Methods for in situ genomic DNA amplification and barcoding

Abstract

Disclosed herein are methods and kits for amplifying and barcoding genomic DNA in situ. In the first step of amplification, primers are employed that that bind a target sequence; and post-amplification, a barcoded primer is ligated onto the newly amplified double-stranded DNA, the barcoded primer having a free end that allows the attachment of additional barcodes. This strategy provides for improved amplification efficiency over methods previously used in the art.

Description

Atty. Dkt. No. 112624.01537METHODS FOR TN STTU GENOMIC DNA AMPLIFICATION AND BARCODINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 718,425 filed on November 8, 2024, the content of which is incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under R35 GM133674 awarded by the National Institutes of Health and 2119963 awarded by the National Science Foundation. The government has certain rights in the invention.BACKGROUND

[0003] With the advent of Next Generation DNA sequencing and the “omics” era, technology for studying the genetic content and function of biological systems has rapidly advanced. Initially, genomics and transcriptomics studies were performed on populations or “batch cultures”. The resulting data represent an average across all cells. However, single-cell techniques permit the study of heterogeneity within populations, and are revealing the extent to which variation contributes to biological behaviors. In order to associate sampled genetic sequences with a given cell, the genetic material is often labelled with a DNA barcode sequence that is unique to each cell. In the first generation of single-cell technologies, barcodes were added after individual cells were sorted into separate containers, such as lOOuL wells or microfluidic droplets. Further methods have been developed for amplifying and barcoding genomic DNA from individual cells without physically separating cells. Further improvements on these methods are desired.SUMMARY

[0004] In an aspect, provided herein is a method comprising: a) contacting a population of fixed and permeabilized cells with at least one set of DNA amplification primers configured to amplify a sequence of interest or a random sequence on genomic DNA within the cell, and a DNA polymerase; b)amplifying the DNA to create a DNA amplicon having a double-stranded primerAtty. Dkt. No. 112624.01537 sequence; c) contacting the cells with an adapter barcode, the adapter barcode comprising: a first double-stranded barcoding sequence (BCS), double-stranded end of the BCS is configured to ligate with the double-stranded primer sequence; and a first annealing sequence (ANS); d) attaching the first BCS to the double-stranded primer sequence using blunt end ligation, thereby creating barcoded amplicons; and e) lysing the cells to release the barcoded amplicons. After step b) and before step c), the method may further comprise quenching the DNA polymerase. Each of the at least one set of DNA amplification primers may be configured to amplify a different sequence of interest or random sequence.

[0005] The method may further comprise: dividing the fixed and permeabilized cells into a plurality of wells before step a); wherein in step d), the adapter barcode contacted to the cells in each well comprises a different first BCS. After step d) and before step e), the method may further comprise: performing at least one round of annealing an additional barcode to the barcoded amplicons; wherein each round comprises: contacting the barcoded amplicons with the additional barcode, wherein the additional barcode of each round comprises: an additional ANS, wherein the additional ANS of each round is different; a complementary annealing sequence (CANS) configured to anneal to the first ANS or the additional ANS of a previous round; and an additional BCS that is different than the first BCS; and annealing the at least one additional barcode to the barcoded amplicon. At least one of the at least one additional barcodes may comprise a different additional BCS. Before each additional round, the method may further comprise pooling and splitting the cells.

[0006] After the at least one additional round and before step e), the method may further comprise performing a final round, the final round comprising: contacting the barcoded amplicons with a terminal barcode, the terminal barcode comprising: the complementary annealing sequence (CANS); the additional BCS; and a terminal primer sequence; and annealing the terminal barcode to the barcoded amplicon. The terminal barcode may further comprise an affinity moiety, and the method may further comprise: f) capturing the barcoded amplicons with a capture reagent that binds to the affinity moiety. The method may further comprise after step d): contacting the cells with: a reverse transcriptase; and at least one set of reverse transcription primers, wherein each set of reverse transcription primers comprises: the first ANS; a single-stranded barcoding sequence (sBCS); wherein each of the at least one set of reverse transcription primers comprises a different sBCS; and a targeting sequence configured to anneal with a sequence of interest or a randomAtty. Dkt. No. 112624.01537 sequence on RNA within the cell; and reverse transcribing the sequence of interest or random sequence on the RNA to generate barcoded cDNAs. Each of the at least one set of reverse transcription primers may be configured to amplify a different sequence of interest or random sequence on the RNA. The fixed and permeabilized cells may be divided into the plurality of wells before step a), wherein the set of reverse transcription primers in each well comprises a different sBCS.

[0007] After step d) and before step e), the method may further comprise: performing at least one round of annealing a cDNA barcode to the barcoded cDNAs; wherein each round comprises: contacting the barcoded cDNAs with the cDNA barcode, wherein the cDNA barcode of each round comprises: the CANS; a double-stranded cDNA BCS (cBCS); and the additional ANS, wherein the additional ANS of each round is different; and annealing the at least one cDNA barcode to the barcoded cDNAs. The cBCS and the BCS in each round may be the same. At least one of the at least one cDNA barcodes may comprise a different cBCS. In the final round, the method may further comprise contacting the barcoded cDNAs with the terminal barcode. The affinity moiety may comprise biotin, and the capture reagent may comprise streptavidin. The affinity reagent may comprise digoxigenin, and the capture reagent may comprise anti-digoxigenin antibody.

[0008] After step e), the method may further comprise: f) ligating a double-stranded DNA sequence comprising a terminal primer sequence to a free end of the barcoded amplicons; and performing a template switch reaction on the barcoded cDNAs; and g) amplifying the barcoded amplicons and barcoded cDNAs off of the affinity moiety and capture reagent to generate free amplification products. The method may further comprise: h) purifying the free amplification products. The method may further comprise: i) sequencing the free amplification products. Step g) may be done by performing polymerase chain reaction (PCR). Step b) may be done by performing an isothermal amplification reaction. The temperature of the isothermal amplification reaction may be about 20-40° C. Step b) may be done for about 12-24 hours. The isothermal amplification reaction may be done using an isothermal polymerase. The isothermal polymerase may be phi29. Step b) may comprise contacting the population of fixed and permeabilized cells with a crowding agent. The crowding agent may comprise one or more of: polyethylene glycol 8000 (PEG-8000), trehalose, and sorbitol. At least one of step d) and step f) may be done using a T4 DNA ligase.Atty. Dkt. No. 112624.01537

[0009] In another aspect, provided herein is a kit for amplifying and barcoding genomic DNA within a cell, the kit comprising: a) at least one set of DNA amplification primers; wherein each set of amplification primers is configured to amplify a sequence of interest or a random sequence on the genomic DNA; b) at least one adapter barcode comprising: a first double-stranded barcoding sequence (BCS), configured to ligate with a double-stranded end of the DNA sequence of interest generated by amplifying the genomic DNA with the DNA amplification primers of a); and a first annealing sequence (ANS). The kit may further comprise: c) at least one additional barcode comprising: an additional ANS, wherein each of the at least one additional barcodes comprises a different additional ANS; a complementary annealing sequence (CANS) configured to anneal to the first ANS or the additional ANS; and an additional BCS that is different than the first BCS, wherein each of the at least one additional barcodes comprises a different additional BCS. The kit may further comprise: d) at least one set of reverse transcription primers, each comprising: the first ANS) wherein each of the at least one set of reverse transcription primers comprises the same first ANS; a single-stranded barcoding sequence (sBCS); wherein each of the at least one set of reverse transcription primers comprises a different sBCS; and a targeting sequence configured to anneal with a sequence of interest or random sequence on RNA; and e) at least one cDNA barcode, each comprising: the CANS; a double-stranded cDNA BCS (cBCS), wherein each cDNA barcode comprises a different additional cBCS; and the additional ANS. The kit may further comprise at least one of: f) a double-stranded DNA sequence comprising a terminal primer sequence configured to ligate with a free end of barcoded products generated by the use of components a) through e); and g) reagents for performing a template switch reaction.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIGS. 1 A-1B. Anatomy of a previous version of a barcoded primer for amplification of DNA, to which this version corrects inefficiencies. A) Anatomy of a barcoded primer for a specific region of interest. An annealing sequence and barcode are incorporated into the primer with homology to the genome. B) Anatomy of a barcoded random hexamer primer. An annealing sequence and barcode are incorporated into the random hexamer primer.

[0011] FIGS. 2A-2F. An in-depth overview of the method for amplifying and single-cell barcoding DNA, including intracellular amplification and split pool combinatorial barcoding. TheAtty. Dkt. No. 112624.01537 left panel of the figure (A-C) shows the steps of intracellular amplification. The right panel of the figure (D-F) shows the steps of combinatorial barcoding.

[0012] FIGS. 3A-3C. Proposed solution to barcode inefficiencies. The method used for barcoding cDNA (A), which involves the use of an annealing sequence overhang on which to ligate further barcodes in combinatorial barcoding steps, potentially will not work for barcoding intracellularly amplified genome (bottom diagram). This could be due to the strand displacement activity of 029 polymerase which generates double-stranded ends. (B). A potential solution to this problem is the use of blunt end ligation to attach the first barcode as well as an annealing sequence with a single-stranded overhang that allows the addition of subsequent barcodes. All barcode ligations are conducted intracellularly after 29 has been quenched (C).DETAILED DESCRIPTION

[0013] Sequencing platforms are capable of delivering enormous amounts of high-quality data. This allows for the possibility of sequencing the genomes of thousands of individual cells. The inventors previously developed methods for sequencing single-cell genomes that do not require cell isolation or specialized equipment beyond typical molecular biology laboratory standards, and thus are more user-friendly and scalable, allowing multiplexing of single cells from many different growth conditions or genetic backgrounds. Sequencing single cells has several advantages over sequencing pools of cells, including, but not limited to: identifying rare or low frequency mutations in a population, gaining a more detailed picture of microbes that inhabit specific environments, characterizing cells that all have unique DNA assortment, such as gametocytes, and determining the distribution of heterogeneous genomes in a population of cells, such as a tumor.

[0014] The inventors’ previous method combines in situ genome amplification with in situ combinatorial barcoding such that many cells from many conditions can all be processed in a single experiment without the need to physically isolate cells using specialized equipment. In other words, the method leverages the cell membrane to contain and separate the DNA from individual cells. To allow for integration with combinatorial barcoding, intracellular amplification was conducted on a multi-well plate, where each well contained primers containing a well-specific barcode, along with a universal annealing sequence at the 5’ end. This universal sequence facilitated the addition of subsequent barcodes post-amplification (see Fig. 1). The well-specific barcode acted as a label that denoted the condition or pool cells originated from and allows forAtty. Dkt. No. 112624.01537 multiplexing. This means cells from numerous samples (e.g., cells from various growth conditions, genotypes, or environmental samples) could all be processed together and later de-multiplexed computationally.

[0015] Once DNA had been amplified in the cell and the initial barcode was present in all newly amplified molecules, whether it was added during or post amplification, additional barcodes were added so that each cell possesses a unique combination. Following genome amplification and initial barcode addition (as depicted in Fig. 2D), cells from all wells were pooled into a single vessel (see Fig. 2E). These cells were evenly redistributed (split) into a second multi-well plate, where each well contained a short, unique barcode with a complimentary adapter to the annealing sequence on the first barcode (Fig. 2F; round 2 barcodes). A ligation reaction covalently bonded these “round 2 barcodes” to the 5’ end of each cell’s amplified DNA. The cells were then pooled and split into a new plate, repeating the process to introduce a third barcode (refer to Fig. 2F; round 3 barcodes). Split pool barcoding worked because of the vast number of possible unique barcodes a cell could acquire during its journey through barcoding plates.

[0016] For instance, if there were 96 starting conditions (assuming the use of a 96-well plate) and cells go through two additional rounds of barcoding, there would be almost 900,000 possible unique barcode combinations. This number increases exponentially with each additional round of barcoding (e g., with 96 barcodes, N split-pools = 96N possible combinations). Because there are so many possible unique combinations of these nonunique barcodes, the probability that two cells follow the same path through the pool and split process can be decreased practically to nothing by processing fewer cells than there are possible barcodes. The final round of barcoding involved the addition of a “terminal barcode,” which is comprised of the barcode sequence, a unique molecular identifier (UMI) to counteract PCR bias, and a biotin molecule to facilitate the isolation of successfully barcoded molecules (see Fig. 2F; round N barcode).

[0017] After the addition of the last barcode, cells from every well were pooled and lysed. Cells were incubated with streptavidin-coated magnetic beads (Fig. 2F; second row) to isolate the successfully barcoded molecules, which bind to the terminal biotin-tagged barcode. To sequence this barcoded DNA, it was copied off the beads using PCR and traditional library preparation steps were performed.

[0018] The inventors attempted to perform combinatorial barcoding on intracellularly amplified DNA using a kit that is made for barcoding RNA-cDNA hybrid molecules. However,Atty. Dkt. No. 112624.01537 they only got back properly barcoded reads for about six cells out of thousands. This inefficiency is likely due to an issue that arises when attempting to integrate intracellular DNA amplification and barcoding. One possible explanation for this is the pieces of DNA that are being made by 029 in the intracellular amplification reaction end up being double-stranded. This makes it difficult to ligate on further barcodes using the established method because doing so requires a single-stranded overhang (Fig. 3B).

[0019] A solution to this issue is provided herein. Instead of using primers that contain barcodes and annealing sequences in the initial amplification step, the primers used for this step are now, in a sense, naked. They contain only what typical primers contain, which is a sequence of DNA that binds either a sequence of interest or a random hexamer. Then, the first barcode is blunt-end ligated onto each piece of newly amplified double-stranded DNA. This barcode will have a free end with a single- stranded overhang that allows the attachment of additional barcodes (Fig, 3C). To facilitate this process, the phi29 polymerase can be quenched with gentle heat, and the first barcode and all subsequent barcodes can be ligated on without the risk of 029 interfering with ligation (Fig. 3C). At this point, the cells can be lysed, and the copied DNA sequence libraries can be prepared using practices outlined in the inventors’ previous patent applications pertaining to barcoding DNA (PCT / US22 / 81578, PCT / US2022 / 04037). The innovation contained herein has the additional advantage of not requiring that the first barcode be present within the primer used for intracellular amplification. This means that the initial amplification primers need not be barcoded, which makes experimental design simpler and less expensive.

[0020] Accordingly, in a first aspect, a method for amplifying and barcoding genomic DNA in situ is provided. The method comprises: a) contacting a population of fixed and permeabilized cells with at least one set of DNA amplification primers configured to amplify a sequence of interest or a random sequence on genomic DNA within the cell, and a DNA polymerase; b) amplifying the DNA to create a DNA amplicon having a double-stranded primer sequence at one end; c) contacting the cells with an adapter barcode, the adapter barcode comprising: a first doublestranded barcoding sequence (BCS), wherein the double-stranded BCS is configured to blunt end ligate with the double-stranded primer sequence; and a first annealing sequence (ANS) comprising a single stranded overhang to allow subsequent annealing of additional barcodes; d) attaching the first BCS to the double-stranded primer sequence using blunt end ligation, thereby creating wellspecific, but not cell-specific barcoded amplicons; optionally ligating on additional barcodes viaAtty. Dkt. No. 112624.01537 multiple rounds of split-pool ligation to create cell-specific barcoded amplicons (as discussed further below); and e) lysing the cells to release the barcoded amplicons for subsequent harvesting, amplification and DNA sequencing. The method may further comprise quenching the DNA polymerase after step b) and before step c). Each ANS comprises a single- stranded overhang to allow subsequent annealing of additional barcodes.

[0021] As used herein, “fixation” or “fixing” refers to the process of chemically stabilizing organic, inorganic, or a combination of organic and inorganic molecules through the use of reagents, known as “fixatives”. Exemplary fixatives for the present disclosure include, but are not limited to, formaldehyde, formaldehyde derived from paraformaldehyde, formalin, phosphate buffered formalin, formal calcium, formal saline, zinc formalin, alcoholic formalin, glutaraldehyde, other organic aldehydes, methanol, ethanol, isopropanol, or other organic alcohols, or solutions containing organic alcohols or aldehydes.

[0022] As used herein, “permeabilization” or “permeabilizing” refer to the process of introducing openings into barriers to allow the penetration of desired molecules past the aforementioned barrier. In some embodiments, the barrier comprises a cell membrane, and or a cell wall. In some embodiments, permeabilization is performed by, for example, ethanol and / or enzymes on biological membranes. Exemplary enzymes for permeabilization of biological membranes include, but are not limited to: proteinase K and zymolyase. In some embodiments, permeabilization is performed by, for example, detergents on biological membranes.

[0023] As used herein, “amplification” refers to the process of conservatively replicating nucleic acid strands by enzyme-catalyzed extension. Exemplary enzymes for amplification of nucleic acids in the current disclosure include, for example, nucleic acid polymerases. In some embodiments, an isothermal polymerase is used to amplify nucleic acids. Isothermal polymerases include, for example, phi29 polymerase, Klenow exo-DNA Polymerase I, Bsu polymerase, Bst polymerase, and Bsm polymerase. In preferred embodiments, the isothermal polymerase is phi29. In some embodiments, amplification is carried out with a high-fidelity polymerase, such as Q5, with the technique known as the polymerase chain reaction (PCR). Amplification can be performed with natural and non-natural nucleotide bases, ribonucleotide bases or deoxyribonucleotide bases, labeled nucleotide bases, and the like.

[0024] As used herein, “isothermal amplification” describes amplification of DNA targets without heat denaturation of DNA. In contrast, polymerase chain reaction (PCR) requires cyclingAtty. Dkt. No. 112624.01537 through different temperatures for denaturation, hybridization, and extension, or, in some cases, different temperatures for (1) denaturation and (2) hybridization / extension. Isothermal amplification may be preceded by a higher temperature hybridization step that does not denature the DNA target. Exemplary polymerases useful for isothermal amplification are referred to herein as isothermal polymerases, and include, but are not limited to phi29 polymerase, Klenow exo- DNA Polymerase I, Bsu polymerase, Bst polymerase, Bsm polymerase. Isothermal amplification may take place at, for example, about 20-40° C, about 20-30° C, about 30-40° C, or about 30° C, or about 40-50, or about 50-60, or about 60-70 deg. C. Isothermal amplification may also take place at about 62-68° C, for example, about 62, about 63, about 64, about 65, about 66, about 67, or about 68° C. Isothermal amplification may require incubation times of about 30 minutes to about 24 hours, or about 12 hours to about 24 hours to complete the reaction.

[0025] As used herein, “primer” refers to a single-stranded nucleotide. A primer may be used to initiate semi-conservative replication of nucleic acids. A primer may also be used to “barcode” nucleic acid sequences. As used herein, “barcode” refers to a nucleotide sequence of any length that is used to identify, for example, nucleotide sequences that are derived from a single sample. An exemplary property of a barcode is the ability to distinguish the sequence of the barcode from any known sequence present in the sample, thereby rendering the barcode sequence informatically distinct and permitting identification or quantification of any nucleotide sequence comprising the barcode. In some embodiments, a barcode may be 6-8 nucleotides in length, though it can be as short or as long as needed for a specific application. A “barcode” may also refer to a longer sequence that comprises a combination of short barcodes, for example, the term “cell-specific barcode” refers to this type of composite sequence. Each barcode must be detected in a single sequencing “read.” Therefore, barcode length is, in principle, dictated by the sequencing platform used to analyze the samples.

[0026] A sequence of interest may be any known sequence, where the amplification primers comprise a sequence complementary to the known sequence in the genomic DNA. A random sequence may be a sequence, where the amplification primers comprise a sequence that may be complementary to a sequence in the genomic DNA. In embodiments, the random sequence may be a random hexamer. As used herein, the term “random hexamer” or “random hexonucleotide” refers to a region of six nucleotides in length comprising sequences that are synthesized at random. The purpose of random hexamers is, in most applications, to bind complementarily to nucleotideAtty. Dkt. No. 112624.01537 sequences of unknown identity. Thus, because random hexamers theoretically cover all possible sequence permutations for a hexameric (6-member) nucleotide, they are likely to bind at many positions to nucleotides of any sequence. It should be understood, however, that a key feature of random hexamers is not that they are six nucleotides in length, but rather that they have random sequence identity. In other words, for many applications it is possible to provide random pentamers (5-member), heptamers (7-member), or other random sequences in place of hexamers. In some embodiments, a random hexamer comprises a part of, or a portion of a larger oligonucleotide, such as an oligonucleotide primer.

[0027] As used herein, “quenched” refers to the process of inactivating the polymerase. In some embodiments, quenching comprises inactivation by heat. In some embodiments, quenching comprises incubating the sample containing the polymerase at 65° C for 10 minutes. Quenching of the phi29 polymerase prevents the enzyme from catalyzing any unwanted extension or amplification in the subsequent steps. Next, the cells may be, in some embodiments, washed to remove the DNA target-specific primers and excess phi29 polymerase.

[0028] As used herein, “ligation” or “blunt-end ligation” refers to the joining of two nucleic acid molecules through the formation of covalent phosphodiester bonds, i.e., by forming phosphodiester bonds between a 3’ OH and a 5’ phosphate molecule on the two nucleic acid molecules. As described herein, 5’ and 3’ refer to the end of the forward strand of double-stranded DNA and cDNA molecules. However, blunt end-ligation joins both the forward and reverse strands. Ligation may involve the joining of double-stranded or single-stranded nicked nucleic acid molecules. In some embodiments, two blunt-ended nucleic acid duplexes are ligated together. In some embodiments, two nucleic acid duplexes that have single- stranded regions that are substantially complementary to one another allowing hybridization of the two nucleic acid duplexes are ligated to one another. Suitable ligase enzymes are known in the art including, but not limited to, T4 DNA ligase and T7 DNA ligase.

[0029] In embodiments, the cells may be divided into a plurality of wells before step a), and the adapter barcode contacted to the cells in each well may comprise a different first BCS. Additionally, the DNA amplification primers in each well may be configured to amplify a different sequence of interest or random sequence.

[0030] The method may further comprise, after step d) and before step e), performing at least one round of annealing an additional barcode to the barcoded amplicons; wherein each roundAtty. Dkt. No. 112624.01537 comprises: contacting the barcoded amplicons with the additional barcode, wherein the additional barcode of each round comprises: an additional ANS, wherein the additional ANS of each round is different; a complementary annealing sequence (CANS) configured to anneal to the first ANS or the additional ANS of the previous round; and an additional BCS that is different than the first BCS; and annealing the at least one additional barcode to the barcoded amplicon. The annealing step in each round includes single-stranded nick ligations. When the method comprises multiple rounds, in at least one round, the additional barcode may comprise a different BCS. The inventors’ methods for annealing additional barcodes to the amplicons are described in International Publication Nos. W02023 / 019024 and WO2023 / 114860, each of which are incorporated herein in their entireties.

[0031] In some embodiments, the ANS and corresponding CANS are between 10 and 20 (inclusive) nucleotides in length. In embodiments, the ANS and CANS are 15 nucleotides in length.

[0032] Before each additional round, the method may further comprise pooling and splitting the cells into different vessels (e.g. wells of a multi-well plate), wherein each vessel contains a unique barcode, such that probabilistically each cell receives a different combinatorial sequence of barcodes. The cells may be “pooled” into a single vessel and re-distributed into new wells for subsequent barcoding, i.e., “split”. In some embodiments, the “pooling" step may involve removing cells from each occupied well of a 96-well plate and “pooling” the cells in a single tube and mixing. In some embodiments, the “splitting” step may involve “splitting” the “pooled” cells into a new multi-well container.

[0033] As used herein, “split and pool” refers to a process for introducing complexity into a group of compounds such that the knowledge of the initial source of each compound is preserved and can be determined after the completion of the split and pool process (see references 1, 2, 4, 5, 6, 7, 8, also see references U.S. Patent Pub. No. US20200263234A1, and U.S. Patent No. US10900065B2 and U.S. Patent App. No. 16 / 949,949 Split and pool relies on probability to ensure that each individual compound has a high statistical likelihood to take a unique path through a set of steps, with each step introducing a new “barcode” which is linked to the compound. A first “barcoding event”, meaning the attachment, such as by ligation, of a barcode to the compound, is performed with a knowledge of the identity of the compound and the identity of the barcode to which each compound is attached. After each barcoding event, all of the individual compoundsAtty. Dkt. No. 112624.01537 are combined, or “pooled”, and “split”, or redistributed into new reaction vessels, with each vessel containing a unique barcode. Thus, a second round of barcoding reduces the chances that two compounds will be split into the same reaction vessel and be attached (e.g., ligated) to the same barcode. Therefore, after successive rounds of splitting and pooling the compounds, each of the compounds is likely to be attached (e.g., ligated) to a unique set of barcodes that correspond to the compounds unique trajectory through the split and pool process. The possible number of unique compounds that can be effectively barcoded using split and pool increases with both the number of reaction vessels, and therefore the number of barcodes, and with the number of successive rounds of barcoding events. Non-limiting examples of potential uses for the split and pool process include the preparation of nucleic acid libraries. As disclosed herein, in an embodiment of the present technology, split and pool may be used to efficiently label nucleic acids that are derived from a single cell with a unique barcode allowing for multiplexed sequencing of nucleic acids derived from many cells.

[0034] As used herein, the noun “well” refers to a single container or reaction vessel. Though the term well is often used when referring to plates or microplates, it is to be understood that the methods of the current disclosure may also be performed using, for example, tubes or other vessels capable of containing and separating liquids.

[0035] The method may comprise performing additional rounds until a final round. In the final round, the method further comprises performing contacting the barcoded amplicons with a terminal barcode, the terminal barcode comprising: the complementary annealing sequence (CANS); the additional BCS; and a terminal primer sequence for post barcoding amplification during sequencing library preparation; and annealing the terminal barcode to the barcoded amplicon.

[0036] The terminal barcode may further comprise an affinity moiety, and the method may further comprise after lysing the cells (step e), f) capturing the barcoded amplicons with a capture reagent that binds to the affinity moiety.

[0037] As used herein, “affinity moiety” refers to a chemical constituent, often attached to a molecule of interest that can be specifically recognized and bound by a “capture reagent” with high affinity, and with binding strength suitable to allow purification of the molecule of interest to which the affinity moiety is attached. The use of affinity moieties with capture reagents is collectively referred to as “affinity capture” in the context of separation of molecules of interestAtty. Dkt. No. 112624.01537 using the pair of reagents (affinity capture reagents). In some embodiments, exemplary affinity capture reagents include, without limitation, for example, biotin and streptavidin, digoxigenin and anti-digoxigenin antibodies, antibody-antigen pairs, and covalent click chemistry. For example, in embodiments, the affinity moiety comprises biotin, and the capture reagent comprises streptavidin. In other embodiments, the affinity reagent comprises digoxigenin, and the capture reagent comprises anti-digoxigenin antibody.

[0038] The methods described herein may further include reverse transcribing RNA and barcoding the resulting cDNA products in the cells concurrently with the DNA amplicons. The method may comprise after step c), contacting the cells with: a reverse transcriptase; and at least one set of reverse transcription primers, wherein each set of reverse transcription primers comprises: the first ANS (which may be the first ANS in the adapter barcode used to prepare the barcoded DNA amplicons); a single-stranded barcoding sequence (sBCS); wherein each of the at least one set of reverse transcription primers comprises a different sBCS; and a targeting sequence configured to anneal with a sequence of interest or a random sequence on RNA within the cell; and reverse transcribing the sequence of interest or random sequence on the RNA to generate barcoded cDNAs. In embodiments, the targeting sequence is the polyA tail of mRNA. The reverse transcription primers are configured in a similar way as depicted in FIG. 1 where the genomic DNA is replaced with an RNA molecule. In embodiments in which the cells are divided into a plurality of wells before step a), the reverse transcription primers in each well comprise a different sBCS.

[0039] Between steps d) and e), the method may further comprise performing at least one round of annealing a cDNA barcode to the barcoded cDNAs; wherein each round comprises: contacting the barcoded cDNAs with the cDNA barcode, wherein the cDNA barcode of each round comprises: the CANS (configured to anneal to the ANS); a double-stranded cDNA BCS (cBCS); and the additional ANS, wherein the additional ANS of each round is different; and annealing the at least one barcode to the barcoded cDNAs. The round of annealing subsequent cDNA barcodes is done concurrently with annealing additional barcodes to the DNA amplicons. In embodiments, in each round, the cBCS of the additional cDNA barcode and the BCS of the additional barcode are the same. In embodiments, at least one of the at least one cDNA barcodes comprises a different cBCS.Atty. Dkt. No. 112624.01537

[0040] In the final round of barcoding, the method may further comprise contacting the barcoded cDNAs with the terminal barcode.

[0041] The inventors’ methods for amplifying DNA and RNA within a cell in situ are described in International Publication Nos. WO2023 / 114860, which is incorporated herein in its entireties.

[0042] The method may further comprise after step e): f) ligating a double-stranded DNA sequence comprising a terminal primer sequence to a free end of the barcoded genomic amplicons; and performing a template switch reaction on the barcoded cDNAs; and g) amplifying the barcoded amplicons and barcoded cDNAs off of the affinity moiety and capture reagent to generate free amplification products. As used herein, “template switch” or “template switch reaction” refers to the use of the intrinsic property of some reverse transcriptases which add non-templated ribocytosines to the cDNA molecule. Therefore, a primer comprising riboguanosines and additional sequences (e.g., terminal primer sequence) can be annealed to the cDNA and added via the reverse transcriptase. The template switch reaction is further described in International Publication No. WO2023 / 114860. The template switch reaction will not affect the double- stranded DNA, but will prepare the cDNA molecules for subsequent amplification.

[0043] The method may further comprise h) purifying the free amplification products, and i) sequencing the free amplification products.

[0044] As used herein, “terminal primer sequence” refers to a sequence that is known and can be used to anneal a primer for amplification. Thus, addition of a terminal primer sequence to an amplicon allows amplification of the amplicon by addition of a primer complementary to the terminal primer sequence.

[0045] During step b), the method may comprise contacting the population of fixed and permeabilized cells with a crowding agent. As used herein, “crowding agent” refers to compounds that decrease the solvent available to macromolecules, thereby increasing the relative concentration of said macromolecules and altering their properties. In some applications, crowding agents have the effect of increasing enzyme activity and accelerating reactions resulting in faster and potentially more specific assays. In some embodiments, crowding agents may include one or more of polyethylene glycol (PEG), polyethylene glycol 8000 (PEG-8000), trehalose, and sorbitol. In some embodiments crowding agents may include ficoll or dextrans.

[0046] In a second aspect, provided herein is a kit for amplifying and barcoding genomic DNA within a cell, the kit comprising: a) at least one set of DNA amplification primers; wherein eachAtty. Dkt. No. 112624.01537 set of amplification primers is configured to amplify a sequence of interest or a random sequence on the genomic DNA; b) at least one adapter barcode comprising: a first double-stranded barcoding sequence (BCS), wherein a double-stranded end of the BCS is configured to ligate with a doublestranded end of DNA sequence of interest generated by amplifying the genomic DNA with the DNA amplification primers of a); and a first annealing sequence (ANS) comprising a singlestranded overhang to facilitate the addition of more barcodes.

[0047] The kit may further comprise c) at least one additional barcode comprising: an additional ANS, wherein each of the at least one additional barcodes comprises a different additional ANS; a complementary annealing sequence (CANS) configured to anneal to the first ANS or the additional ANS; and an additional BCS that is different than the first BCS, wherein each of the at least one additional barcodes comprises a different additional BCS. The kit may comprise one or more multi-well plates where each well contains a unique additional barcode.

[0048] These additional barcodes can be annealed and ligated either to DNA amplicons that already possess a first barcode and annealing sequence (with an overhang) or to cDNA-RNA hybrid molecules generated using other kit components, or both. Thus, the kit may further comprise primers and nucleic acids necessary for reverse transcribing RNA and barcoding cDNA- RNA hybrid molecules, including: d) at least one set of reverse transcription primers, each comprising: the first ANS, wherein each of the at least one set of reverse transcription primers comprises the same ANS sequence; a single-stranded barcoding sequence (sBCS); wherein each of the at least one set of reverse transcription primers comprises a different sBCS; and a targeting sequence configured to anneal with a sequence of interest, or random sequence on RNA. The sequence of interest may be the polyA tail of mRNA. The kit may further comprise: e) at least one cDNA barcode, each comprising: the CANS, a double-stranded cDNA BCS (cBCS), wherein each cDNA barcode comprises a different additional cBCS; and the additional ANS. In embodiments, the kit may comprise pairs of additional adapter barcodes and cDNA barcodes, in which the additional BCS and the cBCS are the same.

[0049] The kit may further comprise: f) a double-stranded DNA sequence comprising a terminal primer sequence configured to ligate with a free end of barcoded DNA products generated by the use of components a) through e). The kit may further comprise g) reagents for performing and g) reagents to perform a template switch reaction as further described in WO2023 / 114860.Atty. Dkt. No. 112624.01537

[0050] The kits described herein may comprise additional reagents used for DNA amplification, reverse transcription, etc. For example, the kits may further comprises a polymerase, reverse transcriptase, a ligase, nucleotides, buffers, etc.

[0051] Additional Definitions

[0052] The disclosed subj ect matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0053] The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human subjects. In some embodiments, subjects may be plants, fish, birds, reptiles, or mammals. In some embodiments, the disclosed methods are performed on fungal, bacterial, archaeal, or protozoal cells.

[0054] The term “hybridization,” as used herein, refers to the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between fully complementary nucleic acid strands or between “substantially complementary” nucleic acid strands that contain minor regions of mismatch. Conditions under which hybridization of fully complementary nucleic acid strands is strongly preferred are referred to as “stringent hybridization conditions” or “sequence-specific hybridization conditions”. Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions; the degree of mismatch tolerated can be controlled by suitable adjustment of the hybridization conditions. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length and base pair composition of the oligonucleotides, ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art (see, e.g., Sambrook et al., 1989, Molecular Cloning- A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol. 26(3 / 4):227-259; and Owczarzy et al., 2008, Biochemistry, 47: 5336-5353, which are incorporated herein by reference).

[0055] As used herein, the terms “complementary” or “complementarity” are used in reference to “polynucleotides” and “oligonucleotides” (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “5'-C-A-G- T,” is complementary to the sequence “5'-A-C-T-G ” Complementarity can be “partial” or “total.” “Partial” complementarity is where one or more nucleic acid bases is not matched according to theAtty. Dkt. No. 112624.01537 base pairing rules. “Total” or “complete” complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules.

[0056] As used herein, the term “specific to” is used to define the relationship between macromolecular binding partners. For example, as used above, two nucleotide sequences that possess total complementarity to one another would be considered “specific” for one another, i.e., each totally complementary nucleotide would be specific to the other.

[0057] The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Nucleic acids generally refer to polymers comprising nucleotides or nucleotide analogs joined together through backbone linkages such as but not limited to phosphodiester bonds. Nucleic acids include deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) such as messenger RNA (mRNA), transfer RNA (tRNA), etc. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g. nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and / or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA / RNA hybrid, or include non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in theAtty. Dkt. No. 112624.015375' to 3' direction unless otherwise indicated. Tn some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine); nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C 5 -iodouridine, C5- propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadeno sine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2- thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0058] Nucleic acids, proteins, and / or other compositions described herein may be purified. As used herein, “purified” means separate from the majority of other compounds or entities, and encompasses partially purified or substantially purified. Purity may be denoted by a weight by weight measure and may be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, spectrophotometer, etc.

[0059] Methods of making polynucleotides of a predetermined sequence are well-known. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press, New York, 1991). Solidphase synthesis methods are preferred for both polyribonucleotides and polydeoxyribonucleotides (the well-known methods of synthesizing DNA are also useful for synthesizing RNA). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases can be incorporated into the polynucleotide, as well. See, e.g., U.S. Pat. No. 7,223,833; Katz, J. Am. Chem. Soc., 74:2238 (1951); Yamane, et al., J. Am. Chem. Soc., 83:2599 (1961); Kosturko, et al., Biochemistry, 13:3949 (1974); Thomas, J. Am. Chem. Soc., 76:6032 (1954); Zhang, et al., J. Am. Chem. Soc., 127:74-75 (2005); and Zimmermann, et al., J. Am. Chem. Soc., 124:13684-13685 (2002).

[0060] In the context of the present disclosure, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenine, “C” refers to cytosine, “G” refers to guanine, “T” refers to thymine, and “U” refers to uracil. The aforementioned abbreviations mayAtty. Dkt. No. 112624.01537 also be used to refer to nucleosides or nucleotides comprising the nucleic acid bases. For example, “G” may refer guanine, guanosine, or guanidine, depending on the context.

[0061] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.

[0062] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0063] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0064] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0065] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood toAtty. Dkt. No. 112624.01537 contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0066] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0067] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0068] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0069] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, orAtty. Dkt. No. 112624.01537 exemplary language (e g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0070] The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

[0071] Materials and methods for embodiments disclosed herein

[0072] Intracellular Amplification. For cells fixed and permeabilized, according to Payne et al. 2021, primers were hybridized by incubating cells in lOOpL of primer hybridization mix (0.5 pM concentration primers in 2x saline-sodium citrate (SSC) buffer and 30% formamide) for 2.5-3 hours at 37 C. Cells were spun down in a 4°C centrifuge at 5000g for 3 min and washed twice with molecular-grade PBS. 5 uL of cells at a concentration of 1 million cells / mL were added into a phi29 master mix (1 mM 10X phi29 buffer, 20mg / mL BSA, 10 mM dNTPs, 2 M sorbitol, lOkU / mL phi29, and 25 uM primers) and incubated at 30 C for 16 hours. For all other fixation and permeabilization permutations, 5ul of cells (at a concentration of 1 million cells / mL) suspended in molecular-grade PBS were added into a phi29 master mix (1 mM 10X phi29 buffer, 20mg / mL BSA, 10 mM dNTPs, 2 M sorbitol, lOkU / mL phi29, and 25 uM primers) and incubated at 30 C for 16 hours.

[0073] Cell Lysis. After amplification, the entire reaction volume was transferred into a 1.5 mb microcentrifuge tube and 1 mL of cold PBS and 5 pL of 10% Triton X-100 was added. Samples were spun down at 4°C, 5000 ref for 3 min. The supernatant was carefully aspirated off, leaving ~ 30 pL to avoid removing the pellet. Cells were then resuspended in 1 mL cold PBS, spun down, and resuspended in 1 mL cold PBS for a total of two washes. After washing cells, the supernatant was aspirated off, and cells were resuspended in 50 pL of cold PBS, 50 pL of 2X lysis buffer [20 mM Tris (pH 8.0), 400 mM NaCl, 100 mM EDTA (pH 8.0), and 4.4% SDS], and 10 pL of proteinase K sol (20 mg / mL). Cells were incubated at 55°C for 2 hours with periodic vortexing to lyse the cells and reverse any remaining formaldehyde cross-links.

[0074] Preparing Streptavidin Beads for Sample Binding. To prepare beads for sample binding, they must first be washed. For each lysate, 44 pL of Dynabeads MyOne Streptavidin ClAtty. Dkt. No. 112624.01537(Tnvitrogen) were washed three times with 800 pL of a IX wash solution of 5 mM Tris-HCl pH 8.0, 1 M NaCl, 500 pM EDTA, and 0.05% Tween-20 using a magnetic 1.5 mL tube rack. The beads were then resuspended in 100 pL per sample of a 2X wash solution containing 10 mM Tris- HCl pH 8.0, 2 M NaCl, and 1 mM EDTA.

[0075] Sample Binding to Streptavidin Beads. Directly after lysates were removed from heat, 5 pL of 100 pM PMSF (resuspended in isopropanol) was added to each tube and incubated at room temperature for 10 minutes to inactivate the proteinase K. To bind DNA to Cl beads, 100 pL of resuspended Cl beads were added to each sample tube and agitated at room temperature for 60 minutes. The samples were then placed on the magnetic rack, and the supernatant was removed. Sampled were removed from the magnetic rack and resuspended in 250 pL of the IX wash solution and agitated at room temperature for 5 min. Samples were replaced on the magnetic rack, and the subsequent steps were repeated for a total of two wash steps. After the two IX wash steps, the supernatant was removed, and the samples were resuspended in 250 pL of 10 mM Tris-HCl pH 8.0 and 0.1% Tween-20. At this point, the beads could be rinsed with 250 uL of molecular-grade water while the beads were still bound to the magnetic rack and moved on to subsequent steps or resuspended in 250 pL of the Tris-HCL Tween-20 buffer and stored at 4°C overnight.

[0076] Size selection bead clean up. The PCR products were then cleaned using a 0.8X SPRI size selection and eluted in 20 pL of molecular-grade water.

[0077] Gel electrophoresis. 5 pL of the product, which was eluted during the bead clean-up, was then run on a 2% agarose gel at 120 V for 15-20 min. A single properly amplified gene should appear as a dark band on the gel at the correct gene size. The genome that has been properly amplified with random hexamers should appear as a smear starting as high as 50 kb and ending at approximately 300 bp on a gel.

[0078] Illumina sequencing. Samples were library-prepped in one of two ways. Intracellularly amplified DNA that was not barcoded was prepped using the DNA flex kit with UPD indexes. Reactions were diluted . from manufacturer recommendations. Samples that were amplified intracellularly and then barcoded were prepped using an in-house library preparation kit48. Briefly, barcoded DNA was fragmented, sequencing adapters were ligated on, and dual indexes for downstream identification were added by PCR. The resulting libraries were sequenced on an Illumina Nextseq 2000 with 300 cycle kits (2x150). This sequencing was generously done by the Lim lab at ASU. Quality control measures were implemented to filter out low-quality reads andAtty. Dkt. No. 112624.01537 adapter contamination before downstream analysis. This approach enabled high-throughput sequencing of amplified DNA samples, providing comprehensive coverage of the genomic regions of interest.

[0079] Computational methods. Following sequencing, the generated files were processed using computational methods. For visualization and analysis of the aligned sequenc data, the software Geneious, a comprehensive platform for sequence data analysis, was employed Geneious Prime 2023.1.1. (https: / / www.geneious.com). Geneious facilitated the visualization of read alignments, identification of variants, and exploration of genomic features. Furthermore, to assess the sequencing depth and coverage across the genome, the read depth at each nucleotide was calculated in Geneious and then exported to make coverage plots using R. These coverage plots provided insights into the distribution of sequencing reads along the genome, allowing for the identification of regions with varying levels of coverage.

[0080] Template switch reaction:

[0081] Because the reverse transcription enzyme poly-cystinates the 3’ end of the cDNA, a primer adapter oligo with ribo-G’s on the 3’ end can be used during a subsequent reverse transcription reaction to add a terminal PCR primer adapter to bead-bound cDNA molecules. The beads were resuspended in the following reverse transcription reaction per sublibrary: 99 uL water, 44 uL 5X buffer, 33 uL PEG8000, 22 uL 10 mM dNTPs, 5.5 uL RNAse inhibitor, 5.5 uL template switch oligo, 11 uL Maxima RNAseH Minus reverse transcriptase. The beads were incubated at room temperature with gentle shaking for 30 minutes and then at 42C with gentle shaking for 90 minutes. They were then placed against a magnetic rack, the supernatant removed, and resuspended in 100 mM Tris-HCL. The beads were stored at 4C for no more than 2 days.

[0082] Embodiments

[0083] Embodiment 1. A method comprising: a) contacting a population of fixed and permeabilized cells with at least one set of DNA amplification primers configured to amplify a sequence of interest or a random sequence on genomic DNA within the cell, and a DNA polymerase; b) amplifying the DNA to create a DNA amplicon having a double-stranded primer sequence; c) contacting the cells with an adapter barcode, the adapter barcode comprising: a first double-stranded barcoding sequence (BCS), double-stranded end of the BCS is configured to ligate with the double-stranded primer sequence; and a first annealing sequence (ANS); d)Atty. Dkt. No. 112624.01537 attaching the first BCS to the double-stranded primer sequence using blunt end ligation, thereby creating barcoded amplicons; and e) lysing the cells to release the barcoded amplicons.

[0084] Embodiment 2. The method of embodiment 1, wherein after step b) and before step c), the method further comprises quenching the DNA polymerase.

[0085] Embodiment 3. The method of embodiment 1 or 2, wherein each of the at least one set of DNA amplification primers is configured to amplify a different sequence of interest or random sequence.

[0086] Embodiment 4. The method of any one of embodiments 1-3, further comprising: dividing the fixed and permeabilized cells into a plurality of wells before step a); wherein in step d), the adapter barcode contacted to the cells in each well comprises a different first BCS.

[0087] Embodiment 5. The method of any one of embodiments 1-4, wherein after step d) and before step e), the method further comprises: performing at least one round of annealing an additional barcode to the barcoded amplicons; wherein each round comprises: contacting the barcoded amplicons with the additional barcode, wherein the additional barcode of each round comprises: an additional ANS, wherein the additional ANS of each round is different; a complementary annealing sequence (CANS) configured to anneal to the first ANS or the additional ANS of a previous round; and an additional BCS that is different than the first BCS; and annealing the at least one additional barcode to the barcoded amplicon.

[0088] Embodiment 6. The method of embodiment 5, wherein at least one of the at least one additional barcodes comprises a different additional BCS.

[0089] Embodiment 7. The method of embodiment 5 or 6, wherein before each additional round, the method further comprises pooling and splitting the cells.

[0090] Embodiment 8. The method of any one of embodiments 5-7, wherein after the at least one additional round and before step e), the method further comprises performing a final round, the final round comprising: contacting the barcoded amplicons with a terminal barcode, the terminal barcode comprising: the complementary annealing sequence (CANS); the additional BCS; and a terminal primer sequence; and annealing the terminal barcode to the barcoded amplicon.

[0091] Embodiment 9. The method of embodiment 8, wherein the terminal barcode further comprises an affinity moiety, and wherein the method further comprises: f) capturing the barcoded amplicons with a capture reagent that binds to the affinity moiety.Atty. Dkt. No. 112624.01537

[0092] Embodiment 10. The method of any one of embodiments 1-6, further comprising after step d): contacting the cells with: a reverse transcriptase; and at least one set of reverse transcription primers, wherein each set of reverse transcription primers comprises: the first ANS; a single-stranded barcoding sequence (sBCS); wherein each of the at least one set of reverse transcription primers comprises a different sBCS; and a targeting sequence configured to anneal with a sequence of interest or a random sequence on RNA within the cell; and reverse transcribing the sequence of interest or random sequence on the RNA to generate barcoded cDNAs.

[0093] Embodiment 11. The method of embodiment 10, wherein each of the at least one set of reverse transcription primers is configured to amplify a different sequence of interest or random sequence on the RNA.

[0094] Embodiment 12. The method of embodiment 10 or 11, wherein the fixed and permeabilized cells are divided into the plurality of wells before step a), and wherein the set of reverse transcription primers in each well comprises a different sBCS.

[0095] Embodiment 13. The method of embodiment 10, wherein after step d) and before step e), the method further comprises: performing at least one round of annealing a cDNA barcode to the barcoded cDNAs; wherein each round comprises: contacting the barcoded cDNAs with the cDNA barcode, wherein the cDNA barcode of each round comprises: the CANS; a doublestranded cDNA BCS (cBCS); and the additional ANS, wherein the additional ANS of each round is different; and annealing the at least one cDNA barcode to the barcoded cDNAs.

[0096] Embodiment 14. The method of embodiment 13, wherein the cBCS and the BCS in each round are the same.

[0097] Embodiment 15. The method of embodiment 13 or 14, wherein at least one of the at least one cDNA barcodes comprise a different cBCS.

[0098] Embodiment 16. The method of any one of embodiments 13-15, wherein in the final round, the method further comprises contacting the barcoded cDNAs with the terminal barcode.

[0099] Embodiment 17. The method of any one of embodiments 9-16, wherein the affinity moiety comprises biotin, and wherein the capture reagent comprises streptavidin.

[0100] Embodiment 18. The method of any one of embodiments 9-16, wherein the affinity reagent comprises digoxigenin, and wherein the capture reagent comprises anti-digoxigenin antibody.Atty. Dkt. No. 112624.01537

[0101] Embodiment 19. The method of any one of embodiments 9-18, wherein after step e), the method further comprises: f) ligating a double-stranded DNA sequence comprising a terminal primer sequence to a free end of the barcoded amplicons; and performing a template switch reaction on the barcoded cDNAs; and g) amplifying the barcoded amplicons and barcoded cDNAs off of the affinity moiety and capture reagent to generate free amplification products.

[0102] Embodiment 20. The method of embodiment 19, further comprising: h) purifying the free amplification products.

[0103] Embodiment 21. The method of embodiment 19 or 20, further comprising: i) sequencing the free amplification products.

[0104] Embodiment 22. The method of embodiment 12, wherein step g) is done by performing polymerase chain reaction (PCR).

[0105] Embodiment 23. The method of any one of embodiments 1-22, wherein step b) is done by performing an isothermal amplification reaction.

[0106] Embodiment 24. The method of embodiment 23, wherein the temperature of the isothermal amplification reaction is about 20-40° C.

[0107] Embodiment 25. The method of embodiment 22 or 24, wherein step b) is done for about 12-24 hours.

[0108] Embodiment 26. The method of any one of embodiments 22-25, wherein the isothermal amplification reaction is done using an isothermal polymerase.

[0109] Embodiment 27. The method of embodiment 26, wherein the isothermal polymerase is phi29.

[0110] Embodiment 28. The method of any one of embodiments 1-27, wherein step b) comprises contacting the population of fixed and permeabilized cells with a crowding agent.

[0111] Embodiment 29. The method of embodiment 28, wherein the crowding agent comprises one or more of: polyethylene glycol 8000 (PEG-8000), trehalose, and sorbitol.

[0112] Embodiment 30. The method of any one of embodiments 1-29, wherein at least one of step d) and step f) is done using a T4 DNA ligase.

[0113] Embodiment 31. A kit for amplifying and barcoding genomic DNA within a cell, the kit comprising: a) at least one set of DNA amplification primers; wherein each set of amplification primers is configured to amplify a sequence of interest or a random sequence on theAtty. Dkt. No. 112624.01537 genomic DNA; b) at least one adapter barcode comprising: a first double-stranded barcoding sequence (BCS), configured to ligate with a double-stranded end of the DNA sequence of interest generated by amplifying the genomic DNA with the DNA amplification primers of a); and a first annealing sequence (ANS).

[0114] Embodiment 32. The kit of embodiment 31, further comprising: c) at least one additional barcode comprising: an additional ANS, wherein each of the at least one additional barcodes comprises a different additional ANS; a complementary annealing sequence (CANS) configured to anneal to the first ANS or the additional ANS; and an additional BCS that is different than the first BCS, wherein each of the at least one additional barcodes comprises a different additional BCS.

[0115] Embodiment 33. The kit of embodiment 31 or 32, further comprising: d) at least one set of reverse transcription primers, each comprising: the first ANS) wherein each of the at least one set of reverse transcription primers comprises the same first ANS; a single-stranded barcoding sequence (sBCS); wherein each of the at least one set of reverse transcription primers comprises a different sBCS; and a targeting sequence configured to anneal with a sequence of interest or random sequence on RNA; and e) at least one cDNA barcode, each comprising: the CANS; a double-stranded cDNA BCS (cBCS), wherein each cDNA barcode comprises a different additional cBCS; and the additional ANS.

[0116] Embodiment 34. The kit of any one of embodiments 31-33, further comprising at least one of: f) a double-stranded DNA sequence comprising a terminal primer sequence configured to ligate with a free end of barcoded products generated by the use of components a) through e); and g) reagents for performing a template switch reaction.

Claims

Atty. Dkt. No. 112624.01537CLAIMS1. A method compri sing : a) contacting a population of fixed and permeabilized cells with at least one set of DNA amplification primers configured to amplify a sequence of interest or a random sequence on genomic DNA within the cell, and a DNA polymerase; b) amplifying the DNA to create a DNA amplicon having a double-stranded primer sequence; c) contacting the cells with an adapter barcode, the adapter barcode comprising: a first double- stranded barcoding sequence (BCS), double-stranded end of the BCS is configured to ligate with the double-stranded primer sequence; and a first annealing sequence (ANS); d) attaching the first BCS to the double-stranded primer sequence using blunt end ligation, thereby creating barcoded amplicons; and e) lysing the cells to release the barcoded amplicons.

2. The method of claim 1, wherein after step b) and before step c), the method further comprises quenching the DNA polymerase.

3. The method of claim 1, wherein each of the at least one set of DNA amplification primers is configured to amplify a different sequence of interest or random sequence.

4. The method of claim 1, further comprising: dividing the fixed and permeabilized cells into a plurality of wells before step a); wherein in step d), the adapter barcode contacted to the cells in each well comprises a different first BCS.

5. The method of claim 1, wherein after step d) and before step e), the method further comprises: performing at least one round of annealing an additional barcode to the barcoded amplicons; wherein each round comprises:Atty. Dkt. No. 112624.01537 contacting the barcoded amplicons with the additional barcode, wherein the additional barcode of each round comprises: an additional ANS, wherein the additional ANS of each round is different; a complementary annealing sequence (CANS) configured to anneal to the first ANS or the additional ANS of a previous round; and an additional BCS that is different than the first BCS; and annealing the at least one additional barcode to the barcoded amplicon.

6. The method of claim 5, wherein at least one of the at least one additional barcodes comprises a different additional BCS.

7. The method of claim 5, wherein before each additional round, the method further comprises pooling and splitting the cells.

8. The method of claim 5, wherein after the at least one additional round and before step e), the method further comprises performing a final round, the final round comprising: contacting the barcoded amplicons with a terminal barcode, the terminal barcode comprising: the complementary annealing sequence (CANS); the additional BCS; and a terminal primer sequence; and annealing the terminal barcode to the barcoded amplicon.

9. The method of claim 8, wherein the terminal barcode further comprises an affinity moiety, and wherein the method further comprises: f) capturing the barcoded amplicons with a capture reagent that binds to the affinity moiety.

10. The method of claim 1, further comprising after step d): contacting the cells with: a reverse transcriptase; andAtty. Dkt. No. 112624.01537 at least one set of reverse transcription primers, wherein each set of reverse transcription primers comprises: the first ANS; a single- stranded barcoding sequence (sBCS); wherein each of the at least one set of reverse transcription primers comprises a different sBCS; and a targeting sequence configured to anneal with a sequence of interest or a random sequence on RNA within the cell; and reverse transcribing the sequence of interest or random sequence on the RNA to generate barcoded cDNAs.

11. The method of claim 10, wherein each of the at least one set of reverse transcription primers is configured to amplify a different sequence of interest or random sequence on the RNA.

12. The method of claim 10, wherein the fixed and permeabilized cells are divided into the plurality of wells before step a), and wherein the set of reverse transcription primers in each well comprises a different sBCS.

13. The method of claim 10, wherein after step d) and before step e), the method further comprises: performing at least one round of annealing a cDNA barcode to the barcoded cDNAs; wherein each round comprises: contacting the barcoded cDNAs with the cDNA barcode, wherein the cDNA barcode of each round comprises: the CANS; a double-stranded cDNA BCS (cBCS); and the additional ANS, wherein the additional ANS of each round is different; and annealing the at least one cDNA barcode to the barcoded cDNAs.

14. The method of claim 13, wherein the cBCS and the BCS in each round are the same.Atty. Dkt. No. 112624.0153715. The method of claim 13, wherein at least one of the at least one cDNA barcodes comprise a different cBCS.

16. The method of claim 13, wherein in the final round, the method further comprises contacting the barcoded cDNAs with the terminal barcode.

17. The method of claim 9, wherein the affinity moiety comprises biotin, and wherein the capture reagent comprises streptavidin.

18. The method of claim 9, wherein the affinity reagent comprises digoxigenin, and wherein the capture reagent comprises anti-digoxigenin antibody.

19. The method of claim 9, wherein after step e), the method further comprises: f) ligating a double-stranded DNA sequence comprising a terminal primer sequence to a free end of the barcoded amplicons; and performing a template switch reaction on the barcoded cDNAs; and g) amplifying the barcoded amplicons and barcoded cDNAs off of the affinity moiety and capture reagent to generate free amplification products.

20. The method of claim 19, further comprising: h) purifying the free amplification products.

21. The method of claim 19, further comprising: i) sequencing the free amplification products.

22. The method of claim 12, wherein step g) is done by performing polymerase chain reaction(PCR).

23. The method of claim 1, wherein step b) is done by performing an isothermal amplification reaction.

24. The method of claim 23, wherein the temperature of the isothermal amplification reaction is about 20-40° C.Atty. Dkt. No. 112624.0153725. The method of claim 22, wherein step b) is done for about 12-24 hours.

26. The method of claim 22, wherein the isothermal amplification reaction is done using an isothermal polymerase.

27. The method of claim 26, wherein the isothermal polymerase is phi29.

28. The method of claim 1, wherein step b) comprises contacting the population of fixed and permeabilized cells with a crowding agent.

29. The method of claim 28, wherein the crowding agent comprises one or more of: polyethylene glycol 8000 (PEG-8000), trehalose, and sorbitol.

30. The method of claim 1, wherein at least one of step d) and step f) is done using a T4 DNA ligase.

31. A kit for amplifying and barcoding genomic DNA within a cell, the kit comprising: a) at least one set of DNA amplification primers; wherein each set of amplification primers is configured to amplify a sequence of interest or a random sequence on the genomic DNA; b) at least one adapter barcode comprising: a first double-stranded barcoding sequence (BCS), configured to ligate with a double-stranded end of the DNA sequence of interest generated by amplifying the genomic DNA with the DNA amplification primers of a); and a first annealing sequence (ANS).

32. The kit of claim 31, further comprising: c) at least one additional barcode comprising: an additional ANS, wherein each of the at least one additional barcodes comprises a different additional ANS;Atty. Dkt. No. 112624.01537 a complementary annealing sequence (CANS) configured to anneal to the first ANS or the additional ANS; and an additional BCS that is different than the first BCS, wherein each of the at least one additional barcodes comprises a different additional BCS.

33. The kit of claim 31, further comprising: d) at least one set of reverse transcription primers, each comprising: the first ANS) wherein each of the at least one set of reverse transcription primers comprises the same first ANS; a single-stranded barcoding sequence (sBCS); wherein each of the at least one set of reverse transcription primers comprises a different sBCS; and a targeting sequence configured to anneal with a sequence of interest or random sequence on RNA; and e) at least one cDNA barcode, each comprising: the CANS; a double-stranded cDNA BCS (cBCS), wherein each cDNA barcode comprises a different additional cBCS; and the additional ANS.

34. The kit of claim 31, further comprising at least one of: f) a double-stranded DNA sequence comprising a terminal primer sequence configured to ligate with a free end of barcoded products generated by the use of components a) through e); and g) reagents for performing a template switch reaction.

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