[go: up one dir, main page]

WO2025247276A1 - Microparticule couplée contenant des amorces de pcr indexées et utilisation associée - Google Patents

Microparticule couplée contenant des amorces de pcr indexées et utilisation associée

Info

Publication number
WO2025247276A1
WO2025247276A1 PCT/CN2025/097790 CN2025097790W WO2025247276A1 WO 2025247276 A1 WO2025247276 A1 WO 2025247276A1 CN 2025097790 W CN2025097790 W CN 2025097790W WO 2025247276 A1 WO2025247276 A1 WO 2025247276A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
indexed
primers
sequence
pcr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/097790
Other languages
English (en)
Chinese (zh)
Inventor
杜金非
陈后
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lantu Biotechnology Suzhou Co Ltd
Original Assignee
Lantu Biotechnology Suzhou Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lantu Biotechnology Suzhou Co Ltd filed Critical Lantu Biotechnology Suzhou Co Ltd
Publication of WO2025247276A1 publication Critical patent/WO2025247276A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • This invention belongs to the field of biotechnology, specifically relating to high-throughput multimodal indexing, labeling, and sequencing detection of nucleic acid materials in single cells or subcellular structures.
  • Nucleic acid target molecules can be the entirety of DNA and/or RNA, or a specific subset. For example, using the Tn5 enzyme to cleave DNA in its chromatin state can selectively enrich open chromatin regions. Using reverse transcription primers containing PolyT can selectively enrich mRNA. Using sequence-specific primer hybridization can target and enrich specific genes and elements.
  • Nucleic acid target molecules can also be non-endogenous, artificially synthesized nucleic acids.
  • Perturb-seq uses viral transfection to deliver sgRNA for embedding in cells for expression, and single-cell transcriptome sequencing simultaneously detects the sgRNA sequence, thus identifying the target gene that is perturbed in each cell.
  • Single-cell multi-omics sequencing refers to the joint detection of different modalities within the same cell.
  • 10X Genomics' Multiome product uses microspheres to conjugate reverse complementary sequences of PolyT and Tn5 inserts to achieve dual-omics detection of RNA and ATAC in the same cell.
  • CITE-seq uses protein-specific antibodies conjugated to pre-encoded nucleic acid molecules, converting the determination of a specific protein modality into the detection of a transcriptome modality, thus enabling simultaneous determination of both RNA and protein information through single-cell transcriptome sequencing.
  • the first type Single-round high-density microreaction systems and coded microbead capture.
  • GEM Global in Emulsion
  • PMID:28091601 the core concept is to form approximately 100,000 or more droplets with a volume of about 1 nanoliter as independent microreaction systems through the physical segmentation of the oil and water phase interfaces in a droplet microfluidic system.
  • Each droplet encapsulates a reaction reagent, a single cell, and microbeads coupled with barcodes to form particles.
  • the microreaction system can also be realized using high-density nanopores.
  • the millions of capture sequences (e.g., PolyT) coupled on the same microbead have the same barcode, while the microbeads have different barcodes. Since there is only one cell in a reaction system, the barcode coupled to the microbeads is both a reaction system-specific barcode and a cell-specific cell barcode. The limitation is that the throughput is almost proportional to the pseudo-single-cell rate, and it is difficult to increase the cell throughput to around 10,000, and the cost is high.
  • the second type is high-throughput single-cell library construction technology using multi-round tag combination labeling.
  • SPLiT-seq technology based on split-pooling established by A.B. Rosenberg's team in 2018, this technology achieves ultra-high throughput, requiring no microfluidic or micropore preparation equipment, nor the preparation of particles.
  • Using fixed cells or the cell nucleus itself as the microreaction system it sequentially adds partial cellular barcodes to the target nucleic acid molecules in situ within the cell through 3 to 4 rounds of reactions (including RT, ligation, PCR, etc.).
  • the complete cellular barcode is formed through the combination of multiple rounds of barcodes.
  • the reaction system-specific barcode is shared by multiple intracellular and target nucleic acid molecules, typically ranging from dozens to hundreds of types. Its limitations include complex operation and inflexible throughput.
  • the present invention provides a microparticle coupled with indexed PCR primers, the microparticle comprising microbeads and indexed PCR primers, wherein the microbeads and primers are coupled by chemical bonds; the microbeads, as a carrier, carry the indexed PCR primers and deliver them to a specific conventional reaction system or microreaction system, and then release the primers in a controlled manner.
  • the material of the microbeads is selected from gels, synthetic polymers, or magnetic beads.
  • the gel is a natural polymer material, including but not limited to alginate, chitosan, agarose, gelatin, fibrinogen and/or peptides.
  • the synthetic polymers include, but are not limited to, polyethylene glycol, polyacrylic acid, polyvinyl alcohol, polyacrylamide, polyhydroxymethyl methacrylate and/or polyacrylamide.
  • the magnetic beads are made of iron(III) oxide magnetic material.
  • the diameter of the microspheres is 5-50 ⁇ m.
  • the diameter of the microspheres is 10 ⁇ m-40 ⁇ m.
  • the indexed PCR primer comprises three parts: a fixed sequence 1, an index sequence, and a fixed sequence 2; wherein the fixed sequence 1 is a sequencing adapter, the fixed sequence 2 is a capture sequence, and the index sequence is a particle-specific barcode sequence.
  • sequencing adapter is selected from the adapters of the Illumina sequencer or the sequencing library combined with the sequencing chip to generate the cluster sequence.
  • sequence of the cluster generated by combining the sequencing library with the sequencing chip is selected from the P5 sequence, the P7 sequence, or a partial sequence of both the P5 and P7 sequences.
  • the capture sequence is selected from the sequencing primer binding site sequence of the Illumina sequencer or any targeted sequencing capture sequence.
  • sequencing primer binding site sequence of the Illumina sequencer is selected from partial sequences of TruSeq read1, TruSeq read2, Nextera read1, Nextera read1, or above.
  • the fixed sequence 1 and fixed sequence 2 can be adjusted according to the sequencer used in subsequent sequencing reactions.
  • nucleotide sequence of the index sequence is random, and each site can be any one of the four bases A, T, C, and G.
  • the length of the index sequence is 1-25 bp.
  • connection methods between the microbeads and the indexed PCR primers include, but are not limited to, links that release chemical bonds, polyacrylamide links cleaved by strong reducing agents, or biotin-streptavidin links.
  • linking of the releasable chemical bonds includes dU that can be released by the USER enzyme or amino modifications that can be released by photolysis.
  • the copy number ratio between the microbeads and the indexed PCR primers is 1:N, where N is greater than or equal to 1, and preferably N is 107 - 108 .
  • PCR primers can be coupled to the same microbead containing indexed PCR primers, preferably 1-5 types of PCR primers.
  • PCR primers can be coupled to the same microbead containing indexed PCR primers.
  • the different types of PCR primers contain at least one different sequence from fixed sequence 1 and fixed sequence 2.
  • index sequences of primers of the same type coupled to the same microbead containing indexed PCR primers are the same, while the index sequences of primers of different types may be the same or different, and the index sequences of primers coupled to different microbeads are different.
  • the fixed sequence 2 of the microparticle coupled with the indexed PCR primer hybridizes with the common sequence of the target nucleic acid molecule.
  • the target nucleic acid molecule refers to a modified target nucleic acid molecule, wherein the modification involves adding a sequence capable of hybridizing with fixed sequence 2 to one or both ends of the target nucleic acid molecule.
  • the present invention provides a method for preparing microparticles coupled with indexed PCR primers as described in the first aspect, the method comprising the following steps:
  • the modified fixed sequence 1 is affinity-linked with specific microbeads, and then random bases of the index sequence are synthesized by split-pooling using a two-step cyclic enzymatic DNA synthesis method or ligation method.
  • the fixed sequence 2 is synthesized by enzymatic method. Specific bases are added to a reaction tank to complete the synthesis of the fixed sequence 2, and the microbeads coupled with the indexed PCR primers of the present invention are obtained.
  • step S1 the chemical synthesis refers to the use of solid-phase phosphoramidite method to fix DNA on a solid support to complete the synthesis of DNA strands, with the synthesis extending from the 3' end to the 5' end of the primer to be synthesized, and adjacent nucleotides being linked by 3' ⁇ 5' phosphodiester bonds.
  • step S1 the chemical modification includes Acrydite or Biotin modification.
  • the fixed sequence 1 is a sequencing adapter, selected from the adapter of the Illumina sequencer or the sequencing library combined with the sequencing chip to generate the cluster sequence.
  • sequence of the cluster generated by combining the sequencing library with the sequencing chip is selected from the P5 sequence, the P7 sequence, or a partial sequence of both the P5 and P7 sequences.
  • the fixed sequence 1 and the specific microbead affinity linking refers to the use of acrylic acid-modified primers to bind to polyacrylamide hydrogel microspheres, and biotin-containing primers to link to gel beads or magnetic beads with streptavidin on their surface.
  • step S2 the microbeads coupled with fixed sequence 1 are divided into 4 reaction chambers, and each reaction chamber contains only one type of synthetic base material.
  • a base is synthesized at the 3' of fixed sequence 1 using TdT enzyme.
  • the microbeads in the 4 reaction chambers are mixed and then evenly distributed into 3-4 reaction chambers containing only one type of synthetic base material. This process is repeated multiple times to generate different throughputs of barcodes.
  • reaction tank contains one of the bases dATP, dTTP, dCTP, or dGTP.
  • step S2 the length of the index sequence is 1-25 bp.
  • step S3 the fixed sequence 2 is selected from a portion of Truseq read1, Truseq read1, Nextera read1, Nextera read2, or more.
  • the 5' end of the fixed sequence 2 is phosphorylated.
  • the present invention provides the application of the microparticles coupled with indexed PCR primers as described in the first aspect in the construction of high-throughput single-cell sequencing libraries for multiple omics modalities; wherein, when cells undergo PCR amplification, at least one amplification is performed on the sequencing cells by indexed-PCR amplification using the microparticles coupled with indexed PCR primers as described in the first aspect.
  • the various omics modalities include, but are not limited to, single modalities and combinations of single modalities such as scRNA-seq, scVDJ-seq, scATAC-seq, sc-whole genome sequencing, and scCUT&Tag, such as scRNA-seq+scATAC-seq single-cell dual-omics modality.
  • the indexed-PCR amplifies nucleic acids while simultaneously labeling them.
  • the fixed sequence 2 of the indexed-PCR primers specifically captures the target sequence, which is a sequence complementary to the fixed sequence 2 introduced into the target RNA and DNA molecules in situ through reverse transcription, ligation, transposition, and other reaction processes.
  • the target sequence is a sequence complementary to the fixed sequence 2 introduced into the target RNA and DNA molecules in situ through reverse transcription, ligation, transposition, and other reaction processes.
  • the cellular origin of molecules in the final sequencing library can be distinguished.
  • the fixed sequence 2 of the microbead-conjugated PCR primer-containing microparticles hybridizes with the target nucleic acid molecule.
  • the target nucleic acid molecule refers to a modified target nucleic acid molecule, wherein the modification involves adding a sequence capable of hybridizing with fixed sequence 2 to one or both ends of the target nucleic acid molecule.
  • this invention provides a method for constructing high-throughput single-cell sequencing libraries with multiple omics modalities, the method comprising the following steps:
  • S2 Perform indexed-PCR amplification on the cells, wherein the primers used for indexed-PCR are microparticles coupled with indexed PCR primers as described in the first aspect of the present invention.
  • the cell index introduced by the indexed-PCR can be a unique cell index or the last index of a multi-round combined index.
  • the target molecule is pre-labeled in situ on the cells/nucleus for multiple rounds, and the number of rounds for introducing partial cell barcodes of the same target molecule can be 1, 2, 3 or more.
  • cell segmentation methods when introducing cell barcodes include, but are not limited to, microfluidics, manual and machine pipetting, or flow cytometry sorting.
  • the cell segmentation carrier when introducing cell barcodes can be water-in-oil microdroplets, nanopores, or microporous PCR plates.
  • the present invention provides a high-throughput single-cell sequencing library construction system with multiple omics modalities, comprising a tissue sample processing module and a cell indexing module;
  • the tissue sample processing module is used to prepare tissue into a single-cell suspension, and then perform cell fixation and permeabilization.
  • the cell indexing module refers to the high-density microreaction system indexed-PCR indexing of the treated cells, and the primers used in the indexed-PCR are the microparticles coupled with indexed PCR primers as described in the first aspect of this invention.
  • the cell indexing is performed in droplet microfluidics using GEM-indexed-PCR indexing or in microplate equipment using indexed-PCR indexing of cells.
  • the target cell library when the target cell library is less than 20,000 cells, it can be achieved independently using the droplet microfluidic GEM indexed-PCR of the present invention.
  • the target cell library when the target cell library is 20,000 to 200,000 cells, it can be achieved by two rounds of labeling using the disclosed droplet microfluidic GEM indexed-RT and the droplet microfluidic GEM indexed-PCR of the present invention.
  • the target cell library is 200,000 to 2,000,000 cells
  • a 96-well plate indexed Tn5 or the disclosed droplet microfluidic GEM indexed-ligation technology can be used, and the droplet microfluidic indexed-PCR of the present invention can be achieved through three rounds of labeling.
  • the present invention provides a reagent kit for single-cell or subcellular indexing, the reagent comprising microparticles coupled with indexed PCR primers as described in the first aspect of the present invention; the microparticles comprising microbeads and indexed PCR primers, wherein the microbeads and primers are coupled by chemical bonds; the microbeads, acting as carriers, carry the indexed PCR primers and deliver them to a specific conventional reaction system or microreaction system, and then release the primers in a controlled manner.
  • This invention develops a novel single-cell sequencing strategy.
  • cells or subcellular structures are immobilized and permeable, or semi-permeable shells are constructed using polymer materials to encapsulate cells or subcellular structures, forming nucleic acid aggregates capable of multi-step reactions. This allows steps prior to PCR to be performed in bulk reactions.
  • microbeads coupled with barcoded PCR primers are designed. These microbeads can be delivered in a single reaction to two to one million or more different reaction systems via microfluidic means, including but not limited to droplet-based methods, adding reaction-specific barcodes to target nucleic acid molecules within the reaction system in a high-throughput, parallel manner.
  • One embodiment of this invention achieves single-cell transcriptome sequencing using GEM-indexed-PCR instead of the existing GEM-indexed-RT, demonstrating the feasibility of this bulk sequencing to single-cell sequencing transformation. Furthermore, since PCR is the final step in all omics library construction, this transformation, by replacing the conventional PCR step with a large-scale indexed PCR step, is also universally applicable, enabling the detection of many modalities that have not yet reached single-cell precision to enter the single-cell era.
  • this invention can be used independently to complete library construction for single-cell sequencing through a single-round indexing process where only one cell is expected in a reaction system.
  • the required cell throughput is high, it can be combined with existing non-indexed-PCR cell barcoding techniques to define the final cell barcode through combined indexing, thus completing ultra-high throughput single-cell sequencing library construction.
  • this invention can be integrated to achieve two or more rounds of indexing through extremely simple steps, thereby significantly increasing cell throughput, effectively reducing the false single-cell rate and cost, while maintaining high data quality and high stability. This solves the problem that existing methods cannot simultaneously achieve high cell throughput, ease of operation, low cost, low false single-cell rate, low empty load rate in microreaction systems, high data quality, and strong applicability.
  • FIG. 1 Schematic diagram of microparticles coupled with indexed PCR primers.
  • a shows the microparticle and sequence structure;
  • b-i are examples of specific primer sequences.
  • Each microparticle can independently perform the function of single-cell sequencing library construction.
  • Figure 2 illustrates a single-cell omics library construction (one-round labeling) scheme based on microparticles coupled with indexed PCR primers.
  • Immobilized and permeabilized cells or nuclei undergo in situ reverse transcription (RT), transposition (Tn5) reactions, or a combination of Tn5 and RT reactions followed by droplet microfluidic techniques to create microreactions.
  • RT reverse transcription
  • Tn5 transposition
  • droplet microfluidic techniques to create microreactions.
  • Each droplet encapsulates a microparticle coupled with an indexed PCR primer and a single cell.
  • RNA-seq single-cell transcriptomics
  • ATAC-seq chromatin accessibility
  • RNA+ATAC-seq co-omics detection can be achieved.
  • Figure 3 shows the library construction process and library structure for conventional high-throughput single-cell omics (one-round labeling) based on droplet microfluidics according to this invention.
  • a Library construction process and library structure for single-cell transcriptomics including 3’ RNA-seq and 5’ RNA;
  • b Library construction process and library structure for single-cell ATAC-seq;
  • c Library construction process and library structure for RNA+ATAC co-omics.
  • Figure 4 shows a scheme for applying microparticles with indexed PCR primers to single-cell omics library construction (2 rounds of labeling).
  • Figure 5 shows the library structure of microparticles based on indexed PCR primers applied to single-cell omics sequencing (2 rounds of labeling).
  • Figure 6 shows a scheme for applying microparticles with indexed PCR primers to single-cell omics library construction (3 rounds of labeling).
  • Figure 7 shows the library structure of a single-cell omics library (3 rounds of labeling) based on microparticles coupled with indexed PCR primers.
  • Figure 8 shows the dimensionality reduction visualization of the single-cell transcriptome species pooling experiment with one round of labeling.
  • the single-cell transcriptome data were pooled according to a 1:1:10 ratio of human Hela:HEK293T:mouse cortical cells.
  • Unsupervised clustering of the obtained data yielded three distinct cell clusters: clusters 1 and 2 represented Hela and HEK293T cells, respectively, while cluster 3 consisted of mouse cortical cells.
  • the present invention relates to microparticles coupled with indexed PCR primers.
  • the microparticles comprise microbeads and indexed PCR primers, wherein the microbeads and primers are chemically coupled.
  • the microbeads act as carriers carrying the indexed PCR primers, delivering them to a specific conventional reaction system or microreaction system, and then releasing the primers in a controlled manner.
  • the indexed PCR primers comprise three parts: a fixed sequence 1, an index sequence, and a fixed sequence 2.
  • Fixed sequence 1 is a sequencing adapter
  • fixed sequence 2 is a capture sequence
  • the index sequence is a microparticle-specific barcode sequence. The specific structure is shown in Figure 1a.
  • the application scenario of this invention is single-cell sequencing, but the smallest unit of the labeled target needs to be a nucleic acid aggregate coupled together to perform multi-step molecular biological reactions.
  • the unit can be a cell, a subcellular component, such as a cell nucleus, organelle, vesicle, exosome, or any artificially defined nucleic acid aggregate.
  • single-cell sequencing will be used consistently.
  • Achieving the coupling of the target nucleic acid includes, but is not limited to, treating natural cells or subcellular components with immobilizing agents, and also includes, but is not limited to, using polymeric materials to form a semi-permeable shell, so that the large nucleic acid molecules of the same cell are encapsulated and cannot flow out, but small molecules and reagents such as enzymes can pass through.
  • the templates for the PCR primers of this invention can be any publicly available omics technology used to convert target nucleic acid molecules into amplifiable nucleic acid fragments through molecular biology techniques. They can be molecules derived from single-modality or multi-modality sources.
  • the particle-specific barcode (index) of this invention can serve as a complete cell barcode, thus expecting only one cell in each reaction system.
  • the PCR primer template is an amplifiable nucleic acid fragment that does not carry any cell barcode.
  • This particle barcode can also serve as a partial cell barcode, allowing 2-1000 or more cells in each reaction system.
  • the PCR primer template is an amplifiable nucleic acid fragment for which partial cell barcodes have been added in previous steps using other disclosed methods.
  • This invention provides a method for preparing primer-coupled gel beads, using highly monodisperse, biodegradable gel beads as a solid-phase carrier to covalently link PCR primers.
  • the specific experimental procedure includes:
  • Aqueous phase a mixture of acrylamide solution, N,N'-bis(acryloyl)cysteine BAC solution (cross-linking agent), ammonium persulfate APS solution (catalyst), TBSET buffer, and modified fixed sequence 1 (fixed sequence is Illumina P5/P7, the 5' end of the sequence is modified with acrydite followed by Int HS-SH C6 modification), to achieve a final concentration of 0.392% acrylamide, 0.6% cross-linking agent, and 25 ⁇ M Oligo DNA.
  • cross-linking agent cross-linking agent
  • ammonium persulfate APS solution catalyst
  • TBSET buffer and modified fixed sequence 1 (fixed sequence is Illumina P5/P7, the 5' end of the sequence is modified with acrydite followed by Int HS-SH C6 modification), to achieve a final concentration of 0.392% acrylamide, 0.6% cross-linking agent, and 25 ⁇ M Oligo DNA.
  • Oil phase Add 12 ⁇ L of catalytic accelerator: tetramethylethylenediamine (TEMED) to 3 mL of drop-surf oil and shake to mix.
  • catalytic accelerator tetramethylethylenediamine (TEMED)
  • the microdroplet preparation apparatus was installed and connected to the gas source, power supply, and PDMS chip (PDMS-FF-50 superhydrophobic chip). Aqueous and oil phases were added to their respective storage tanks.
  • the gas source was turned on to purge air from the system, and the flow rates were set to 20 ⁇ L/min for the oil phase and 10 ⁇ L/min for the aqueous phase.
  • the microdroplets were stably generated, they were allowed to stand, transferred to centrifuge tubes, sealed with mineral oil, and cured overnight in an oven.
  • Example 1 the fixed sequence 1 of the PCR primer was covalently bound to the polyacrylamide backbone of the microbeads via 5’-Acrydite modification, and then random sequences and fixed sequences 2 were generated on the microbeads using various methods.
  • Method 1 Random bases of index sequences are gradually added to microbeads through multiple rounds of enzymatic DNA synthesis reactions. Specifically, equal amounts of microbeads are added to four TDT enzyme synthesis reaction solutions, each containing only one type of nucleotide (3'-ONH2-dATP, 3'-ONH2-dGTP, 3'-ONH2-dCTP, or 3'-ONH2-dTTP). After one base extension, the reaction is terminated, and the reaction solution is washed away. The microbeads from the four reactions are then mixed and divided equally among four synthesis reactions containing only one type of nucleotide.
  • a 12bp random sequence is generated, enabling combinations of over 700,000 tag sequences.
  • a ligation reaction is performed to ligate a fixed sequence 2 (Fixed sequence 2 is Truseq read1, Truseq read1, Nextera read1, Nextera read2, or a portion thereof, with phosphorylation modification at the 5' end) to the random sequence.
  • Method 2 A step-by-step, modular construction of the tag sequence is achieved through multi-round connection reactions.
  • the random sequence is actually a combination of 96*96*96 tag sequences and 2 bridging sequences.
  • the specific operation is as follows:
  • Primer 1 contains the 6nt terminal of fixed sequence 1 and an 8nt tag sequence 1 (e.g., 5’p-CCGATCT[tag sequence 1]-3’); sequence 2 includes a 14nt complementary sequence to the terminal of fixed sequence 1, the complementary sequence of the 8nt tag sequence 1 of primer 1, and the complementary sequence of the 4nt bridging sequence 1 (e.g., 3’-GCGAGAAGGCTAGA[tag sequence 1]CAGT-5’p); primer 3 consists of a 4nt bridging sequence 1 and an 8nt tag sequence 2 (e.g., 5’p-GTCA).
  • Primer 4 contains the complementary sequence of 8nt tag sequence 2 and the complementary sequence of 4nt bridging sequence 2 (e.g., 3’-[Tag sequence 2]TGTC-5’p);
  • Primer 5 contains the 4nt bridging sequence 2, the 8nt tag sequence 3, and the fixed sequence 2 or part of the fixed sequence 2 (e.g., 5’p-ACAG[tag sequence 3][fixed sequence 2: ACACTCTTTCCCTACACGACGCTCTTCCGATCT]);
  • Primer 6 is the complementary sequence of tag sequence 3.
  • primers 1 and 2 are annealed in pairs so that their complementary regions are bound by base pairing.
  • Example 2 The microbeads containing fixed sequence 1 obtained in Example 1 were divided into 96-well plates. T4 ligase reaction system was added to the 96-well plates, and an annealing product of primers 1 and 2 was added to each well for ligation reaction.
  • microparticles of the present invention coupled with indexed PCR primers, are introduced as tags for single cells using droplet microfluidic GEM technology, enabling single-cell omics sequencing of multiple omics modalities ( Figures 2 and 4).
  • N represents any bit of ATCG
  • V represents any bit of ACG
  • rG represents riboguanosines
  • +G represents LNA-modified guanosine.
  • the method of the present invention can greatly simplify the experimental operation and improve the data quality and stability.
  • the specific experimental procedures include: in situ reverse transcription of immobilized and permeabilized cells/nuclei (scRNA-seq, scVDJ-seq for RNA); or in situ transposition (scATAC-seq for open chromatin regions); or in situ transposition followed by RT reaction (dual-omics capture for both RNA and open chromatin regions).
  • the cells/nuclei are aliquoted with the indexed PCR primers of this invention using droplet microfluidics, ensuring that each effective microdroplet, microwell, or PCR plate contains one cell, one microbead, and the reaction components required for PCR amplification (including PCR enzyme, buffer, dNTPs, and paired primers).
  • the number of microreactions can be adjusted according to the actual required cell throughput, achieving labeling of 1-10,000 cells. Different types of primers are used for different omics modalities (see Figures 2 and 3 for details).
  • the target nucleic acid molecule of each cell is introduced into the microparticles of the indexed PCR primers of this invention as a cell tag and at least one sequencing adapter. Therefore, the products of all cells can be mixed and purified thereafter.
  • the purified scRNA-seq is now a full-length cDNA product with both a cell tag and a sequencing adapter at one end. Further library construction is required, and after random fragmentation, another sequencing adapter is loaded at the other end of the sequence with the cell tag.
  • TCR/BCR can be enriched from the full-length cDNA product using specific primers or probes and a library can be constructed to achieve sequencing of paired immune receptor sequences of the same cell (Figure 3.a).
  • the purified scATAC-seq has a complete next-generation sequencing library structure and can be used for subsequent sequencing ( Figure 3.b).
  • the scRNA-seq and scATAC-seq dual-omics approach further enriches the full-length cDNA product and the chromatin open region library separately from the PCR pre-amplified product using specific primers.
  • the scRNA-seq is further used for library construction, ultimately achieving single-cell dual-omics sequencing (Figure 3.c).
  • Tn5 transposition reaction solution including TN5 enzyme and buffer containing Mg2 +
  • the cells are centrifuged, the supernatant is removed, and the cells are resuspended in reverse transcription solution (including reverse transcriptase, buffer, dNTP, RT primer, TSO sequence). After thorough mixing, the reverse transcription reaction is carried out.
  • reverse transcription solution including reverse transcriptase, buffer, dNTP, RT primer, TSO sequence.
  • PCR reaction solution including NEBNext Ultra IIQ5 PCR enzyme, buffer, dNTP, TSO primer, P7 primer
  • the product introduced by the read2 sequencing adapter was purified with 1x XPbeads (Beckman Coulter, A63881), and then further amplified by PCR with P5 primers and P7 sample index primers. The product was purified with 0.8x XPbeads to obtain the final scRNA-seq library.
  • a species pooling test was conducted using human cell lines HeLa and HEK293T with mouse cerebral cortex cells at a ratio of 1:1:10. The analysis results showed that 5753 cells were detected, with a cell capture rate of approximately 57%. Unsupervised clustering was sufficient to clearly distinguish the three cell types (see Figure 6 for details). The number of genes detected by scRNA-seq was greater than 1500; the scATAC-seq TSS enrichment score was greater than 6.5, and the reads in peak/cell ratio was greater than 8000.
  • Example 4 Combining well-indexed RT with the GEM-indexed PCR of this invention to achieve high-throughput single-cell transcriptomics library construction with two rounds of tagging.
  • the cells and the microbead primers of this invention are subjected to indexed PCR reaction in droplet microfluidics, thereby achieving high-throughput single-cell transcriptome sequencing.
  • Example 5 Combining existing GEM-indexed RT technology with the Plate-indexed PCR of this invention to achieve high-throughput single-cell transcription library assembly with two rounds of tagging.
  • the present invention uses microparticles coupled with indexed PCR primers to introduce the cell tags in the second round through a well plate, achieving ultra-high throughput, sample multiplexing, and extremely low pseudo-single cell rate in single-cell transcriptomics sequencing (see Figure 4).
  • RNA-seq instructions immobilized and permeabilized cells/nuclei, along with relevant reagents and gel beads, were loaded into a microfluidic chip to prepare a water-in-oil emulsion.
  • the number of cells loaded could be, but was not limited to, 10,000 to 1,000,000 (300,000 cells were loaded in this example).
  • the obtained water-in-oil product underwent reverse transcription, allowing the nucleic acid sequence on the 10x Genomics gel beads to be loaded onto the 3’ end of the cDNA through reverse transcription and template substitution reactions, achieving the first round of in situ cell tagging.
  • the structure of the water-in-oil was broken to separate the aqueous and oil phases, releasing the cells from the water-in-oil droplets.
  • the thoroughly mixed cell suspension was aliquoted into one 96-well plate, fewer than one 96-well plate, or multiple 96-well plates (each well containing 1,000-3,000 cells) according to the number of cells loaded.
  • Each well plate contained microbeads of type-specific random sequence primers (primer structures are detailed in Figure 1b) and PCR reaction solution. After the PCR reaction, all products from the 96-well plate were collected, mixed, and purified to obtain full-length cDNA amplification products with two rounds of tags.
  • the human cell line HeLa was tested. Data analysis showed that 150,000 cells were detected, with a cell capture rate of about 50%; the number of genes detected by scRNA-seq was greater than 2,500 (see Table 1 for details).
  • Example 6 Combining existing GEM-indexed RT & Tn5 technology with the Plate-indexed PCR of this invention to achieve high-throughput single-cell RNA + ATAC dual-omics library construction with two rounds of tagging.
  • the first round of cell tagging was performed using the 10x Genomics microfluidic platform and scRNA-seq and scATAC-seq Multiome reagents. Then, the cells and microparticles coupled with indexed PCR primers of this invention were introduced into a 96-well plate for the second round of tagging, achieving ultra-high throughput scMultiome dual-omics (library structure shown in Figure 5).
  • the specific experimental steps are as follows:
  • Example 7 Combining existing GEM-indexed RT & Tn5 technology with the GEM-indexed PCR of this invention to achieve high-throughput single-cell multi-omics library construction with two rounds of tagging.
  • the first round of cell tag loading was performed using the 10x Genomics microfluidic platform and Multiome reagents. Then, the cells and microparticles containing indexed PCR primers of this invention were introduced into microfluidic droplets for the second round of tagging, achieving ultra-high throughput single-cell transcription library assembly (library structure shown in Figure 5). Specific experimental steps are as follows:
  • Example 6 cells labeled using the 10x Genomics Multiome manual were demulsified and washed from a GEM. The cells, PCR reaction solution, and PCR primer beads were then used to prepare water-in-oil microdroplets. Each droplet contained a microbead with a specific random sequence primer (primer structure detailed in Figure 1h) and the PCR reaction solution. After the PCR reaction, the emulsion was demulsified and the nucleic acid products were purified to obtain full-length amplified cDNA with two rounds of labeling, and gDNA from the chromatin development region. The cDNA and gDNA were then enriched and amplified using specific primers (which could be biotin-modified). scATAC-seq completed library construction. The transcriptome portion followed the same procedure as in the first application scenario. After sequencing, the two rounds of molecular tags were used to define a single cell.
  • Example 8 Combining existing GEM technology with the GEM-indexed PCR of this invention to achieve high-throughput single-cell multi-omics library construction with three rounds of tagging.
  • microparticles coupled with indexed PCR primers of this invention are introduced into a third round of cell tagging through a well plate, achieving ultra-high throughput, sample multiplexing, and extremely low pseudo-single-cell rate single-cell sequencing (Figure 6).
  • the specific experimental steps are as follows:
  • indexedTn5s can be, but are not limited to, 2-96 types. This allowed for the in-situ loading of different tags onto open chromatin regions of cells in different reaction chambers.
  • the pre-indexed cells/nuclei were then used for 10x Genomics Multiome GEM preparation and cell labeling. The demulsified cells were then subjected to droplet microfluidic preparation again.
  • Each droplet generated a microparticle containing one PCR primer (primer structure shown in Figure 1f) and PCR reaction solution. Multiple cells were allowed to exist in each droplet ( Figure 6 right, Figure 7 library structure). Subsequent library construction was the same as in Example 7. After sequencing, three rounds of molecular tags were used to define a single cell.
  • cells from different sample sources can be traced back based on the first round of indexed Tn5 pre-indexing. 300,000 cells were captured, with a cell capture rate greater than 35%; the number of genes detected by scRNA-seq was greater than 1500; the scATAC-seq TSS enrichment score was greater than 5.5, and the reads inpeak/cell ratio was greater than 7000.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)

Abstract

La présente invention concerne une microparticule couplée à une bille contenant des amorces de PCR à code-barres. La microparticule peut être administrée à une pluralité de systèmes de réaction différents en une seule réaction par des moyens tels que la microfluidique en gouttelettes et des codes-barres spécifiques à un système de réaction sont ajoutés à des molécules d'acide nucléique cibles dans les systèmes de réaction d'un mode parallèle à haut débit, de façon à achever la construction de bibliothèques de séquençage monocellulaire ou de séquençage multiomique.
PCT/CN2025/097790 2024-05-28 2025-05-28 Microparticule couplée contenant des amorces de pcr indexées et utilisation associée Pending WO2025247276A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202410667777 2024-05-28
CN202410667777.7 2024-05-28

Publications (1)

Publication Number Publication Date
WO2025247276A1 true WO2025247276A1 (fr) 2025-12-04

Family

ID=97048128

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2025/097790 Pending WO2025247276A1 (fr) 2024-05-28 2025-05-28 Microparticule couplée contenant des amorces de pcr indexées et utilisation associée

Country Status (2)

Country Link
CN (1) CN120666033A (fr)
WO (1) WO2025247276A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180080021A1 (en) * 2016-09-17 2018-03-22 The Board Of Trustees Of The Leland Stanford Junior University Simultaneous sequencing of rna and dna from the same sample
CN110199019A (zh) * 2016-05-02 2019-09-03 Encodia有限公司 采用核酸编码的大分子分析
CN111172257A (zh) * 2020-01-16 2020-05-19 南方科技大学 一种带编码的凝胶微粒及其制备方法和应用
CN113025695A (zh) * 2019-12-25 2021-06-25 苏州绘真生物科技有限公司 高通量单细胞染色质可及性的测序方法
US20240018584A1 (en) * 2021-02-04 2024-01-18 Illumina, Inc. Long indexed-linked read generation on transposome bound beads

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110199019A (zh) * 2016-05-02 2019-09-03 Encodia有限公司 采用核酸编码的大分子分析
US20180080021A1 (en) * 2016-09-17 2018-03-22 The Board Of Trustees Of The Leland Stanford Junior University Simultaneous sequencing of rna and dna from the same sample
CN113025695A (zh) * 2019-12-25 2021-06-25 苏州绘真生物科技有限公司 高通量单细胞染色质可及性的测序方法
CN111172257A (zh) * 2020-01-16 2020-05-19 南方科技大学 一种带编码的凝胶微粒及其制备方法和应用
US20240018584A1 (en) * 2021-02-04 2024-01-18 Illumina, Inc. Long indexed-linked read generation on transposome bound beads

Also Published As

Publication number Publication date
CN120666033A (zh) 2025-09-19

Similar Documents

Publication Publication Date Title
AU2022203968B2 (en) Hydrogel Beads For Nucleotide Sequencing
AU2022204100B2 (en) Methods Of Encapsulating Single Cells, The Encapsulated Cells And Uses Thereof
CN113272449B (zh) 调整聚合物小珠以进行dna处理
WO2021046232A1 (fr) Codes-barres lisibles optiquement et systèmes et procédés pour caractériser des interactions moléculaires
US20230049664A1 (en) Droplet microfluidics-based single cell sequencing and applications
CN114096678A (zh) 多种核酸共标记支持物及其制作方法与应用
CN115261389A (zh) 一种功能序列、包含其的标签群以及应用
WO2025247276A1 (fr) Microparticule couplée contenant des amorces de pcr indexées et utilisation associée
CN115478098A (zh) 一种单细胞转录组及染色质可及性双组学测序文库构建方法及测序方法
WO2024077439A1 (fr) Procédé de construction d'une banque de séquençage à cellule unique de transcriptome et d'accessibilité à la chromatine en doubleomique
CN115948518A (zh) 一种用于单细胞检测的编码微珠及其制备方法
CN118389494B (zh) 细胞标签微珠及其制备方法和应用
CN115747301B (zh) 一种测序文库的构建方法、构建测序文库的试剂盒及基因测序方法
AU2020314224B2 (en) Compositions and methods for preparing nucleic acid sequencing libraries using CRISPR/Cas9 immobilized on a solid support
CN118272490A (zh) 用于同时分析单个细胞内基因组和转录组的功能化微球、其制备方法及试剂盒
Alam et al. Microfluidics in Genomics
CN115595356A (zh) 用于分析单个细胞内核酸样本的功能化凝胶微珠及其制备方法
HK40029073A (en) Methods of encapsulating single cells, the encapsulated cells and uses thereof
WO2023114190A1 (fr) Établissement de profils épigénomiques unicellulaires à l'aide de la fluidique et des hydrogels
CN120945011A (zh) 一种分池式超高通量单细胞空间染色质可及性分析的建库方法及其应用
HK40019988A (en) Hydrogel beads for nucleotide sequencing
HK40019988B (en) Hydrogel beads for nucleotide sequencing