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NZ760038B2 - High-throughput single-cell sequencing with reduced amplification bias - Google Patents
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NZ760038B2 - High-throughput single-cell sequencing with reduced amplification bias - Google Patents

High-throughput single-cell sequencing with reduced amplification bias Download PDF

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Publication number
NZ760038B2
NZ760038B2 NZ760038A NZ76003819A NZ760038B2 NZ 760038 B2 NZ760038 B2 NZ 760038B2 NZ 760038 A NZ760038 A NZ 760038A NZ 76003819 A NZ76003819 A NZ 76003819A NZ 760038 B2 NZ760038 B2 NZ 760038B2
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NZ
New Zealand
Prior art keywords
nuclei
cells
indexed
nucleic acids
nucleic acid
Prior art date
Application number
NZ760038A
Other versions
NZ760038A (en
Inventor
Jay Shendure
Frank J Steemers
Yi Yin
Original Assignee
Illumina Inc
University Of Washington
Filing date
Publication date
Application filed by Illumina Inc, University Of Washington filed Critical Illumina Inc
Priority claimed from PCT/US2019/032966 external-priority patent/WO2019222688A1/en
Publication of NZ760038A publication Critical patent/NZ760038A/en
Publication of NZ760038B2 publication Critical patent/NZ760038B2/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
    • 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
    • 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
    • C12Q1/6869—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
    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/101—Linear amplification, i.e. non exponential
    • 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
    • C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
    • C12Q2535/122—Massive parallel 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
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/159—Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
    • 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
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/179—Nucleic acid detection characterized by the use of physical, structural and functional properties the label being a nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
    • C40B50/10—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support involving encoding steps

Abstract

Provided herein are methods for preparing a sequencing library that includes nucleic acids from a plurality of single cells. In one embodiment, the methods include linear amplification of the nucleic acids. In one embodiment, the sequencing library includes whole genome nucleic acids from the plurality of single cells. In one embodiment, the nucleic acids include three index sequences. Also provided herein are compositions, such as compositions that include the nucleic acids having three index sequences

Claims (34)

1. A method for preparing a sequencing library comprising nucleic acids from a plurality of single nuclei or cells, the method comprising: providing a plurality of isolated nuclei or cells in a first plurality of compartments, wherein each compartment comprises a subset of isolated nuclei or cells, wherein nuclei or cells comprise nucleic acid fragments; introducing a linear amplification mediator to the cells or nuclei; amplifying the nucleic acid fragments by linear amplification; processing each subset of nuclei or cells to generate indexed nuclei or cells, wherein the processing comprises adding to nucleic acid fragments present in the isolated nuclei or cells a first compartment specific index sequence to result in indexed nucleic acids present in isolated nuclei or cells, wherein the processing comprises ligation, primer extension, hybridization, amplification, or transposition; combining the indexed nuclei or cells to generate pooled indexed nuclei or cells,; distributing subsets of the pooled indexed nuclei or cells into a second plurality of compartments; and introducing a second compartment specific index sequence to indexed nucleic acids to generate dual indexed nuclei or cells comprising dual indexed nucleic acids, wherein the introducing comprises ligation, primer extension, amplification, or transposition thereby producing a sequencing library from the plurality of nuclei or cells.
2. The method of claim 1 wherein the amplifying occurs before the processing.
3. The method of claim 1 wherein the processing occurs before the amplifying.
4. A method for preparing a sequencing library comprising nucleic acids from a plurality of single nuclei or cells, the method comprising: providing a plurality of isolated nuclei or cells, wherein nuclei or cells comprise nucleic acid fragments; introducing a linear amplification mediator to the isolated nuclei or cells; distributing the isolated nuclei or cells into a first plurality of compartments, wherein each compartment comprises a subset of isolated nuclei or cells; amplifying the nucleic acid fragments by linear amplification; processing each subset of isolated nuclei or cells to generate indexed nuclei or cells, wherein the processing comprises adding to nucleic acid fragments present in the isolated nuclei or cells a first compartment specific index sequence to result in indexed nucleic acids present in isolated nuclei or cells, wherein the processing comprises ligation, primer extension, amplification, or transposition; combining the indexed nuclei or cells to generate pooled indexed nuclei or cells, distributing subsets of the pooled indexed nuclei or cells into a second plurality of compartments; and introducing a second compartment specific index sequence to indexed nucleic acids to generate dual-indexed nuclei or cells comprising dual-indexed nucleic acids, wherein the introducing comprises ligation, primer extension, amplification, or transposition, thereby producing a sequencing library from the plurality of nuclei or cells.
5. A method for preparing a sequencing library comprising nucleic acids from a plurality of single nuclei or cells, the method comprising: providing a plurality of isolated nuclei or cells in a first plurality of compartments, wherein each compartment comprises a subset of isolated nuclei or cells, wherein nuclei or cells comprise nucleic acid fragments; processing each subset of nuclei or cells to generate indexed nuclei or cells, wherein the processing comprises adding to nucleic acid fragments present in the isolated nuclei or cells (i) a first compartment specific index sequence to result in indexed nucleic acids present in isolated nuclei or cells and (ii) a nucleotide sequence recognized by a linear amplification mediator, wherein the processing comprises ligation, primer extension, hybridization, amplification, or transposition; introducing a linear amplification mediator to the cells or nuclei; amplifying the nucleic acid fragments by linear amplification; combining the indexed nuclei or cells to generate pooled indexed nuclei or cells, distributing subsets of the pooled indexed nuclei or cells into a second plurality of compartments; and introducing a second compartment specific index sequence to indexed nucleic acids to generate dual-indexed nuclei or cells comprising dual-indexed nucleic acids, wherein the introducing comprises ligation, primer extension, amplification, or transposition, thereby producing a sequencing library from the plurality of nuclei or cells.
6. The method of any one of claims 1, 4, or 5, wherein the linear amplification mediator comprises a phage RNA polymerase or a linear amplification primer.
7. The method of claim 6, wherein the nucleic acid fragments comprise a T7 promoter and the phage RNA polymerase comprises a T7 RNA polymerase.
8. The method of claim 6, wherein introducing the linear amplification mediator comprises adding to nucleic acid fragments present in the isolated nuclei or cells the linear amplification primer.
9. The method of claim 1 or 5, further comprising exposing the plurality of isolated nuclei or cells of each compartment of the first plurality of compartments to a predetermined condition.
10. The method of claim 9, further comprising isolating nuclei from the plurality of cells after the exposing.
11. The method of claim 4, further comprising exposing the plurality of isolated nuclei or cells to a predetermined condition.
12. The method of any one of claims 1, 4, or 5, further comprising subjecting the isolated nuclei to conditions to generate nucleosome-depleted nuclei while maintaining integrity of the isolated nuclei.
13. The method of any one of claims 1, 4, or 5, wherein the processing comprises: contacting each subset with a transposome complex, wherein the transposome complex in each compartment comprises the first index sequence that is different from first index sequences in the other compartments; and fragmenting nucleic acids in the subsets into a plurality of nucleic acids and incorporating the first index sequences into at least one strand of the nucleic acids to generate the indexed nuclei or cells comprising the indexed nucleic acids.
14. The method of any one of claims 1, 4, or 5, wherein the processing comprises: contacting each subset with reverse transcriptase and a primer that anneals to RNA molecules in the isolated nuclei, wherein the primer in each compartment comprises the first index sequence that is different from first index sequences in the other compartments to generate the indexed nuclei or cells comprising the indexed nucleic acids.
15. The method of claim 14, wherein the contacting further comprises a target specific primer that anneals to a specific nucleotide sequence.
16. The method of any one of claims 1, 4, or 5, wherein the processing to add the first compartment specific index sequence comprises a two step process of adding a nucleotide sequence comprising a universal sequence to the nucleic acid fragments and then adding the first compartment specific index sequence to the nucleic acid fragments.
17. The method of claim 16, wherein the adding comprises a transposome complex that comprises the universal sequence.
18. The method of any one of claims 1, 4, or 5, wherein the processing comprises adding a first index to DNA nucleic acids present in the isolated nuclei or cells, a first index to RNA nucleic acids present in the isolated nuclei or cells, or a combination thereof.
19. The method of claim 18, wherein the adding a first index sequence to RNA nucleic acids comprises: contacting each subset with a reverse transcriptase and a primer that anneals to RNA molecules in the isolated nuclei or cells, wherein the primer in each compartment comprises the first compartment specific index sequence to generate the indexed nuclei or cells comprising the indexed nucleic acids.
20. The method of claim 18, wherein the adding a first index sequence to DNA nucleic acids comprises: contacting each subset with a transposome complex, wherein the transposome complex in each compartment comprises the first compartment specific index sequence; and fragmenting nucleic acids in the subsets into a plurality of nucleic acids and incorporating the first compartment specific index sequences into at least one strand of the nucleic acids to generate the indexed nuclei or cells comprising the indexed nucleic acids.
21. The method of claim 19, wherein the first index sequence added to DNA nucleic acids and the first index sequence added to RNA nucleic acids in each compartment are identical.
22. The method of claim 19, wherein the first index sequence added to DNA nucleic acids and the first index sequence added to RNA nucleic acids in each compartment are not identical.
23. The method of any one of claims 1, 4, or 5, further comprising a re-amplification of the nucleic acid fragments, wherein the re-amplification comprises a target specific primer that anneals to a specific nucleotide sequence.
24. The method of any one of claims 1, 4 or 5, further comprising combining the dual-indexed nuclei or cells to generate pooled dual-indexed nuclei or cells, distributing subsets of the pooled dual-indexed nuclei or cells into a third plurality of compartments; and introducing a third compartment specific index sequence to indexed nucleic acids to generate triple-indexed nuclei or cells comprising triple-indexed nucleic acids, wherein the introducing comprises ligation, primer extension, amplification, or transposition.
25. The method of any one of claims 1, 4, or 5, further comprising treating the indexed nuclei or cells for methylation analysis to generate nucleic acid fragments suitable for methylation analysis.
26. The method of any one of claims 1, 4, or 5, further comprising subjecting the indexed nuclei or cells to proximity ligation to generate nucleic acid fragments suitable for analysis of chromatin conformation.
27. The method of any one of claims 1, 4, or 5, further comprising amplifying the nucleic acid fragments of the sequencing library to produce DNA nanoballs.
28. The method of any one of claims 1-27, wherein the compartment of the first plurality of compartments or of the second plurality of compartments comprises a well or a droplet.
29. The method of any one of claims 1-28, wherein each compartment of the first plurality of compartments comprises from 50 to 100,000,000 nuclei or cells.
30. The method of any one of claims 1-28, wherein each compartment of the second plurality of compartments comprises from 50 to 100,000,000 nuclei or cells.
31. The method of any one of claims 1-30, further comprising: providing a surface comprising a plurality of amplification sites, wherein the amplification sites comprise at least two populations of attached single stranded capture oligonucleotides having a free 3’ end, and contacting the surface comprising amplification sites with the indexed nucleic acid fragments under conditions suitable to produce a plurality of amplification sites that each comprise a clonal population of amplicons from an individual fragment comprising a plurality of indexes.
32. A method of preparing a sequencing library comprising nucleic acids from a plurality of single cells, the method comprising: (a) providing isolated nuclei from a plurality of cells; (b) subjecting the isolated nuclei to a chemical treatment to generate nucleosome-depleted nuclei, while maintaining integrity of the isolated nuclei; (c) distributing subsets of the nucleosome-depleted nuclei into a first plurality of compartments and contacting each subset with a transposome complex, wherein the transposome complex in each compartment comprises a transposase and a first index sequence that is different from first index sequences in the other compartments; (d) fragmenting nucleic acids in the subsets of nucleosome-depleted nuclei into a plurality of nucleic acid fragments and incorporating the first index sequences into at least one strand of the nucleic acid fragments to generate indexed nuclei comprising indexed nucleic acid fragments, wherein the indexed nucleic acid fragments remain attached to the transposases; (e) combining the indexed nuclei to generate pooled indexed nuclei; (f) distributing subsets of the pooled indexed nuclei into a second plurality of compartments and contacting each subset with a hairpin ligation duplex under conditions suitable for ligation of the hairpin ligation duplex to one or both ends of indexed nucleic acid fragments to result in dual-indexed nucleic acid fragments, wherein the hairpin ligation duplex comprises a second index sequence that is different from second index sequences in the other compartments; (g) combining the dual-indexed nuclei to generate pooled indexed nuclei; (h) distributing subsets of the pooled dual-indexed nuclei into a third plurality of compartments; (i) lysing the dual-indexed nuclei and amplifying the dual-indexed nucleic acids fragments by linear amplification; (j) processing the dual-indexed nucleic acid fragments to include a third index sequence that is different from third index sequences in the other compartments; and (k) combining the triple-index fragments, thereby producing a sequencing library comprising whole genome nucleic acids from the plurality of single cells.
33. The method of any one of claims 1, 4, or 5, wherein the nucleic acid fragments comprise a phage promoter at one or both ends and wherein the linear amplification mediator comprises a phage RNA polymerase.
34. The method of any one of claims 1, 4 or 5, wherein the linear amplification mediator comprises either one primer or two primers with one in excess.
NZ760038A 2019-05-17 High-throughput single-cell sequencing with reduced amplification bias NZ760038B2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201862673023P 2018-05-17 2018-05-17
US201962821864P 2019-03-21 2019-03-21
PCT/US2019/032966 WO2019222688A1 (en) 2018-05-17 2019-05-17 High-throughput single-cell sequencing with reduced amplification bias

Publications (2)

Publication Number Publication Date
NZ760038A NZ760038A (en) 2024-11-29
NZ760038B2 true NZ760038B2 (en) 2025-03-04

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