as described herein.In some cases, the amplicon produced by PTA is connected to an adapter (optionally in the case of removing terminator nucleotides).In some cases, the amplicon produced by PTA comprises a homology region produced by the fragmentation based on transposase, which is used as a priming site.In some cases, the library is prepared by mechanical or enzymatic mode fragmentation nucleic acid.In some cases, labeling is used to prepare the library via transposome.In some cases, the library is prepared via an adapter, such as the connection of a Y-adapter, a universal adapter or a circular adapter.

[0120] The non-complementary portion of the primer used in PTA can include a sequence that can be used to further manipulate and / or analyze the amplified sequence. An example of such a sequence is a "detection tag". The detection tag has a sequence that is complementary to the detection probe and is detected using its cognate detection probe. There can be one, two, three, four or more detection tags on the primer. Except for the size of the primer, there is no basic limit to the number of detection tags that may be present on the primer. In some cases, there is only one detection tag on the primer. In some cases, there are two detection tags on the primer. When there are multiple detection tags, they can have the same sequence or different sequences, each different sequence being complementary to a different detection probe. In some cases, multiple detection tags have the same sequence. In some cases, multiple detection tags have different sequences.

[0121] Another example of a sequence that can be included in the non-complementary portion of a primer is an "address tag," which can encode other details of the amplicon, such as the location in a tissue section. In some cases, the cell barcode includes an address tag. The address tag has a sequence that is complementary to the address probe. The address tag is incorporated into the end of the amplified chain. If present, there can be one or more address tags on the primer. There is no basic limit to the number of address tags that may be present on the primer, except for the size of the primer. When there are multiple address tags, they can have the same sequence or different sequences, each of which is complementary to a different address probe. The address tag portion can be any length that supports specific and stable hybridization between the address tag and the address probe. In some cases, nucleic acids from more than one source can be incorporated into a variable tag sequence. The length of the tag sequence can be up to 100 nucleotides, preferably 1 to 10 nucleotides in length, most preferably 4, 5 or 6 nucleotides, and includes a combination of nucleotides. In some cases, the length of the tag sequence is 1-20, 2-15, 3-13, 4-12, 5-12 or 1-10 nucleotides. For example, if six base pairs are chosen to form the tag and arrangements of four different nucleotides are used, a total of 4096 nucleic acid anchors (eg, hairpins) can be made, each with a unique 6-base tag.

[0122] Primers described herein may be present in solution or immobilized on a solid support. In some cases, primers with sample barcodes and / or UMI sequences may be immobilized on a solid support. For example, the solid support may be one or more beads. In some cases, individual cells are contacted with one or more beads having a unique set of sample barcodes and / or UMI sequences to identify individual cells. In some cases, lysates from individual cells are contacted with one or more beads having a unique set of sample barcodes and / or UMI sequences to identify individual cell lysates. In some cases, extracted nucleic acids from individual cells are contacted with one or more beads having a unique set of sample barcodes and / or UMI sequences to identify extracted nucleic acids from individual cells. The beads may be manipulated in any suitable manner known in the art, for example, using a droplet actuator described herein. The beads may be of any suitable size, including, for example, microbeads, microparticles, nanobeads, and nanoparticles. In some embodiments, the beads are magnetically responsive; in other embodiments, the beads have no apparent magnetic response. Non-limiting examples of suitable beads include flow cytometry microbeads, polystyrene microparticles and nanoparticles, functionalized polystyrene microparticles and nanoparticles, coated polystyrene microparticles and nanoparticles, silica microbeads, fluorescent microspheres and nanospheres, functionalized fluorescent microspheres and nanospheres, coated fluorescent microspheres and nanospheres, color-dyed microspheres and nanoparticles, magnetic microspheres and nanoparticles, superparamagnetic microspheres and nanoparticles (e.g., ATCC®, available from Invitrogen Group, Carlsbad, CA). ), fluorescent microparticles and nanoparticles, coated magnetic microparticles and nanoparticles, ferromagnetic microparticles and nanoparticles, coated ferromagnetic microparticles and nanoparticles, and described in U.S. Patent Application Publication Nos. US20050260686, US20030132538, US20050118574, 20050277197, 20060159962. The beads may be pre-coupled with antibodies, proteins or antigens, DNA / RNA probes, or any other molecule with affinity for the desired target. In some embodiments, primers with sample barcodes and / or UMI sequences may be in solution. In certain embodiments, a plurality of droplets may be provided, wherein each droplet in the plurality of droplets has a sample barcode unique to the droplet and a UMI unique to the molecule, such that the UMI is repeated multiple times within a collection of droplets. In some embodiments, individual cells are contacted with droplets having a unique set of sample barcodes and / or UMI sequences to identify the individual cells. In some implementations, a lysate from an individual cell is contacted with a droplet having a unique set of sample barcodes and / or UMI sequences to identify the individual cell lysate. In some implementations, an extracted nucleic acid from an individual cell is contacted with a droplet having a unique set of sample barcodes and / or UMI sequences to identify the extracted nucleic acid from the individual cell.

[0123] PTA primers may include sequence-specific or random primers, cell barcodes, and / or unique molecular identifiers (UMIs) (see, e.g., Fig. 10A (Linear Primer) and Fig. 10B In some cases, the primers include sequence-specific primers. In some cases, the primers include random primers. In some cases, the primers include cell barcodes. In some cases, the primers include sample barcodes. In some cases, the primers include unique molecular identifiers. In some cases, the primers include two or more cell barcodes. In some cases, the barcodes identify a unique sample source or a unique workflow. In some cases, the barcodes or UMIs are 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, or more than 30 bases in length. In some cases, the primers include at least 1000, 10,000, 50,000, 100,000, 250,000, 500,000, 10 6 , 10 7 , 10 8 , 10 9 or at least 10 10In some cases, the primers include at least 8, 16, 96, or 384 unique barcodes or UMIs. In some cases, standard adapters are then ligated to the amplified products prior to sequencing; after sequencing, reads are first assigned to specific cells based on the cell barcodes. Suitable adapters that can be used with the PTA method include, for example, the adapters available from Integrated DNA Technologies (IDT). DualIndex UMI adapters. UMIs are then used to group reads from each cell and reads with the same UMI are merged into a common read. Using cell barcodes allows all cells to be merged before library preparation, as they can then be identified by their cell barcodes. In some cases, using UMIs to form a common read corrects for PCR bias, thereby improving copy number variation (CNV) detection ( Fig.11A and Fig. 11B ). In addition, sequencing errors can be corrected by requiring a fixed percentage of reads from the same molecule to have the same detected base changes at each position. This approach has been used to improve CNV detection and correct sequencing errors in a large number of samples. In some cases, UMIs are used with the methods described herein, for example, U.S. Pat. No. 8,835,358 discloses a digital counting principle after attaching a random amplifiable barcode. Schmitt. et al. and Fan et al. disclose similar methods for correcting sequencing errors. In some cases, a library is generated for sequencing using primers. In some cases, the library includes fragments of 200-700 bases, 100-1000, 300-800, 300-550, 300-700, or 200-800 bases in length. In some cases, the library includes fragments of at least 50, 100, 150, 200, 300, 500, 600, 700, 800, or at least 1000 bases in length. In some cases, the library includes fragments that are about 50, 100, 150, 200, 300, 500, 600, 700, 800, or about 1000 bases in length.

[0124] The methods described herein may also include additional steps, including steps performed on the sample or template. In some cases, these samples or templates are subjected to one or more steps before PTA. In some cases, a pretreatment step is performed on the sample including cells. For example, a combination of freeze-thaw, Triton X-100, Tween 20 and proteinase K is used to lyse and proteolyse the cells to increase the accessibility of chromatin. Other lysis strategies are also applicable to the implementation of the methods described herein. These strategies include but are not limited to the use of detergents and / or lysozyme and / or protease treatments, and / or other combinations of cell physical destruction such as ultrasound and / or alkaline lysis and / or hypotonic lysis for lysis. In some cases, a pretreatment step is performed on the primary template or one or more target molecules. In some cases, sodium hydroxide is used to denature the primary template (or target), and then the solution is neutralized. Other denaturation strategies may also be applicable to the implementation of the methods described herein. These strategies may include but are not limited to combining alkaline lysis with other alkaline solutions, increasing the sample temperature and / or changing the salt concentration in the sample, adding additives such as solvents or oils, other modifications or any combination thereof. In some cases, additional steps include sorting, filtering or separating samples, templates or amplicons by size. In some cases, cells are mechanically (e.g., high pressure homogenizer, bead grinding) or non-mechanically (physical, chemical or biological) lysed. In some cases, physical lysis methods include heating, osmotic shock and / or cavitation. In some cases, chemical lysis includes alkali and / or detergent. In some cases, biological lysis includes the use of enzymes. The combination of lysis methods is also compatible with the methods described herein. Non-limiting examples of lysing enzymes include recombinant lysozymes, serine proteases and bacterial lysins. In some cases, lysis with enzymes includes the use of lysozymes, lysostaphin, digestive enzymes, cellulose, proteases or polysaccharidase. For example, after amplification with the methods described herein, the amplicon library is enriched with amplicons of desired length. In some cases, the amplicon library is enriched in amplicons of 50-2000, 25-1000, 50-1000, 75-2000, 100-3000, 150-500, 75-250, 170-500, 100-500, or 75-2000 bases in length. In some cases, the amplicon library is enriched in amplicons of no more than 75, 100, 150, 200, 500, 750, 1000, 2000, 5000, or no more than 10,000 bases in length. In some cases, the amplicon library is enriched in amplicons of at least 25, 50, 75, 100, 150, 200, 500, 750, 1000, or at least 2000 bases in length.

[0125] The methods and compositions described herein may include buffers or other preparations. In some cases, such buffers are used for PTA, RT or other methods described herein. In some cases, these buffers include surfactants / detergents or denaturants (Tween-20, DMSO, DMF, PEGylated polymers including hydrophobic groups or other surfactants), salts (potassium phosphate or sodium phosphate (mono or dibasic), sodium chloride, potassium chloride), TrisHCl, magnesium chloride or magnesium sulfate, ammonium salts, such as phosphates, nitrates or sulfates, EDTA), reducing agents (DTT, THP, DTE, β-mercaptoethanol, TCEP or other reducing agents) or other components (glycerol, hydrophilic polymers, such as PEG). In some cases, buffers are used in combination with components such as polymerases, chain displacement factors, terminators or other reaction components described herein. In some cases, buffers are used in combination with components such as polymerases, chain displacement factors, terminators or other reaction components described herein. Buffers may include one or more crowding agents. In some cases, crowding agents include polymers. In some cases, crowding agents include polymers such as polyols. In some cases, the crowding agent includes a polyethylene glycol polymer (PEG). In some cases, the crowding agent includes a polysaccharide. Without limitation, examples of crowding agents include ficoll (e.g., ficoll PM 400, ficoll PM 70 or other molecular weight ficoll), PEG (e.g., PEG1000, PEG 2000, PEG4000, PEG6000, PEG8000 or other molecular weight PEG), dextran (dextran 6, dextran 10, dextran 40, dextran 70, dextran 6000, dextran 138k or other molecular weight dextran).

[0126] Nucleic acid molecules amplified according to the methods described herein can be sequenced and analyzed using methods known to those skilled in the art. In some cases, non-limiting examples of sequencing methods used include, for example, hybridization sequencing (SBH), ligation sequencing (SBL) (Shendure et al. (2005) Science 309: 1728), quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer (FRET), molecular beacons, TaqMan reporter gene probe digestion, pyrophosphate sequencing, fluorescence in situ sequencing (FISSEQ), FISSEQ beads (U.S. Patent No. 7,425,431), wobble sequencing (International Patent Application Publication No. WO2006 / 073504), multiple sequencing (U.S. Patent Application Publication No. US2008 / 0269068; Porreca et al., 2007, Nat. Methods 4:931), polymerase clone (POLONY) sequencing (U.S. Pat. Nos. 6,432,360, 6,485,944 and 6,511,803, and International Patent Application Publication No. WO2005 / 082098), nanogrid rolling circle sequencing (ROLONY) (U.S. Pat. No. 9,624,538), allele-specific oligonucleotide ligation assays (e.g., oligonucleotide ligation assays (OLA), single template molecule OLA using ligated linear probes and rolling circle amplification (RCA) readout, ligated padlock probes and / or single template molecule OLA using ligated circular padlock probes and rolling circle amplification (RCA) readout), high-throughput sequencing methods, for example, methods using Roche 454, Illumina Solexa, AB-SOLiD, Helicos, Polonator platforms, and the like, and light-based sequencing technologies (Landegren et al. (1998) Genome Res. 8:769-76; Kwok (2000) Pharmacogenomics 1:95-100; and Shi (2001) Clin. Chem. 47:164-172). In some cases, the amplified nucleic acid molecules are subjected to shotgun sequencing. In some cases, sequencing of the sequencing library is performed using any appropriate sequencing technology, including but not limited to single molecule real-time (SMRT) sequencing, Polony sequencing, ligation sequencing, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electronic sequencing, pyrophosphate sequencing, Maxam-Gilbert sequencing, chain termination (e.g., Sanger) sequencing, +S sequencing or synthetic sequencing (array / colony-based or nanoball-based).

[0127] The sequencing library produced using the methods described herein (e.g., PTA or RNAseq) can be sequenced to obtain the desired number of sequencing reads. In some cases, a library (alone or part of a multi-omics workflow) is produced from a single cell or a sample comprising a single cell. In some cases, the library is sequenced to obtain at least 0.1, 0.2, 0.4, 0.5, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.5, 2, 5 or at least 10 million reads. In some cases, the library is sequenced to obtain no more than 0.1, 0.2, 0.4, 0.5, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.5, 2, 5 or no more than 10 million reads. In some cases, the library is sequenced to obtain about 0.1, 0.2, 0.4, 0.5, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.5, 2, 5 or about 10 million reads. In some cases, the library is sequenced to obtain 0.1-10, 0.1-5, 0.1-1, 0.2-1, 0.3-1.5, 0.5-1, 1-5 or 0.5-5 million reads per sample. In some cases, the number of reads depends on the size of the genome. In some cases, the sample comprising the bacterial genome is sequenced to obtain 0.5-1 million reads. In some cases, the library is sequenced to obtain at least 2, 4, 10, 20, 50, 100, 200, 300, 500, 700 or at least 900 million reads. In some cases, the library is sequenced to obtain no more than 2, 4, 10, 20, 50, 100, 200, 300, 500, 700 or no more than 900 million reads. In some cases, the library is sequenced to obtain about 2, 4, 10, 20, 50, 100, 200, 300, 500, 700 or about 900 million reads. In some cases, the sample comprising the mammalian genome is sequenced to obtain 500-600 million reads. In some cases, the type of sequencing library (cDNA library or genomic library) is identified during sequencing. In some cases, cDNA libraries and genomic libraries are identified with unique barcodes during sequencing.

[0128] When used with respect to polymerase-mediated amplification reaction, the term "cycle" is used in this article to describe the following steps: dissociation (denaturation) of at least a portion of a double-stranded nucleic acid (e.g., a template or double-stranded template from an amplicon), hybridization (annealing) of at least a portion of a primer with a template, and extension of the primer to produce an amplicon. In some cases, the temperature remains constant (e.g., isothermal reaction) during the amplification cycle. In some cases, the number of cycles is directly related to the number of amplicon produced. In some cases, the number of cycles of isothermal reaction is controlled by the amount of time that the reaction is carried out.

[0129] Methods and Applications

[0130] Described herein is the method for the mutation in the PTA method such as single cell identification cell with multi-omics analysis.Using the PTA method in some cases can cause the improvement to known method (for example, MDA).In some cases, compared with the MDA method, the false positive and false negative variant determination rate of PTA is lower.In some cases, by genome, such as NA12878 platinum genome, whether the larger genome coverage and uniformity of PTA can cause lower false negative variant determination rate.Not subject to theoretical constraints, it can be determined that the missing of error propagation in PTA reduces the false positive variant determination rate.In some cases, the amplification balance between alleles with these two methods is estimated by comparing the allele frequency of heterozygous mutation determined by known positive locus.In some cases, the amplicon library produced by PTA is further amplified by PCR.In some cases, PTA is used in workflow together with other analytical methods, such as RNAseq, methylation group analysis or additives as described herein.

[0131] In some cases, the cells analyzed using the methods described herein include tumor cells. For example, circulating tumor cells can be separated from body fluids taken from patients, such as but not limited to blood, bone marrow, urine, saliva, cerebrospinal fluid, pleural fluid, pericardial fluid, ascites or aqueous humor. Then, cells are subjected to the methods described herein (e.g., PTA) and sequenced to determine the mutation load and mutation combination of each cell. In some cases, these data are used to diagnose specific diseases or as a tool for predicting treatment response. Similarly, in some cases, cells with unknown malignant potential are separated from body fluids taken from patients, such as but not limited to blood, bone marrow, urine, saliva, cerebrospinal fluid, pleural fluid, pericardial fluid, ascites, aqueous humor, blastocyst cavity fluid or cultured cells around the collection medium. In some cases, samples are obtained from the collection medium around embryonic cells. After using the methods described herein and sequencing, these methods are also used to determine the mutation load and mutation combination of each cell. In some cases, these data are used to diagnose specific diseases or as a tool for predicting that pre-malignant states develop into dominant malignancies. In some cases, cells can be separated from primary tumor samples. Then, the cells can be subjected to PTA and sequencing to determine the mutation load and mutation combination of each cell. These data can be used to diagnose specific diseases or as a tool to predict the resistance of a patient's malignant tumor to available anticancer drugs. By exposing the sample to different chemotherapeutic drugs, it has been found that the main and secondary clones have different sensitivities to specific drugs, and these sensitivities are not necessarily related to the presence of known "driver mutations", which indicates that the combination of mutations in the clonal population determines its sensitivity to specific chemotherapeutic drugs. Without being bound by theory, these findings suggest that if a precancerous lesion that has not yet expanded and can evolve into a clone with an increased number of genomic modifications that makes it more likely to be resistant to treatment is detected, it may be easier to eradicate the malignancy. See, Ma et al., 2018, "Pan-cancer genome and transcriptome analyses of 1,699 pediatric leukemias and solid tumors". In some cases, single-cell genomics protocols are used to detect somatic genetic variant combinations in a single cancer cell or clonal type within a mixture of normal and malignant cells isolated from a patient sample. In some cases, this technique is also used to identify clonotypes that have undergone positive selection following exposure to a drug in vitro and / or in vivo in patients. Figure 6As shown in A, by comparing the surviving clones exposed to chemotherapy with the clones identified at diagnosis, a cancer clonal type catalog can be created that records their resistance to specific drugs. In some cases, the PTA method detects the sensitivity of specific clones in a sample composed of multiple clonal types to existing drugs or new drugs and their combinations, wherein the method can detect the sensitivity of specific clones to drugs. In some cases, this method shows the efficacy of drugs to specific clones, while the current drug sensitivity measurement takes into account the sensitivity of all cancer clones in one measurement, so this efficacy may not be detected. When the PTA described herein is applied to patient samples collected at diagnosis to detect cancer clonal types in a given patient's cancer, the drug sensitivity catalog can then be used to find these clones, thereby informing oncologists which drugs or drug combinations are ineffective and which drugs or drug combinations are most likely to be effective for the patient's cancer. PTA can be used to analyze samples containing clustered cells. In some cases, the sample contains neurons or glial cells. In some cases, the sample contains nuclei.

[0132] This paper describes the method of gene expression change when measuring and environmental factor mutagenicity combination.For example, cell (single cell or cell colony) is exposed to potential environmental conditions.For example, in some cases, the method uses cells such as being derived from organs (liver, pancreas, lung, colon, thyroid or other organs), tissues (skin or other tissues), blood or other biological sources.In some cases, environmental conditions include heat, light (for example, ultraviolet), radiation, chemical substances or any combination thereof.After a certain amount of environmental conditions are exposed, in some cases, this is a few minutes, a few hours, a few days or longer, separates single cells and performs PTA method.In some cases, molecular barcodes and unique molecular identifiers are used to mark samples.Samples are sequenced and then analyzed to identify gene expression changes and or exposure to the mutation caused by environmental conditions.In some cases, this mutation is compared with control environmental conditions, such as known non-mutagenic substances, vehicles / solvents or lack of environmental conditions.In some cases, this analysis not only provides the total number of mutations caused by environmental conditions, but also provides the position and properties of this mutation.In some cases, pattern is identified from data, and can be used to diagnose disease or illness. In some cases, patterns are used to predict future disease states or conditions. In some cases, the methods described herein measure the mutation load, location, and pattern of cells after exposure to environmental agents, e.g., potential mutagens or teratogens. In some cases, the method is used to assess the safety of a given agent, including the likelihood that it induces mutations that may lead to disease development. For example, the method can be used to predict the carcinogenicity or teratogenicity of a particular agent to a particular cell type after exposure to a particular concentration of a particular agent.

[0133] Described herein is the method for identifying the gene expression changes during the combination of mutations in animals, plants or microbial cells that have undergone genome editing (e.g., using CRISPR technology). In some cases, these cells can be separated and PTA and sequencing can be performed to determine the mutation load and mutation combination of each cell. In some cases, the mutation rate per cell and the mutation position produced by the genome editing scheme are used to assess the safety of a given genome editing method.

[0134] Described herein are methods for determining changes in gene expression when combined with mutations in cells used for cell therapy, such as, but not limited to, transplantation of induced pluripotent stem cells, transplantation of hematopoietic or other cells that have not been manipulated, or transplantation of hematopoietic or other cells that have undergone genome editing. The cells can then be subjected to PTA and sequencing to determine the mutation load and combination of mutations in each cell. The per-cell mutation rate and mutation location in a cell therapy product can be used to assess the safety and potential efficacy of the product.

[0135] The cell used for use with the PTA method can be a fetal cell, such as an embryonic cell. In some embodiments, PTA is used in combination with non-invasive pre-implantation genetic testing (NIPGT). In another embodiment, cells can be separated from blastomeres produced by in vitro fertilization. Then, cells can undergo PTA and sequencing to determine the load and combination of potential disease-susceptible genetic variants in each cell. Then, the combination of gene expression changes in cells and mutation spectrum can be used to infer the genetic susceptibility of blastomeres to specific diseases before implantation. In some cases, the embryo in the culture sheds nucleic acid, and the nucleic acid is used to assess the health status of the embryo using low-throughput genome sequencing. In some cases, the embryo is frozen and thawed. In some cases, nucleic acid is obtained from embryonic cell culture conditioned medium (BCCM), blastocyst cavity fluid (BF) or its combination. In some cases, chromosome aberrations are detected using the PTA analysis of fetal cells, such as fetal aneuploidy. In some cases, diseases such as Down's or Patau syndrome are detected using PTA. In some cases, before obtaining nucleic acid for analysis (e.g., culture medium, BF or cell biopsy), frozen embryonic cells are thawed and cultured for a period of time. In some cases, before obtaining nucleic acid for analysis, embryonic cells are cultured for no more than 4, 6, 8, 12, 16, 24, 36, 48 hours or no more than 64 hours.

[0136] In another embodiment, microbial cells (e.g., bacteria, fungi, protozoa) can be isolated from plants or animals (e.g., microbiota samples [e.g., GI microbiota, skin microbiota, etc.] or body fluids, e.g., blood, bone marrow, urine, saliva, cerebrospinal fluid, pleural fluid, pericardial fluid, ascites, or aqueous humor). In addition, microbial cells can be isolated from indwelling medical devices, such as, but not limited to, intravenous catheters, urinary catheters, cerebrospinal shunts, prosthetic valves, artificial joints, or endotracheal catheters. The cells can then undergo PTA and sequencing to determine the identity of the specific microorganism and detect the presence of microbial genetic variants that predict response (or resistance) to specific antimicrobial agents. These data can be used to diagnose specific infectious diseases and / or as a tool to predict treatment response.

[0137] This article describes the method of using the PTA method described herein to produce an amplicon library from a sample including short nucleic acids. In some cases, PTA can improve the fidelity and uniformity of short nucleic acid amplification. In some cases, the length of the nucleic acid is no more than 2000 bases. In some cases, the length of the nucleic acid is no more than 1000 bases. In some cases, the length of the nucleic acid is no more than 500 bases. In some cases, the length of the nucleic acid is no more than 200, 400, 750, 1000, 2000 or 5000 bases. In some cases, the sample including the short nucleic acid fragment includes but is not limited to ancient DNA (age is hundreds of years, thousands of years, millions or even billions of years), FFPE (formalin-fixed paraffin-embedded) sample, cell-free DNA or other samples including short nucleic acids.

[0138] Implementation

[0139] Described herein is a method for amplifying a target nucleic acid molecule, the method comprising: a) contacting a sample comprising a target nucleic acid molecule, one or more amplification primers, a nucleic acid polymerase, and a nucleotide mixture comprising one or more terminator nucleotides, the terminator nucleotides terminating nucleic acid replication by the polymerase, and b) incubating the sample under conditions that promote replication of the target nucleic acid molecule to obtain a plurality of terminated amplification products, wherein replication is performed by strand displacement replication. In one embodiment of any of the above methods, the method further comprises separating products having a length of about 50 to about 2000 nucleotides from a plurality of terminated amplification products. In one embodiment of any of the above methods, the method further comprises separating products having a length of about 400 to about 600 nucleotides from a plurality of terminated amplification products. In one embodiment of any of the above methods, the method further comprises: c) repairing the ends and adding A-tails, and d) connecting the molecules obtained in step (c) to adapters, thereby producing a library of amplification products. In some embodiments, the method further comprises removing terminator nucleotides from the terminated amplification products. In one embodiment of any of the above methods, the method further comprises sequencing the amplification products. In one embodiment of any of the above methods, the amplification is performed under substantially isothermal conditions. In one embodiment of any of the above methods, the nucleic acid polymerase is a DNA polymerase.

[0140] In one embodiment of any of the above methods, the DNA polymerase is a strand displacement DNA polymerase. In one embodiment of any of the above methods, the nucleic acid polymerase is selected from bacteriophage phi29 (Φ29) polymerase, genetically modified phi29 (Φ29) DNA polymerase, Klenow fragment of DNA polymerase I, bacteriophage M2 DNA polymerase, bacteriophage phiPRD1 DNA polymerase, Bst DNA polymerase, Bst large fragment DNA polymerase, exo(-)Bst polymerase, exo(-)Bca DNA polymerase, BsuDNA polymerase, Vent R DNA polymerase, Vent R(exo-)DNA polymerase, Deep Vent DNA polymerase, Deep Vent (exo-)DNA polymerase, IsoPol DNA polymerase, DNA polymerase I, Therminator DNA polymerase, T5 DNA polymerase, Sequenase, T7 DNA polymerase, T7-Sequenase and T4 DNA polymerase. In one embodiment of any of the above methods, the nucleic acid polymerase has 3'->5' exonuclease activity, and the terminator nucleotide inhibits this 3'->5' exonuclease activity. In a specific embodiment, the terminator nucleotide is selected from a nucleotide modified with an α group (e.g., an α-thiodideoxynucleotide that produces a thiophosphate bond), a C3 spacer nucleotide, a locked nucleic acid (LNA), a reverse nucleic acid, a 2'fluoro nucleotide, a 3'phosphorylated nucleotide, a 2'-O-methyl modified nucleotide and a trans nucleic acid. In one embodiment of any of the above methods, the nucleic acid polymerase does not have 3'->5' exonuclease activity. In a specific embodiment, the polymerase is selected from Bst DNA polymerase, exo(-)Bst polymerase, exo(-)Bca DNA polymerase, Bsu DNA polymerase, Vent R(exo-)DNA polymerase, Deep Vent (exo-)DNA polymerase, Klenow fragment (exo-)DNA polymerase and Therminator DNA polymerase. In a specific embodiment, the terminator nucleotide includes a modification of the r group of the 3' carbon of the deoxyribose. In a specific embodiment, the terminator nucleotide is selected from a reversible terminator including a 3' sealed terminator of a nucleotide, a reversible terminator including a 3' unsealed terminator of a nucleotide, a terminator including a 2' modified terminator of a deoxynucleotide, a terminator including a modification of the nitrogenous base of a deoxynucleotide and a combination thereof. In a specific embodiment, the terminator nucleotide is selected from dideoxynucleotides, reverse dideoxynucleotides, 3' biotinylated nucleotides, 3' aminonucleotides, 3'-phosphorylated nucleotides, 3'-O-methyl nucleotides, 3' C3 spacer nucleotides, 3' C18 nucleotides, 3' hexanediol spacer nucleotides 3' carbon spacer nucleotides, acyclic nucleotides, and combinations thereof. In one embodiment of any of the above methods, the length of the amplification primer is 4 to 70 nucleotides. In one embodiment of any of the above methods, the length of the amplified product is about 50 to about 2000 nucleotides. In one embodiment of any of the above methods, the target nucleic acid is DNA (e.g., cDNA or genomic DNA). In one embodiment of any of the above methods, the amplification primer is a random primer. In one embodiment of any of the above methods, the amplification primer includes a barcode. In a specific embodiment, the barcode includes a cell barcode. In a specific embodiment, the barcode includes a sample barcode. In one embodiment of any of the above methods, the amplification primer includes a unique molecular identifier (UMI). In one embodiment of any of the above methods, the method includes denaturing the target nucleic acid or genomic DNA before the initial primer annealing. In a specific embodiment, the denaturation is performed under alkaline conditions and then neutralized. In one embodiment of any of the above methods, the sample, amplification primer, nucleic acid polymerase and nucleotide mixture are contained in a microfluidic device. In one embodiment of any of the above methods, the sample, amplification primer, nucleic acid polymerase and nucleotide mixture are contained in a droplet. In one embodiment of any of the above methods, the sample is selected from a sample of one or more tissues, a cell, a biological fluid sample (e.g., blood, urine, saliva, lymph, cerebrospinal fluid (CSF), amniotic fluid, pleural fluid, pericardial fluid, ascites, aqueous humor), a bone marrow sample, a semen sample, a biopsy sample, a cancer sample, a tumor sample, a cell lysate sample, a forensic sample, an archaeological sample, a paleontological sample, an infection sample, a production sample, a whole plant, a plant part, a microbiome sample, a viral preparation, a soil sample, a marine sample, a freshwater sample, a household or industrial sample, and combinations and isolates thereof. In one embodiment of any of the above methods, the sample is a cell (e.g., an animal cell [e.g., a human cell], a plant cell, a fungal cell, a bacterial cell, and a protozoan cell).In a specific embodiment, the cell is lysed before replication. In a specific embodiment, cell lysis is accompanied by proteolysis. In a specific embodiment, the cell is selected from cells from preimplantation embryos, stem cells, fetal cells, tumor cells, suspected cancer cells, cancer cells, cells subjected to gene editing procedures, cells from pathogenic organisms, cells obtained from forensic samples, cells obtained from archaeological samples, and cells obtained from paleontological samples. In one embodiment of any of the above methods, the sample is from cells from preimplantation embryos (e.g., blastomeres [e.g., blastomeres obtained from eight-cell embryos produced by in vitro fertilization]). In a specific embodiment, the method also includes determining the presence of disease-susceptible germline or somatic variants in embryonic cells. In one embodiment of any of the above methods, the sample is a cell from a pathogenic organism (e.g., bacteria, fungi, protozoa). In a specific embodiment, pathogenic organism cells are obtained from body fluids, microbial samples (e.g., GI microbial samples, vaginal microbial samples, skin microbial samples, etc.) or indwelling medical devices (e.g., intravenous catheters, urinary catheters, cerebrospinal shunts, prosthetic valves, artificial joints, tracheal tubes, etc.) taken from patients. In a specific embodiment, the method further includes a step of determining the identity of the pathogenic organism. In a specific embodiment, the method further includes determining the presence of genetic variants that cause the pathogenic organism to be resistant to treatment. In one embodiment of any of the above methods, the sample is a tumor cell, a suspected cancer cell, or a cancer cell. In a specific embodiment, the method further includes determining the presence of one or more diagnostic or prognostic mutations. In a specific embodiment, the method further includes determining the presence of germline or somatic variants that cause resistance to treatment. In one embodiment of any of the above methods, the sample is a cell that has undergone a gene editing procedure. In a specific embodiment, the method further includes determining the presence of unplanned mutations caused by the gene editing process. In one embodiment of any of the above methods, the method further includes determining the cell lineage history. In a related aspect, the present invention provides the use of any of the above methods in identifying low-frequency sequence variants (e.g., variants that constitute ≥0.01% of the total sequence).

[0141] In a related aspect, the present invention provides a kit comprising a nucleic acid polymerase, one or more amplification primers, a nucleotide mixture including one or more terminator nucleotides, and optional instructions for use. In one embodiment of the kit of the present invention, the nucleic acid polymerase is a strand displacement DNA polymerase. In one embodiment of the kit of the present invention, the nucleic acid polymerase is selected from bacteriophage phi29 (Φ29) polymerase, genetically modified phi29 (Φ29) DNA polymerase, Klenow fragment of DNA polymerase I, bacteriophage M2 DNA polymerase, bacteriophage phiPRD1 DNA polymerase, Bst DNA polymerase, Bst large fragment DNA polymerase, exo(-)Bst polymerase, exo(-)Bca DNA polymerase, Bsu DNA polymerase, Vent R DNA polymerase, Vent R (exo-)DNA polymerase, Deep Vent DNA polymerase, Deep Vent (exo-)DNA polymerase, IsoPol DNA polymerase, DNA polymerase I, Therminator DNA polymerase, T5 DNA polymerase, Sequenase, T7 DNA polymerase, T7-sequenase and T4 DNA polymerase. In one embodiment of the kit of the present invention, the nucleic acid polymerase has 3'->5' exonuclease activity, and the terminator nucleotide inhibits this 3'->5' exonuclease activity (e.g., nucleotides modified with an α group [e.g., α-thiodideoxynucleotides], C3 spacer nucleotides, locked nucleic acids (LNA), reverse nucleic acids, 2' fluoronucleotides, 3' phosphorylated nucleotides, 2'-O-methyl modified nucleotides, trans nucleic acids). In one embodiment of the kit of the present invention, the nucleic acid polymerase does not have 3'->5' exonuclease activity (e.g., Bst DNA polymerase, exo(-)Bst polymerase, exo(-)Bca DNA polymerase, Bsu DNA polymerase, Vent R(exo-)DNA polymerase, Deep Vent (exo-)DNA polymerase, Klenow fragment (exo-)DNA polymerase, Therminator DNA polymerase). In a specific embodiment, the terminator nucleotide includes a modification of the r group of the 3' carbon of the deoxyribose. In a specific embodiment, the terminator nucleotide is selected from a reversible terminator including a 3' blocked terminator of the nucleotide, a reversible terminator including a 3' unblocked terminator of the nucleotide, a terminator including a 2' modified terminator of the deoxynucleotide, a terminator including a modification of the nitrogenous base of the deoxynucleotide, and a combination thereof. In a specific embodiment, the terminator nucleotide is selected from dideoxynucleotides, reverse dideoxynucleotides, 3'biotinylated nucleotides, 3'aminonucleotides, 3'-phosphorylated nucleotides, 3'-O-methyl nucleotides, 3'C3 spacer nucleotides, 3'C18 nucleotides, 3'hexanediol spacer nucleotides 3'carbon spacer nucleotides, acyclic nucleotides, and combinations thereof.

[0142] Described herein is a method for amplifying a genome, the method comprising: a) contacting a sample comprising a genome, a plurality of amplification primers (e.g., two or more primers), a nucleic acid polymerase, and a nucleotide mixture comprising one or more terminator nucleotides, the terminator nucleotides terminating nucleic acid replication of the polymerase, and b) incubating the sample under conditions that promote genome replication to obtain a plurality of terminated amplification products, wherein replication is performed by strand displacement replication. In one embodiment of any of the above methods, the method further comprises separating a product having a length of about 50 to about 2000 nucleotides from a plurality of terminated amplification products. In one embodiment of any of the above methods, the method further comprises separating a product having a length of about 400 to about 600 nucleotides from a plurality of terminated amplification products. In one embodiment of any of the above methods, the method further comprises: c) repairing the ends and adding A-tails, and d) connecting the molecules obtained in step (c) to adapters, thereby producing an amplification product library. In one embodiment of any of the above methods, the method further comprises sequencing the amplification products. In one embodiment of any of the above methods, the amplification is performed under substantially isothermal conditions. In one embodiment of any of the above methods, the nucleic acid polymerase is a DNA polymerase.

[0143] In one embodiment of any of the above methods, the DNA polymerase is a strand displacement DNA polymerase. In one embodiment of any of the above methods, the nucleic acid polymerase is selected from bacteriophage phi29 (Φ29) polymerase, genetically modified phi29 (Φ29) DNA polymerase, Klenow fragment of DNA polymerase I, bacteriophage M2 DNA polymerase, bacteriophage phiPRD1 DNA polymerase, Bst DNA polymerase, Bst large fragment DNA polymerase, exo(-)Bst polymerase, exo(-)Bca DNA polymerase, BsuDNA polymerase, Vent R DNA polymerase, Vent R (exo-)DNA polymerase, Deep Vent DNA polymerase, Deep Vent (exo-)DNA polymerase, IsoPol DNA polymerase, DNA polymerase I, Therminator DNA polymerase, T5 DNA polymerase, Sequenase, T7 DNA polymerase, T7-Sequenase and T4 DNA polymerase. In one embodiment of any of the above methods, the nucleic acid polymerase has 3'->5' exonuclease activity, and the terminator nucleotide inhibits this 3'->5' exonuclease activity. In a specific embodiment, the terminator nucleotide is selected from a nucleotide modified with an α group (e.g., an α-thiodideoxynucleotide that produces a thiophosphate bond), a C3 spacer nucleotide, a locked nucleic acid (LNA), a reverse nucleic acid, a 2'fluoro nucleotide, a 3'phosphorylated nucleotide, a 2'-O-methyl modified nucleotide and a trans nucleic acid. In one embodiment of any of the above methods, the nucleic acid polymerase does not have 3'->5' exonuclease activity. In a specific embodiment, the polymerase is selected from Bst DNA polymerase, exo(-)Bst polymerase, exo(-)Bca DNA polymerase, Bsu DNA polymerase, Vent R(exo-)DNA polymerase, Deep Vent (exo-)DNA polymerase, Klenow fragment (exo-)DNA polymerase and Therminator DNA polymerase. In a specific embodiment, the terminator nucleotide includes a modification of the r group of the 3' carbon of the deoxyribose. In a specific embodiment, the terminator nucleotide is selected from a reversible terminator including a 3' sealed terminator of a nucleotide, a reversible terminator including a 3' unsealed terminator of a nucleotide, a terminator including a 2' modified terminator of a deoxynucleotide, a terminator including a modification of the nitrogenous base of a deoxynucleotide and a combination thereof. In a specific embodiment, the terminator nucleotide is selected from dideoxynucleotides, reverse dideoxynucleotides, 3' biotinylated nucleotides, 3' aminonucleotides, 3'-phosphorylated nucleotides, 3'-O-methyl nucleotides, 3' C3 spacer nucleotides, 3' C18 nucleotides, 3' hexanediol spacer nucleotides 3' carbon spacer nucleotides, acyclic nucleotides, and combinations thereof. In one embodiment of any of the above methods, the length of the amplification primer is 4 to 70 nucleotides. In one embodiment of any of the above methods, the length of the amplified product is about 50 to about 2000 nucleotides. In one embodiment of any of the above methods, the target nucleic acid is DNA (e.g., cDNA or genomic DNA). In one embodiment of any of the above methods, the amplification primer is a random primer. In one embodiment of any of the above methods, the amplification primer includes a barcode. In a specific embodiment, the barcode includes a cell barcode. In a specific embodiment, the barcode includes a sample barcode. In one embodiment of any of the above methods, the amplification primer includes a unique molecular identifier (UMI). In one embodiment of any of the above methods, the method includes denaturing the target nucleic acid or genomic DNA before the initial primer annealing. In a specific embodiment, the denaturation is performed under alkaline conditions and then neutralized. In one embodiment of any of the above methods, the sample, amplification primer, nucleic acid polymerase and nucleotide mixture are contained in a microfluidic device. In one embodiment of any of the above methods, the sample, amplification primer, nucleic acid polymerase and nucleotide mixture are contained in a droplet. In one embodiment of any of the above methods, the sample is selected from tissue samples, cells, biological fluid samples (e.g., blood, urine, saliva, lymph, cerebrospinal fluid (CSF), amniotic fluid, pleural fluid, pericardial fluid, ascites, aqueous humor), bone marrow samples, semen samples, biopsy samples, cancer samples, tumor samples, cell lysate samples, forensic samples, archaeological samples, paleontological samples, infection samples, production samples, whole plants, plant parts, microbial samples, viral preparations, soil samples, marine samples, freshwater samples, household or industrial samples, and combinations and isolates thereof. In one embodiment of any of the above methods, the sample is a cell (e.g., animal cells [e.g., human cells], plant cells, fungal cells, bacterial cells, and protozoan cells).In a specific embodiment, the cell is lysed before replication. In a specific embodiment, cell lysis is accompanied by proteolysis. In a specific embodiment, the cell is selected from cells from preimplantation embryos, stem cells, fetal cells, tumor cells, suspected cancer cells, cancer cells, cells subjected to gene editing procedures, cells from pathogenic organisms, cells obtained from forensic samples, cells obtained from archaeological samples, and cells obtained from paleontological samples. In one embodiment of any of the above methods, the sample is from cells from preimplantation embryos (e.g., blastomeres [e.g., blastomeres obtained from eight-cell embryos produced by in vitro fertilization]). In a specific embodiment, the method also includes determining the presence of disease-susceptible germline or somatic variants in embryonic cells. In one embodiment of any of the above methods, the sample is a cell from a pathogenic organism (e.g., bacteria, fungi, protozoa). In a specific embodiment, pathogenic organism cells are obtained from body fluids, microbial samples (e.g., GI microbial samples, vaginal microbial samples, skin microbial samples, etc.) or indwelling medical devices (e.g., intravenous catheters, urinary catheters, cerebrospinal shunts, prosthetic valves, artificial joints, tracheal tubes, etc.) taken from patients. In a specific embodiment, the method further includes a step of determining the identity of the pathogenic organism. In a specific embodiment, the method further includes determining the presence of genetic variants that cause the pathogenic organism to be resistant to treatment. In one embodiment of any of the above methods, the sample is a tumor cell, a suspected cancer cell, or a cancer cell. In a specific embodiment, the method further includes determining the presence of one or more diagnostic or prognostic mutations. In a specific embodiment, the method further includes determining the presence of germline or somatic variants that cause resistance to treatment. In one embodiment of any of the above methods, the sample is a cell that has undergone a gene editing procedure. In a specific embodiment, the method further includes determining the presence of unplanned mutations caused by the gene editing process. In one embodiment of any of the above methods, the method further includes determining the cell lineage history. In a related aspect, the present invention provides the use of any of the above methods in identifying low-frequency sequence variants (e.g., variants that constitute ≥0.01% of the total sequence).

[0144] In a related aspect, the invention provides a kit comprising a reverse transcriptase, a nucleic acid polymerase, one or more amplification primers, a nucleotide mixture comprising one or more terminator nucleotides, and optional instructions for use. In one embodiment of the kit of the invention, the nucleic acid polymerase is a strand displacement DNA polymerase. In some cases, the reverse transcriptase performs template switching. In some cases, the reverse transcriptase is a variant of MMLV (Moloney murine leukemia virus), HIV-1, AMV (avian myeloblastosis virus), telomerase RT, FIV (feline immunodeficiency virus) or XMRV (heterophilic murine leukemia virus-related virus). Non-limiting examples of reverse transcriptases include SuperScript I (Thermo), SuperScript II (Thermo), SuperScript III (Thermo), SuperScript IV (Thermo), OmniScript (Qiagen), SensiScript (Qiagen), PrimeScript (Takara), Maxima H- (Thermo), AcuuScript Hi-Fi (Agilent), iScript (Bio-Rad), eAMV (Merck KGaA), qScript (Quanta Biosciences), SmartScribe (Clontech), or GoScript (Promega). In one embodiment of the kit of the present invention, the nucleic acid polymerase is selected from bacteriophage phi29 (Φ29) polymerase, genetically modified phi29 (Φ29) DNA polymerase, Klenow fragment of DNA polymerase I, bacteriophage M2 DNA polymerase, bacteriophage phiPRD1 DNA polymerase, Bst DNA polymerase, Bst large fragment DNA polymerase, exo (-) Bst polymerase, exo (-) Bca DNA polymerase, Bsu DNA polymerase, Vent R DNA polymerase, Vent R(exo-)DNA polymerase, Deep Vent DNA polymerase, Deep Vent (exo-)DNA polymerase, IsoPol DNA polymerase, DNA polymerase I, Therminator DNA polymerase, T5 DNA polymerase, Sequenase, T7 DNA polymerase, T7-sequenase and T4 DNA polymerase. In one embodiment of the kit of the present invention, the nucleic acid polymerase has 3'->5' exonuclease activity, and the terminator nucleotide inhibits this 3'->5' exonuclease activity (e.g., nucleotides modified with an α group [e.g., α-thiodideoxynucleotides], C3 spacer nucleotides, locked nucleic acids (LNA), reverse nucleic acids, 2' fluoronucleotides, 3' phosphorylated nucleotides, 2'-O-methyl modified nucleotides, trans nucleic acids). In one embodiment of the kit of the present invention, the nucleic acid polymerase does not have 3'->5' exonuclease activity (e.g., Bst DNA polymerase, exo(-)Bst polymerase, exo(-)Bca DNA polymerase, Bsu DNA polymerase, Vent R In one embodiment, the terminator nucleotide comprises a modification of the r group of the 3' carbon of the deoxyribose. In one embodiment, the terminator nucleotide is selected from a reversible terminator including a 3' sealed terminator of the nucleotide, a reversible terminator including a 3' unsealed terminator of the nucleotide, a terminator including a 2' modified terminator of the deoxynucleotide, a terminator including a modification of the nitrogenous base of the deoxynucleotide, and a combination thereof. In one embodiment, the terminator nucleotide is selected from dideoxynucleotides, reverse dideoxynucleotides, 3' biotinylated nucleotides, 3' aminonucleotides, 3'-phosphorylated nucleotides, 3'-O-methyl nucleotides, 3' C3 spacer nucleotides, 3' C18 nucleotides, 3' hexanediol spacer nucleotides, acyclic nucleotides, and combinations thereof. In some cases, the test kit comprises at least one enzyme stabilizer, a neutralizing buffer, a denaturing buffer, or a combination thereof. In some cases, the test kit comprises one or more modules. In some cases, a kit comprises a genome module and a transcriptome module.

[0145] Numbering Implementation Plan

[0146] The following numbered embodiments 1-46 are described herein. 1. Embodiments are described herein, comprising a method for multi-omics single-cell analysis, the method comprising: a. isolating a single cell from a cell population; b. sequencing a cDNA library comprising polynucleotides amplified from mRNA transcripts from the cell; and c. sequencing the genome of the cell, wherein sequencing the genome of the cell comprises: i. providing a genome from a single cell; ii. contacting the genome with at least one amplification primer, at least one nucleic acid polymerase, and a nucleotide mixture, wherein the nucleotide mixture comprises at least one terminator nucleotide, the at least one terminator nucleotide terminating nucleic acid replication of the polymerase; and iii. amplifying at least some of the genome to produce a plurality of terminated amplification products, wherein the replication is performed by strand displacement replication; iv. ligating the molecule obtained in step (ii) to an adapter, thereby producing a genomic DNA library; and v. sequencing the genomic DNA library. 2. Also provided herein is a method as in embodiment 1, wherein the method further comprises identifying at least one protein on the surface of the cell. 3. Also provided herein is a method as in embodiment 1, wherein the mRNA transcript comprises a polyadenylated mRNA transcript. 4. Also provided herein is a method as in embodiment 1, wherein the mRNA transcripts do not include polyadenylated mRNA transcripts. 5. Also provided herein is a method as in any one of embodiments 1-4, wherein sequencing the cDNA library comprises amplifying the mRNA transcripts with a template switching primer. 6. Also provided herein is a method as in any one of embodiments 1-4, wherein at least some of the polynucleotides in the cDNA library comprise a barcode. 7. Also provided herein is a method as in any one of embodiments 1-4, wherein at least some of the polynucleotides in the cDNA library comprise at least two barcodes. 8. Also provided herein is a method as in embodiment 6 or 7, wherein the barcode comprises a cell barcode. 9. Also provided herein is a method as in embodiment 6 or 7, wherein the barcode comprises a sample barcode.10. A method for multi-omics single-cell analysis, the method comprising: a. isolating a single cell from a cell population; b. identifying at least one protein on the cell surface; and c. sequencing the genome of the cell, wherein sequencing the genome of the cell comprises: i. providing a genome from a single cell; ii. contacting the genome with at least one amplification primer, at least one nucleic acid polymerase, and a nucleotide mixture, wherein the nucleotide mixture comprises at least one terminator nucleotide, the at least one terminator nucleotide terminating nucleic acid replication of the polymerase; iii. amplifying at least some of the genome to produce a plurality of terminated amplification products, wherein the replication is performed by strand displacement replication; iv. ligating the molecule obtained in step (ii) to an adapter, thereby producing a genomic DNA library; and v. sequencing the genomic DNA library. 11. Also provided herein is a method as in embodiment 10, wherein identifying at least one protein on the cell surface comprises contacting the cell with a labeled antibody that binds to the at least one protein. 12. Also provided herein is a method as in embodiment 11, wherein the labeled antibody comprises at least one fluorescent label. 13. Also provided herein is a method as in embodiment 11, wherein the labeled antibody comprises at least one mass tag. 14. Also provided herein is a method as in embodiment 11, wherein the labeled antibody comprises at least one nucleic acid barcode. 15. A method for multi-omics single-cell analysis, the method comprising: a. isolating a single cell from a cell population; b. sequencing the genome of the cell, wherein sequencing the genome of the cell comprises: i. providing a genome from a single cell; ii. digesting the genome with a methylation-sensitive restriction enzyme to produce genomic fragments; iii. contacting at least some of the genomic fragments with at least one amplification primer, at least one nucleic acid polymerase and a nucleotide mixture, wherein the nucleotide mixture comprises at least one terminator nucleotide, the at least one terminator nucleotide terminating nucleic acid replication of the polymerase; iv. amplifying at least some of the genome to produce a plurality of terminated amplification products, wherein the replication is performed by strand displacement replication; v. amplifying at least some of the genomic fragments with methylation-specific PCR; vi. connecting the molecules obtained in steps (iv and v) to adapters to produce a genomic DNA library and a methylome DNA library; and vii. sequencing the genomic DNA library and the methylome library. 16. Also provided herein is a method as in embodiment 15, wherein identifying at least one protein on the surface of the cell comprises contacting the cell with a labeled antibody that binds to the at least one protein. 17. The present invention also provides a method as described in embodiment 16, wherein the labeled antibody comprises at least one fluorescent label.18. Also provided herein is a method as in embodiment 16, wherein the labeled antibody comprises at least one mass tag. 19. Also provided herein is a method as in embodiment 16, wherein the labeled antibody comprises at least one nucleic acid barcode. 20. Also provided herein is a method as in any one of embodiments 1-19, wherein the single cell is a mammalian cell. 21. Also provided herein is a method as in any one of embodiments 1-19, wherein the single cell is a human cell. 22. Also provided herein is a method as in any one of embodiments 1-19, wherein the single cell is derived from liver, skin, kidney, blood or lung. 23. Also provided herein is a method as in any one of embodiments 1-19, wherein the single cell is a primary cell. 24. Also provided herein is a method as in any one of embodiments 1-23, wherein the method further comprises removing at least one terminator nucleotide from the terminated amplification product. 25. Also provided herein is a method as in any one of embodiments 1-23, wherein at least some of the amplification products comprise a barcode. 26. Also provided herein is a method as in any one of embodiments 1-23, wherein at least some of the amplification products comprise at least two barcodes. 27. Also provided herein is a method as in embodiment 24 or 26, wherein the barcode comprises a cell barcode. 28. Also provided herein is a method as in embodiment 24 or 26, wherein the barcode comprises a sample barcode. 29. Also provided herein is a method as in any one of embodiments 1-28, wherein at least some of the amplification primers comprise a unique molecular identifier (UMI). 30. Also provided herein is a method as in any one of embodiments 1-28, wherein at least some of the amplification primers comprise at least two unique molecular identifiers (UMIs). 31. Also provided herein is a method as in any one of embodiments 1-30, wherein the method further comprises an additional amplification step using PCR. 32. Also provided herein is a method as in any one of embodiments 1-30, wherein at least one mutation is identified in the genome of the cell, wherein the mutation is different from the corresponding position in the reference sequence. 33. Also provided herein is a method as in embodiment 32, wherein the at least one mutation occurs in less than 50% of the cell population. 34. Also provided herein is a method as in embodiment 32, wherein the at least one mutation occurs in less than 25% of the cell population. 35. Also provided herein is a method as in embodiment 32, wherein the at least one mutation occurs in less than 1% of the cell population. 36. Also provided herein is a method as in embodiment 32, wherein the at least one mutation occurs in no more than 0.1% of the cell population. 37. Also provided herein is a method as in embodiment 32, wherein the at least one mutation occurs in no more than 0.01% of the cell population.38. Also provided herein is a method as in embodiment 32, wherein the at least one mutation occurs in no more than 0.001% of the cell population. 39. Also provided herein is a method as in embodiment 32, wherein the at least one mutation occurs in no more than 0.0001% of the cell population. 40. Also provided herein is a method as in embodiment 32, wherein the at least one mutation occurs in no more than 50% of the amplified product sequences. 41. Also provided herein is a method as in embodiment 32, wherein the at least one mutation occurs in no more than 25% of the amplified product sequences. 42. Also provided herein is a method as in embodiment 32, wherein the at least one mutation occurs in no more than 1% of the amplified product sequences. 43. Also provided herein is a method as in embodiment 32, wherein the at least one mutation occurs in no more than 0.1% of the amplified product sequences. 44. Also provided herein is a method as in embodiment 32, wherein the at least one mutation occurs in no more than 0.01% of the amplified product sequences. 45. Also provided herein is a method as in embodiment 32, wherein the at least one mutation occurs in no more than 0.001% of the amplified product sequences. 46. ​​The present invention also provides a method as described in embodiment 32, wherein the at least one mutation occurs in no more than 0.0001% of the sequence of the amplified product.

[0147] Example

[0148] The following examples are presented to more clearly illustrate the principles and practices of the embodiments disclosed herein to those skilled in the art, and should not be construed as limiting the scope of any claimed embodiments. Unless otherwise indicated, all parts and percentages are by weight.

[0149] Example 1: Primary template directed amplification (PTA)

[0150] Although PTA can be used for any nucleic acid amplification, it is particularly useful for whole genome amplification because it can capture a greater percentage of the cellular genome in a more uniform and reproducible manner and with lower error rates than currently used methods such as multiple displacement amplification (MDA), avoiding the shortcomings of currently used methods, such as exponential amplification where the polymerase first extends random primers, which can lead to random overrepresentation of loci and alleles and propagation of mutations (see Figure 1G ). PTA is also used with other analysis techniques, such as transcriptome analysis.

[0151] Cell culture

[0152] Human NA12878 (Coriell Institute) cells were maintained in RPMI medium supplemented with 15% FBS and 2 mM L-glutamine, 100 units / mL penicillin, 100 μg / mL streptomycin, and 0.25 μg / mL amphotericin B (Gibco, Life Technologies). 5 The cultures were split every 3 days and maintained in a humidified incubator at 37°C with 5% CO2.

[0153] Single cell isolation and WTA

[0154] The general protocol of WTA (whole transcriptome analysis) is shown in Figure 2F Resuspend the cells at a concentration of 150-500 cells / μL. Wash the cell suspension with 20 μL of freshly prepared staining buffer (1.25 mL of cell buffer from Life Technology containing 1X PBS and 0.05% tween-20). The cells were then sorted using a FACS Aria III sorter to deposit cells in each of the 96 wells. The following reaction mixture was added to each well: 5x RT buffer, PEG4000, RT primer (100uM), TS oligonucleotide (20uM), reverse transcriptase, RNase inhibitor, gelatin, Tween-20, Triton-X, dNTP mixture, TMAC (1M), betaine (5M), MgCl2 (50mM), ERCC incorporation. The sample was then placed on a thermal cycler at 42°C for 90min, at 50°C for 30min, and then kept at 4°C until the sample can be processed for pre-amplification. After thermal cycling for RT, the sample was processed for DNA amplification or pre-amplification of the first-strand cDNA generated by the RT reaction. Pre-amplification of the samples was done using the following protocol for amplifying cDNA products using a single primer (semi-inhibitory PCR). Briefly, 5uL of RT reaction was added to a 30 microliter reaction containing 2X master mix, 1 micromolar primer, and 5X pre-amplification buffer using the following thermal cycling conditions: 95°C-1min, 21 cycles of 95°C-15s, 60°C-30s, 68°C-4min, and then a ten-minute period at 72°C. The samples were then converted into sequencing libraries using the Nextera XT library preparation kit using the manufacturer's instructions ( Figure 2G ). The results of the RT experiments are shown in Table 1 for the six samples.

[0155] Table 1

[0156]

[0157] Single cell isolation and WGA

[0158] 3.5×10 5 After inoculation at a density of 10 cells / ml, NA12878 cells were cultured for at least three days, after which 3 mL of the cell suspension was pelleted at 300 x g for 10 minutes. The culture medium was then discarded and 1 mL of cell wash buffer (containing 2% FBS, without Mg) was used. 2 or Ca 2 1X PBS) was washed 3 times, centrifuged at 300xg, 200xg and finally 100xg for 5 minutes. The cells were then resuspended in 500 μL cell washing buffer. Subsequently, 100nM calcein AM (molecular probes) and 100ng / ml propidium iodide (PI; Sigma-Aldrich) were used to distinguish the living cell population. The cells were loaded on a BD FACScan flow cytometer (FACSAria II) (BD Biosciences) that had been thoroughly washed with ELIMINase (DeconLabs) and calibrated using Accudrop fluorescent beads (BD Biosciences) to perform cell sorting. In cells to be subjected to PTA (Sigma-Aldrich), single cells from the fractions that were positive for calcein AM and negative for PI were sorted into each well of a 96-well plate, containing 3 μL of PBS (Qiagen, REPLI-g SC kit) with 0.2% Tween 20 in the well. Several wells were intentionally left empty to serve as no template controls (NTCs). Immediately after sorting, the plate was briefly centrifuged and placed on ice. The cells were then frozen at -20°C for at least overnight. The next day, WGA reactions were performed on a pre-PCR workstation that provided constant positive pressure of HEPA-filtered air and was sterilized with UV light for 30 minutes before each experiment.

[0159] MDA was performed with modifications that have previously been shown to improve amplification uniformity. Specifically, exonuclease-resistant random primers (ThermoFisher) were added to the lysis buffer / mixture to a final concentration of 125 μM. 4 μL of the resulting lysis / denaturation mixture was added to the tube containing the single cells, vortexed, briefly centrifuged, and incubated on ice for 10 minutes. The cell lysate was neutralized by adding 3 μL of quenching buffer, vortexed, briefly centrifuged, and placed at room temperature. 40 μl of amplification mixture was then added, followed by incubation at 30°C for 8 hours, and then amplification was terminated by heating to 65°C for 3 minutes.

[0160] PTA is performed by further lysing the cells first after freeze-thaw, and the lysis is by adding 2 μl pre-cooled solutions of a 1:1 mixture of 5% Triton X-100 (Sigma-Aldrich) and 20mg / ml proteinase K (Promega). The cells are then vortexed and briefly centrifuged, and then placed at 40 degrees for 10 minutes. Then 4 μl lysis buffer / mixture and 1 μl 500 μM random primers against exonucleases are added to the lysed cells to denature the DNA, then vortexed, centrifuged and placed at 65 degrees for 15 minutes. Then 4 μl room temperature quenching buffer is added, and the sample is vortexed and centrifuged. 56 μl amplification mixture (primers, dNTPs, polymerase, buffer) contains equal ratios of α-sulfo-ddNTPs, which are 1200 μM in the final amplification reaction. The sample is then placed at 30°C for 8 hours, and then heated to 65°C for 3 minutes to terminate amplification.

[0161] After the amplification step, DNA from the MDA and PTA reactions was purified using AMPure XP magnetic beads (Beckman Coulter) at a 2:1 bead to sample ratio, and yield was measured using the Qubit dsDNA HS Assay Kit and Qubit 3.0 Fluorometer according to the manufacturer's instructions (Life Technologies).

[0162] Library preparation

[0163] The MDA reaction results in the production of 40 μg of amplified DNA. According to the standard protocol, 1 μg of product was fragmented for 30 minutes. The sample was then subjected to standard library preparation using 15 μM dual index adapters (end repair of T4 polymerase, T4 polynucleotide kinase, and Taq polymerase for adding A tail) and 4 PCR cycles. Each PTA reaction produces 40-60 ng of material, all of which are used for standard DNA sequencing library preparation without fragmentation. 2.5 μM adapters with UMI and dual indexes were used for connection, and 15 PCR (hot start polymerase) cycles were used in the final amplification. The library was then cleaned up using double-sided SPRI, with the ratios used for right-side selection and left-side selection being 0.65X and 0.55X, respectively. Before sequencing on the Illumina NextSeq platform, the final library was quantified using the Qubit dsDNA BR assay kit and 2100 bioanalyzer (Agilent Technologies). All Illumina sequencing platforms, including NovaSeq, are also compatible with this protocol.

[0164] Data analysis

[0165] Sequencing reads were demultiplexed based on cellular barcodes using Bcl2fastq. Reads were then trimmed using trimmomatic and subsequently aligned to hg19 using BWA. Reads were duplicate labeled by Picard followed by local realignment and base realignment using GATK 4.0. All files used to calculate quality metrics were downsampled to 20 million reads using PicardDownSampleSam. Quality metrics were obtained from the final bam files using qualimap as well as PicardAlignmentSummaryMetrics and CollectWgsMetrics. Total genome coverage was also estimated using Preseq.

[0166] Variant determination

[0167] Single nucleotide variants and insertions / deletions were determined using GATK UnifiedGenotyper from GATK 4.0. Standard filtering conditions using GATK best practices were used for all steps in the process (https: / / software.broadinstitute.org / gatk / best-practices / ). Copy number variants were determined using Control-FREEC (Boeva ​​et al., Bioinformatics, 2012, 28(3):423-5). Structural variants were also detected using CREST (Wang et al., Nat Methods, 2011, 8(8):652-4).

[0168] result

[0169] like Figure 3A and Figure 3B As shown, the mapping rate and mapping quality score of amplification using only dideoxynucleotides ("reversible") were 15.0 + / - 2.2 and 0.8 + / - 0.08, respectively, while the mapping rate and mapping quality score of amplification incorporating exonuclease-resistant α-thio dideoxynucleotide terminators ("irreversible") were 97.9 + / - 0.62 and 46.3 + / - 3.18, respectively. Experiments were also performed using reversible ddNTPs and different concentrations of terminators. ( Figure 2A ,bottom).

[0170] Figure 2B-2EComparative data generated by NA12878 human single cells after MDA (according to Dong, X. et al., Nat Methods.2017, 14 (5): 491-493) or PTA are shown. Although both schemes produce a relatively low PCR repetition rate (MDA is 1.26% + / - 0.52, and PTA is 1.84% + / - 0.99) and GC% (MDA is 42.0 + / - 1.47, and PTA is 40.33 + / - 0.45), the amplicon produced by PTA is smaller. Compared with MDA, the percentage of mapped reads and the mapping quality score of PTA are also significantly higher (PTA 97.9 + / - 0.62 and MDA 82.13 + / - 0.62, and PTA 46.3 + / - 3.18 and MDA 43.2 + / - 4.21). Overall, PTA generated more usable mapping data compared to MDA. Figure 4A It was shown that PTA significantly improved the uniformity of amplification compared to MDA, with a wider coverage range and fewer regions with coverage close to 0. Low-frequency sequence variants in nucleic acid populations can be identified using PTA, including variants that account for ≥ 0.01% of the total sequence. PTA can be successfully used for single-cell genome amplification.

[0171] Example 2: Comparative Analysis of PTA

[0172] Benchmarking PTA and SCMDA cell maintenance and isolation

[0173] Lymphoblastoid cells from the 1000 Genomes Project object NA12878 (Coriell Institute, Camden, NJ, USA) were maintained in RPMI medium supplemented with 15% FBS, 2 mM L-glutamine, 100 units / mL penicillin, 100 μg / mL streptomycin, and 0.25 μg / mL amphotericin B. Cells were grown at 3.5 × 10 5 Cells were seeded at a density of 10 cells / ml and split every 3 days. They were maintained in a humidified incubator at 37°C, 5% CO2. Before single cell isolation, 3 mL of the suspension of cells that had been expanded in the past 3 days was centrifuged at 300xg for 10 minutes. The precipitated cells were washed with 1 mL of cell wash buffer (containing 2% FBS, without Mg). 2+ or Ca 2+The cells were washed three times with 1X PBS (10% PBS), then centrifuged at 300xg, 200xg and finally 100xg for 5 minutes, to remove dead cells. The cells were then resuspended in 500uL cell washing buffer, then stained with 100nM calcein AM and 100ng / ml propidium iodide (PI) to distinguish live cell populations. The cells were loaded onto a BD FACScan flow cytometer (FACSAriaII) that had been thoroughly cleaned by ELIMINase and calibrated with Accudrop fluorescent beads. Single cells from calcein AM positive and PI negative fractions were sorted into each well of a 96-well plate containing 3uL PBS with 0.2% Tween20. Multiple wells were intentionally left blank to serve as a template-free control. After sorting, the plate was briefly centrifuged and placed on ice immediately. Then, the cells were frozen at -80°C for at least overnight.

[0174] PTA and SCMDA experiments

[0175] The WGA reaction was assembled on a pre-PCR workstation that provided constant positive pressure using HEPA-filtered air and was sterilized with ultraviolet light for 30 minutes before each experiment. MDA was performed according to the SCMDA method according to a published protocol (Dong et al., Nat. Meth. 2017, 14, 491-493). Specifically, random primers resistant to exonucleases were added to the lysis buffer at a final concentration of 12.5uM. 4uL of the resulting lysis mixture was added to a tube containing a single cell, pipetted three times to mix, centrifuged briefly and incubated on ice for 10 minutes. The cell lysate was neutralized by adding 3uL of quenching buffer, pipetted 3 times to mix, centrifuged briefly, and placed on ice. 40ul of the amplification mixture was subsequently added, then incubated at 30°C for 8 hours, and then amplification was terminated by heating to 65°C for 3 minutes. PTA was performed by first further lysing the cells after freeze-thaw, and the lysis was performed by adding 2μl of a 1:1 mixture of 5% Triton X-100 and 20mg / ml proteinase K in a precooled solution. The cells were then vortexed and briefly centrifuged before being placed at 40 degrees for 10 minutes. 4 μl of denaturation buffer and 1 μl of 500 μM exonuclease-resistant random primers were then added to the lysed cells to denature the DNA, followed by vortexing, centrifugation and placement at 65°C for 15 minutes. 4 μl of room temperature quenching solution was then added, and the samples were vortexed and centrifuged. The 56 μl amplification mixture contained an equal proportion of α-thio-ddNTPs, which were at a concentration of 1200 μM in the final amplification reaction. The samples were then placed at 30°C for 8 hours and then heated to 65°C for 3 minutes to terminate the amplification. After SCMDA or PTA amplification, DNA was purified using AMPure XP beads at a 2:1 bead-to-sample ratio, and the yield was measured using the Qubit dsDNA HS assay kit and Qubit 3.0 fluorometer according to the manufacturer's instructions.

[0176] Library preparation

[0177] After adding the conditioning solution, 1ug ​​SCMDA product was fragmented for 30 minutes according to the HyperPlus protocol. The samples were then subjected to standard library preparation using 15uM unique dual index adapters and 4 PCR cycles. The entire product of each PTA reaction was used for DNA sequencing library preparation using a standard amplification protocol without fragmentation. 2.5uM unique dual index adapters were used for connection, and 15 PCR cycles were used in the final amplification. The libraries from SCMDA and PTA were then visualized on 1% agarose E-Gel. Fragments between 400-700bp were cut from the gel and recovered using a Gel DNA recovery kit. The final library was quantified using a Qubit dsDNA BR assay kit and an Agilent 2100 bioanalyzer before sequencing on the NovaSeq6000.

[0178] Data analysis

[0179] Data were trimmed using trimmomatic and subsequently aligned to hg19 using BWA. Reads were duplicate marked by Picard followed by local realignment and base realignment using GATK 3.5 best practices. All files were downsampled to the specified number of reads using PicardDownSampleSam. Quality metrics were obtained from the final bam files using qualimap along with PicardAlignmentMetricsAummary and CollectWgsMetrics. Lorentz curves were plotted and the Gini index was calculated using htSeqTools. SNV calling was performed using UnifiedGenotyper followed by filtering using standard recommended criteria (QD<2.0||FS>60.0||MQ<40.0||SOR>4.0||MQRankSum<-12.5||ReadPosRankSum<-8.0). No regions were excluded from the analysis and no other data normalization or manipulation was performed. Sequencing metrics for the methods tested are listed in Table 2.

[0180] Table 2: Comparison of sequencing metrics between the tested methods.

[0181]

[0182]

[0183] CV = coefficient of variation; SNV = single nucleotide variation; values ​​refer to 15X coverage.

[0184] Genome coverage and uniformity

[0185] PTA was comprehensively compared with all common single-cell WGA methods. To achieve this goal, 10 NA12878 cells were subjected to PTA and a modified version of MDA, called single-cell MDA (Dong et al. Nat. Meth. 2017, 14, 491-493) (SCMDA). In addition, data generated as part of the LIANTI study were used to compare results from cells expanded by DOP-PCR (Zhang et al., PNAS 1992, 89, 5847-5851), MDA kit 1 (Dean et al., PNAS 2002, 99, 5261-5266), MDA kit 2, MALBAC (Zong et al., Science 2012, 338, 1622-1626), LIANTI (Chen et al., Science 2017, 356, 189-194), or PicoPlex (Langmore, Pharmacogenomics 3, 557-560 (2002)).

[0186] To normalize between samples, raw data from all samples were aligned and preprocessed for variant calling using the same pipeline. The bam files were then downsampled to 300 million reads before comparison. Importantly, PTA and SCMDA products were not screened before further analysis, whereas all other methods were screened for genome coverage and uniformity before selecting the highest quality cells used in subsequent analyses. Notably, SCMDA and PTA were compared to a bulk diploid NA12878 sample, whereas all other methods were compared to a bulk BJ1 diploid fibroblast cell population used in the LIANTI study. Figure 3C-Figure 3F As shown, PTA had the highest percentage of reads aligned to the genome, as well as the highest mapping quality. PTA, LIANTI, and SCMDA had similar GC content, all lower than the other methods. PCR replication rates were similar in all methods. In addition, the PTA method enabled smaller templates (such as the mitochondrial genome) to provide higher coverage (similar to larger typical chromosomes) relative to the other methods tested ( Figure 3G ).

[0187] The coverage and uniformity of all methods were then compared. An example of the coverage plot of SCMDA and PTA on chromosome 1 is shown, showing that PTA has significantly improved coverage uniformity and allele frequency ( Figure 4B The increased number of reads was then used to calculate coverage for all methods. PTA approached both bulk samples at each depth, a significant improvement over all other methods ( Figure 5AWe then used two strategies to measure coverage uniformity. The first approach was to calculate the coverage variation coefficient when increasing sequencing depth, where PTA was found to be more uniform than all other methods ( Figure 5B The second strategy is to compute the Lorentz curve for each downsampled bam file, where PTA is again found to have the greatest uniformity ( Figure 5C To measure the reproducibility of amplification uniformity, the Gini index was calculated to estimate the difference between each amplification reaction and complete uniformity (de Bourcy et al., PloS one 9, e105585 (2014)). PTA again showed higher reproducibility than other methods ( Figure 5D ).

[0188] SNV sensitivity

[0189] To determine the impact of these differences in amplification method performance on SNV calling, the variant call ratios of each method and the corresponding bulk samples were compared at increasing sequencing depths. To estimate sensitivity, the percentage of variants found in each cell that were called in the corresponding bulk samples downsampled to 650 million reads was compared at each sequencing depth. Figure 5E The improved coverage and uniformity of PTA resulted in the detection of 45.6% more variants than MDA Kit 2, the second most sensitive method. Examination of sites judged to be heterozygous in a large number of samples showed that PTA significantly reduced allelic skew at those heterozygous sites ( Fig. 5F ). This finding supports the idea that PTA not only has more uniform amplification across the genome, but also more uniform amplification of both alleles in the same cell.

[0190] SNV specificity

[0191] To estimate the specificity of mutation calls, variants called in each single cell that were not found in the corresponding bulk sample were considered false positives. The lower temperature lysis of SCMDA significantly reduced the number of false positive variant calls ( Figure 5G ). Methods using thermostable polymerases (MALBAC, PicoPlex, and DOP-PCR) showed that the specificity of SNV calls decreased further with increasing sequencing depth. Without being bound by theory, this is likely a result of the significantly increased error rates of these polymerases compared to phi29 DNA polymerase. In addition, the base change patterns seen in false positive calls also appear to be polymerase dependent ( Figure 5H ).like Figure 5GAs shown, the lower false-positive SNV call rate in PTA compared to the standard MDA protocol supports the model that error propagation is suppressed in PTA. In addition, the allele frequency of false-positive variant calls in PTA is the lowest, which is again consistent with the model that error propagation is suppressed in PTA ( Fig.5I ).

[0192] Example 3: Massively parallel single-cell DNA sequencing

[0193] Using PTA, a protocol for massively parallel DNA sequencing was established. First, the cellular barcodes were added to random primers. Two strategies were used to minimize any bias in amplification introduced by the cellular barcodes: 1) extending the size of the random primers and / or 2) creating primers that loop back on themselves to prevent the cellular barcodes from binding to the template ( Fig. 10B ). Once an optimal primer strategy is established, it can be scaled up to 384 sorted cells using, for example, the Mosquito HTS liquid handler, which can move viscous liquids down to 25 nL volumes with high precision. This liquid handler also reduces reagent costs by about 50-fold by using 1 μL PTA reactions instead of the standard 50 μL reaction volumes.

[0194] By delivering primers with cell barcodes to droplets, the amplification scheme is converted into droplets. Optionally, solid supports are used, such as beads created using a split pool strategy. For example, suitable beads can be obtained from ChemGenes. In some cases, oligonucleotides contain random primers, cell barcodes, unique molecular identifiers, and cleavable sequences or spacers to release oligonucleotides after encapsulating beads and cells in the same droplet. In this process, the template, primer, dNTP, α-thio-ddNTP and polymerase concentrations of low nanoliter volumes in the droplet are optimized. In some cases, optimization includes using larger droplets to increase the reaction volume. As shown in Figure 9, this process requires two consecutive reactions to lyse cells, and then WGA is performed. The first droplet containing lysed cells and beads is combined with the second droplet containing the amplification mixture. Alternatively or in combination, cells are encapsulated in hydrogel beads before lysis, and then the two beads can be added to the oil droplets. See Lan, F. et al., Nature Biotechnol., 2017, 35: 640-646).

[0195] Other approaches involve the use of microwells, which in some cases capture 140,000 single cells in a 20 picoliter reaction chamber on a 3" x 2" microscope slide-sized device. Similar to droplet-based approaches, these wells bind cells to beads containing cellular barcodes, allowing for massively parallel processing. See Gole et al., Nature Biotechnol., 2013, 31: 1126-1132.

[0196] Example 4: Parallel analysis of genomes and transcriptomes in single cells

[0197] Single cells from a cell population are sorted, with one cell placed per well. Each well contains an antibody fixed to a surface area, where the antibody binds to the cell nucleus. The outer membrane of the cell is lysed, thereby releasing the mRNA into the solution in the well, while the nuclease remains intact and bound to the area of ​​the well. RT is performed using the mRNA in the solution as a template to use Fig. 8A cDNA is produced using primers in. Optionally, an rRNA (ribosomal RNA) depletion step is performed. A first template comprising a TSS region (transcription start site), an anchor region, an RNA BC region, and a poly-dT tail from 5' to 3'; and a second template comprising a TSS region, an anchor region, and a poly-G region from 5' to 3' are used for RT PCR. After taking out the RT PCR product (cDNA library) for subsequent sequencing, any remaining RNA in the cell is removed by UNG. RNA libraries are prepared using Nextera / transposon-based sequencing methods and reagents ( Figure 8B ). The cDNA library contains short cDNAs with approximately 1000-fold amplification. The nuclei are then lysed and the released genomic DNA is subjected to the PTA method using random primers with a length of 6-9 bases and an isothermal polymerase. Amplification conditions for PTA are selected to produce amplicons with a length of 250-1500 bases. The PTA products are optionally further amplified and sequenced. The RNA sequencing data and the DNA sequencing data are compiled into a database for analysis.

[0198] Example 5: Single-cell multi-omics analysis

[0199] The cell colony is contacted with an antibody library, wherein the antibody is labeled. The antibody is labeled with a fluorescent marker, a nucleic acid barcode or both. The labeled antibody is combined with at least one cell in the colony, and such cells are sorted, and one cell is placed in each hole. Some labeled antibodies provide specific information about cell surface protein markers after binding, which is obtained by fluorescence microscopy or reading of the barcode labeled to the antibody. Each hole includes an antibody fixed to a surface area, wherein the antibody is combined with the nucleus. The outer membrane of the cell is lysed, thereby releasing mRNA into the solution in the hole, and the nuclease remains intact and is bound to the area of ​​the hole. Optionally, an rRNA (ribosomal RNA) depletion step is performed. Next, RT is performed using the mRNA in the solution as a template to produce cDNA. A first template comprising a TSS region (transcription start site), an anchor region, an RNA BC region and a poly-dT tail from 5' to 3'; and a second template comprising a TSS region, an anchor region and a poly-G region from 5' to 3' is used for RT PCR. After taking out RT PCR product (cDNA library) to carry out subsequent sequencing, remove any remaining RNA in the cell by UNG.CDNA library comprises short cDNA with about 1000 times of amplification.Then by nuclear lysis, and use random primers and isothermal polymerase to carry out PTA method to the genomic DNA of release, wherein the length of random primers is 6-9 base.The amplification condition that is selected for PTA is the amplicon of 250-1500 base to produce length.Optionally PTA product is carried out other amplification, and is sequenced.Protein data, RNA sequencing data and DNA sequencing data are compiled into database to analyze.

[0200] Example 6: Single-cell analysis of methylome and transcriptome

[0201] Single cells from a cell population are sorted, and one cell is placed in each hole. Each hole includes an antibody fixed to a surface area, wherein the antibody is bound to the nucleus. The outer membrane of the cell is lysed, thereby releasing mRNA into the solution in the hole, while the nuclease remains intact and is bound to the area of ​​the hole. The mRNA transcript is contacted with a terminal transferase to add riboguanine to the 5' end of the mRNA chain. Next, RT is performed using the mRNA in the solution as a template to produce cDNA. Optionally, an rRNA (ribosomal RNA) depletion step is performed. The first template comprising the TSS region (transcription start site), anchor region, RNA BC region, and poly-dT tail from 5' to 3'; and the second template comprising the TSS region, anchor region, and poly-G region from 5' to 3' is used for RTPCR. After taking out the RT PCR product (cDNA library) for subsequent sequencing, any remaining RNA in the cell is removed by UNG. The cDNA library comprises short cDNAs with approximately 1000 times amplification. Then the nucleus is lysed, and the released genomic DNA is fragmented using a methylation-sensitive nuclease. Use random primers and isothermal polymerase to carry out PTA method to genomic fragment, wherein the length of random primers is 6-9 base.Selection is used for the amplification condition of PTA to produce the amplicon of 250-1500 base in length.Optionally the PTA product is carried out other amplification, and order-checking. RNA sequencing data and DNA sequencing data are compiled into the database to analyze, and identify the methylation sensitive nuclease cleavage site.Use these sites to map the position of methylation on the original genomic DNA.

[0202] Example 7: Single-cell analysis of methylome and genome

[0203] Single cells from cell colony are sorted, and one cell is placed in each hole. Each hole includes an antibody fixed to the surface area, wherein the antibody is combined with the nucleus. Cells are methylation-sensitive enzyme cracked, and random primers and isothermal polymerase are used to carry out PTA method to genome, wherein the length of random primers is 6-9 bases. The amplification conditions selected for PTA are to produce an amplicon of 250-1500 bases in length. Reaction mixture is split, wherein exon group enrichment, whole genome sequencing or other targeted sequencing methods are carried out to half mixture. Methylation-sensitive PCR conditions are carried out to the other half reaction mixture. Methylation and DNA sequencing data are compiled into the database for analysis.

[0204] Example 8: Single-cell analysis of surface proteome and genome

[0205] Cells from samples comprising cell colonies are contacted with libraries of baits, such as libraries of antibodies, polynucleotides or other small molecules. In some cases, the bait is barcoded (such as barcoded antibodies) to allow pull-down and identification of the combination of baits with proteins on the cell surface. Alternatively or in combination, the bait is labeled with other markers (such as fluorescent markers or mass tags). Single cells from cell colonies are sorted, and one cell is placed in each hole. Optionally, the bait combined with the cell surface is taken out to sequence or identify before the genomic library is prepared. Cells are lysed, the genome is released into the solution, and fragments are produced. PTA methods are performed on genomic fragments using random primers and isothermal polymerases, wherein the length of the random primers is 6-9 bases. Alternatively, the genome is not fragmented before amplification with PTA. The amplification conditions selected for PTA are selected to produce amplicons with a length of 250-1500 bases. Optionally, the PTA product is further amplified and sequenced. Cell surface protein and DNA sequencing data were compiled into a database for analysis.

[0206] Example 9: Multi-omics for measuring drug resistance

[0207] In AML (acute myeloid leukemia), monotherapy with small molecule inhibitors targeting FLT3 has shown clinical benefits, but resistance always occurs. The FLT3 inhibitor Quizartinib (AC220) is a kind of such inhibitor, in which the drug produces about 50% of compound complete remission in patients with relapsed or refractory AML. Despite this success, secondary FLT3 mutations at the gatekeeper residue F691 in the activation loop (D835) have been identified in FLT3-ITD patients who relapsed when receiving Quizartinib therapy. It is determined that clinical resistance to multikinase inhibitor PKC412 is the result of secondary mutations in the FLT3 kinase domain. Other FLT3 independence resistance patterns for targeted therapy have been identified in FLT3-ITD AML, including bypass pathway activation of AXL, and NRAS, TET2 and IDH1 / 2 mutations. Mutations in epigenetic modification enzymes and transcription factors have also been observed, highlighting the complexity and diversity of the mechanism of resistance to FLT3 inhibition.

[0208] Quizartinib-resistant and matched parental MOLM-13 AML cell lines, as well as cell lines with heterozygous FLT3-ITD mutations, were generated. The PTA method is a combined RNAseq chemistry and is used to probe these drug-resistant single cells in a genomic and transcriptional manner to gain insight into the resistance mechanisms after FLT3 inhibition in AML. In brief, the workflow includes (1) generation of resistant cells, (2) isolation of resistant cells, (3) cytoplasmic lysis to release mRNA, (4) reverse transcription to generate cDNA from mRNA, (5) nuclear lysis to release genomic DNA, (6) PTA amplification, (7) isolation of DNA / RNA enrichment, (8) cDNA pre-amplification of enriched mRNA, (9) library preparation, QC and pooling, (10) next generation sequencing, and (11) data analysis.

[0209] Cell culture.MOLM-13 acute myeloid leukemia cells with heterozygous FLT3 internal tandem repeat (ITD) 1 were obtained from DSMZ-German Collection of Microorganisms and Cell Cultures (ACC 554).Cells were maintained in RPMI 1640 (Gibco 11875-093) supplemented with 10% FBS and penicillin / streptomycin, and subcultured every 2-3 days while maintaining a density range of 2.5E5-1.5E6 cells / ml.In order to produce quizartinib-resistant MOLM-13 lines, cells were continuously treated with 2nM quizartinib, and supplemented with drugs under each subculture until resistant clones ( Fig.9A ). Genomic DNA or total RNA was isolated from quizartinib-resistant and matched parental MOLM-13 cells upon FACS sorting to generate bulk sequencing control libraries for comparison with single-cell datasets.

[0210] FACS.For single cell analysis, about 2.0E6 MOLM-13 quizartinib resistance or the parental cells of matching were rinsed twice in the Dulbecco's phosphate buffered saline (Gibco) lacking calcium and enzyme, supplemented with 2% FBS, and remained on ice until BD FACSAria III FACS sorting.After calcein AM, propidium iodide and DAPI dyeing, set up live cell gating (DAPI / PI negative, first 70% calcein-AM positive) and single cell sorting (130 micron nozzle assemblies) was sorted into the low binding 96-well PCR plates (semi-skirt) containing cell buffer and frozen on dry ice immediately after short vortex and centrifugation.

[0211] Combined genome / transcriptome analysis.First, oligo dT primers conjugated with biotin are utilized in template switching reverse transcription reaction to produce the first strand cDNA from single MOLM-13 parent or quizartinib resistant cells. Primary template directional amplification (PTA) is continuously performed after reverse transcription.Then the first strand cDNA is affinity purified using streptavidin M-280 beads, and twice high salt washing is performed, followed by a low salt washing.Pre-amplification of 20 cycles is performed to produce the 2nd strand cDNA, and RNA sequencing libraries are prepared using Nextera DNA Flex library preparation kit.In order to prepare PTA libraries, the PTA products that are not bound to streptavidin beads are purified using beads and connected to TruSeq adapters.The amplified products from PTA reactions are first purified by beads, measured by Qubit and analyzed by electrophoresis.The typical yield of mammalian cells (about 6pg DNA) is 1-3ug, wherein a single bacterial genome (2-4fg) up to 50ng is produced. The amplicon product size of the samples amplified by PTA ranged from 0.2-4 kB (average 1.5 Kb). PTA libraries were prepared without fragmentation for WGS methods and yielded approximately 500 ng of yield with a size range of 300-550 bases. Whole genomes from mammalian cells were analyzed by NovaSeq with a target of approximately 550 million reads. The sequencing files were then transferred for trimming alignment and VCF file creation and were analyzed by Trailblazer-based TM Cloud-based bioinformatics platform solution analysis. QC and library preparation time is 4-6 hours. Parallel experiments are performed using separate RNASeq for comparison.

[0212] Results. RNA expression from both parental and resistant cultures demonstrated the ability to generate cDNA pools using one-pot RNAseq chemistry ( Fig. 9B ), and the genes expressed in these cells produce distinct patterns that can be visualized in cell populations by gene expression on an average of about 10K genes detected per cell. In a separate workflow, single-cell genomes were amplified using the PTA method. The two protocols were then combined (yields in Fig.9D The low throughput (~5 million reads / cell) demonstrated efficient amplification and library preparation for both resistant and parental lines, with low mitochondrial chromosome amounts and high complete PreSeq genome estimates ( Figures 10A-10C The data demonstrate that transcripts generated during the RT step are not efficiently amplified by the PTA reaction compared to DNA, and that DNA in single cells is efficiently amplified using the combined protocol compared to standard PTA amplified genomes from single cells ( Fig.9D). The combined RNASeq / PTA approach produced similar results to the standard PTA protocol ( Fig. 10A ), where ChrM and repeat percentages are typically less than 2%, and the estimated genome size is greater than 3 billion bases ( Figures 10A-10C Evaluation of the genome revealed over 90% mapping and coverage, and over 75% specific calls for single nucleotide variants in each cell. More variation was observed in the dual protocol compared to the standard PTA genomic chemistry. For the transcriptome, the prototype chemistry appeared to detect approximately 3000-5000 genes containing exon-exon junctions. Compared to the RNAseq-only protocol ( Fig. 9C ) compared to about 30% of the genes detected in the dual protocol ( Fig. 10D ). In addition, the dual / combined RNASeq / PTA protocol was used with a second resistant cell line, SUM159 (a triple-negative breast cancer cell line). RNAseq data run in both protocols produced similar PCA distributions, indicating that combinatorial chemistry is capable of detecting differential gene expression in single cell types that are not limited to parental and resistant cells.

[0213] ( Figures 10E-10F ).

[0214] Deep sequencing of 7 parental and 5 resistant molm13 cells was performed to an approximate depth of 25x (Figure 11). Reads were aligned to Hg38 using bwamem. Quality control and SNV determination were performed using GATK4 optimal operation. SNVs were considered only if they were limited to at least 2 resistant cells, alternative alleles were not determined in any parental cells, and at least 6 parental cells were genotyped. All cells covered at least 96% of the genome at 1x coverage, and at least 76% at 10x. The illustration shows the known Flt3 indels detected in molm13 cells in all cells (4 are shown for clarity).

[0215] RNAseq and PTA methods were generally comparable, with both mapping rates and coverage exceeding 95%, and ChrM and PCR duplications generally below 2.0%. In addition, more than 95% of the genome was recovered in select samples of sum 159 parental and resistant cell lines. For the Molm13 cell line, an overexpressed gene GAS6 (L) was identified, which is a known mechanism of resistance to quizartinib. Gas6 is a ligand for AXL, a clinically relevant resistance mechanism in relapsed patients who have failed quizartinib treatment ( Fig. 11B). The deep genome sequencing of the parental and resistant MOLM13 cell lines by the dual scheme detected mutations distributed on all chromosomes. In summary, in all single cells, 5675 SNVs unique to the quizartinib resistance population were identified. Coding sequence variations were detected, however, most of the observed variants were in the intergenic space. Not bound by theory, although the follower mutation is undoubtedly present in the variant cohort, this shows that the regulation of gene expression under the enhancer or promoter level is helpful for resistance and potentially helpful for the regulation of non-coding RNA. Dual mRNAseq transcriptome chemistry / PTA has the ability to detect more than 10K genes in a single cell, which can be enriched by FACS. The PTA method has the ability to reclaim more than 97% of the complete genome of a single cell. The ability to reclaim both transcriptomes and genomes does not significantly affect the sensitivity of the ability to reclaim most of the genome. When comparing only transcriptomes or combined transcriptome / genomic amplification chemistry, more than 70% of the expressed genes can be detected in many cells.

[0216] Example 10: PTA single cell analysis using exome capture

[0217] The general PTA method of Example 3 was used with the following modifications: an additional exome capture step was utilized to enrich the amplicons generated by PTA. 60 million reads were obtained for both single cell samples (27 samples) and bulk samples (112 samples). Exome capture sequencing results from single cells were compared to those of bulk samples ( Figures 12A-12D , 13A, 14A and 14B). The sequencing results were consistent across multiple samples ( Fig.13A ), and the average size of the captured amplicons was 623 bases ( Fig. 13B ).

[0218] Example 11: Exome capture + multi-omics

[0219] The general method of any one of Examples 5-8 is used with the following modifications: an additional capture step is used to enrich the PTA-generated amplicons generated from genomic DNA. The capture step includes an exome panel or other panels targeting specific genes. In some cases, such panels involve cancer hotspots, viral genomes, or mitochondrial DNA.

[0220] The embodiments described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art will now expect multiple variations, changes and substitutions without departing from the present invention. It should be understood that the various alternatives of the embodiments of the present invention described herein can be used to implement the present invention. The following claims are intended to define the scope of the present invention, and thus encompass methods and structures and their equivalents within the scope of these claims.

Claims

1. A method for multi-omics single cell analysis, the method comprising: a. Isolation of single cells from a cell population; b. sequencing a cDNA library comprising polynucleotides amplified from mRNA transcripts from said single cell; as well as c. sequencing the genome of the single cell, wherein sequencing the genome comprises: i. contacting the genome with at least one amplification primer, at least one nucleic acid polymerase, and a mixture of nucleotides, wherein the mixture of nucleotides comprises at least one terminator nucleotide that terminates nucleic acid replication by the polymerase, and wherein the at least one terminator nucleotide is an alpha-thiodideoxynucleotide; and ii. amplifying at least some of the genome to produce a plurality of terminated amplification products, wherein the replication is by strand displacement replication, and wherein termination of nucleic acid replication is performed prior to strand displacement; iii. ligating the molecules obtained in step (ii) to adaptors, thereby generating a genomic DNA library; as well as iv. sequencing the genomic DNA library.

2. The method of claim 1, wherein the mRNA transcript comprises a polyadenylated mRNA transcript.

3. The method of claim 1, wherein the mRNA transcripts do not include polyadenylated mRNA transcripts.

4. The method of claim 1, wherein sequencing the cDNA library comprises amplifying mRNA transcripts using template switching primers.

5. The method of claim 1, wherein at least some of the polynucleotides in the cDNA library comprise barcodes. The method of claim 5 , wherein the barcode comprises a cell barcode or a sample barcode.

7. The method of claim 1, wherein the cDNA library and the genomic DNA library are combined prior to sequencing.

8. The method of claim 1, wherein the single cell is a primary cell.

9. The method of claim 1, wherein the single cell is derived from liver, skin, kidney, blood or lung.

10. The method of claim 1, wherein the single cell is a cancer cell, a neuron, a glial cell, or a fetal cell.

11. The method of claim 1, wherein the single cells are isolated by flow cytometry.

12. The method of claim 1, wherein the method further comprises removing at least one terminator nucleotide from the terminated amplification product.

13. The method of claim 1, wherein the lengths of the plurality of terminated amplification products comprise an average of 1000-2000 bases.

14. The method of claim 1, wherein the plurality of terminated amplification products are 250-1500 bases in length.

15. The method of claim 1, wherein the plurality of terminated amplification products comprise at least 97% of the genome of the single cell.

16. The method of claim 1, wherein at least some of the amplification products comprise a cell barcode or a sample barcode.

17. The method of claim 1, wherein sequencing the cDNA library comprises cytoplasmic lysis of the single cell and reverse transcription.

18. The method of claim 1, wherein the mRNA transcript is amplified via template switching reverse transcription.

19. The method of claim 1, wherein the cDNA library comprises at least 10,000 genes.

20. The method of claim 1, wherein sequencing the genome of the single cell further comprises lysing the nucleus of the single cell.

21. The method of claim 1, wherein the method further comprises an additional amplification step using PCR.

22. The method of claim 1, wherein at least one mutation is identified in the genome of the cell, wherein the mutation differs from a corresponding position in a reference sequence.

23. The method of claim 1, wherein the at least one mutation occurs in less than 1% of the cell population.

24. The method of claim 1, wherein the at least one mutation occurs in no more than 0.1% of the cell population.

25. The method of claim 1, wherein the at least one mutation occurs in no more than 0.001% of the cell population.

26. The method of claim 1, wherein the at least one mutation occurs in no more than 1% of the sequences of the amplification products.

27. The method of claim 1, wherein the at least one mutation occurs in no more than 0.1% of the sequences of the amplification products.

28. The method of claim 1, wherein the at least one mutation occurs in no more than 0.001% of the sequences of the amplification products.

29. The method of claim 1, wherein after a maximum of 200 million sequencing reads, the proportion of the genome covered reaches 0.

9.

30. A method for multi-omics single cell analysis, the method comprising: a. Isolation of single cells from a cell population; b. identifying at least one protein on the surface of the single cell; as well as c. sequencing the genome of the single cell, wherein sequencing the genome comprises: i. contacting the genome with at least one amplification primer, at least one nucleic acid polymerase, and a mixture of nucleotides, wherein the mixture of nucleotides comprises at least one terminator nucleotide that terminates nucleic acid replication by the polymerase, and wherein the at least one terminator nucleotide is an alpha-thiodideoxynucleotide; ii. amplifying at least some of the genome to produce a plurality of terminated amplification products, wherein the replication is by strand displacement replication, and wherein termination of nucleic acid replication is performed prior to strand displacement; iii. ligating the molecules obtained in step (ii) to adaptors, thereby generating a genomic DNA library; and iv. sequencing the genomic DNA library.

31. The method of claim 30, wherein identifying at least one protein on the surface of the cell comprises contacting the cell with a labeled antibody that binds to the at least one protein.

32. The method of claim 31, wherein the labeled antibody comprises at least one fluorescent label or mass tag.

33. The method of claim 31, wherein the labeled antibody comprises at least one nucleic acid barcode.

34. A method for multi-omics single cell analysis, the method comprising: a. Isolation of single cells from a cell population; b. sequencing the genome of the single cell, wherein sequencing the genome of the cell comprises: i. digesting the genome with a methylation-sensitive restriction enzyme to generate genomic fragments; ii. contacting at least some of the genomic fragments with at least one amplification primer, at least one nucleic acid polymerase, and a mixture of nucleotides, wherein the mixture of nucleotides comprises at least one terminator nucleotide that terminates nucleic acid replication by the polymerase, and wherein the at least one terminator nucleotide is an alpha-thiodideoxynucleotide; iii. amplifying at least some of the genome to produce a plurality of terminated amplification products, wherein the replication is by strand displacement replication, and wherein termination of nucleic acid replication is performed prior to strand displacement; iv. amplifying at least some of the genomic fragments using methylation-specific PCR; v. ligating the molecules obtained in steps (iii and iv) to adaptors, thereby generating a genomic DNA library and a methylome DNA library; and vi. Sequencing the genomic DNA library and the methylome library.

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