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WO2025019701A1 - Procédés et compositions pour résoudre des cloisons à billes multiples - Google Patents

Procédés et compositions pour résoudre des cloisons à billes multiples Download PDF

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Publication number
WO2025019701A1
WO2025019701A1 PCT/US2024/038585 US2024038585W WO2025019701A1 WO 2025019701 A1 WO2025019701 A1 WO 2025019701A1 US 2024038585 W US2024038585 W US 2024038585W WO 2025019701 A1 WO2025019701 A1 WO 2025019701A1
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Prior art keywords
partitions
bead
sequence
detection
partition
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English (en)
Inventor
Duc Do
Zachary Burkett
Chong YIP
Andres REYNOSO
Efren ORTEGA VILLALOBOS
Xiuling CHI
Eden JOHNSON
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Bio Rad Laboratories Inc
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Bio Rad Laboratories Inc
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    • 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
    • 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
    • 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

Definitions

  • the beads for example deliver many copies of the same oligonucleotide to a partition.
  • the oligonucleotide copies linked to a bead will have barcode sequences unique or nearly unique for the bead to which the oligonucleotides are linked.
  • bead concentrations are typically adjusted so that only about 1 out of 10 partitions are occupied by a bead. This results in low utilization of the partitions and increases the amount of sample and reagents that is needed for detection of samples. Increasing bead concentrations would result in higher partition occupancy and greater utilization of partitions.
  • the method comprises providing a plurality of partitions, wherein different partitions comprise (i) a different dsDNA detection oligonucleotide, wherein the detection oligonucleotide comprises 5’-3’: a promoter, a partition barcode sequence specific for the partition in which the detection oligonucleotide resides, and a poly A 3’ sequence; (ii) an RNA polymerase that recognizes the promoter; (iii) a reverse transcriptase; (iv) a single lysed cell or single lysed nucleus and cellular RNA; and (v) a bead linked to multiple copies of a barcoding oligonucleotide comprising 5’-3’: a first universal adapter sequence, a bead-specific barcode sequence and a poly T 3’ end sequence; wherein at least one partition contains (a) a first bead linked to a first barcoding oligonucleotide comprising a first bead
  • the methods further comprise in the partitions, releasing the barcoding oligonucleotides from the beads; in the partitions performing reverse transcription, wherein the performing comprises: (i) annealing some of the detection RNA poly A tail sequences to some copies of the barcoding oligonucleotides and forming first strand detection cDNAs by extending the barcoding oligonucleotides with the reverse transcriptase using the detection RNAs as a template, wherein the first strand detection cDNAs comprise a bead-specific barcode sequence and a partition barcode sequence, wherein in the at least one partition (a) a first first strand cDNA in the partition comprises the first bead-specific barcode sequence and a specific partition barcode sequence and (b) a second first strand cDNA in the partition comprises the second bead-specific barcode sequence and the specific partition barcode sequence, and (ii) annealing cellular RNAs to some copies of the barcoding oli
  • the different partitions comprise two or more different dsDNA detection oligonucleotides having different partition barcode sequences specific for the partition in which the detection oligonucleotide resides.
  • the detection oligonucleotide comprises a sequencing adapter sequence between the promoter and the barcode sequence.
  • the providing comprises providing partitions comprising intact cells and subsequently lysing the cells in the partitions.
  • the intact cells are fixed cells.
  • the providing comprises providing partitions comprising isolated cell nuclei comprising cellular RNA and subsequently lysing the nuclei in the partitions.
  • the method further comprises size selecting nucleic acids in the bulk mixture to separate detection cDNAs from cellular cDNAs.
  • the nucleotide sequence after generating the bulk mixture and before determining the nucleotide sequence: further comprising generating random breaks in the double-stranded cellular cDNAs and randomly inserting at the breaks adaptor oligonucleotides, thereby forming cellular cDNA fragments comprising at least one 5’ end linked to an adaptor oligonucleotide, wherein the adaptor oligonucleotide comprises a second universal sequence, wherein some of the cellular cDNA fragments also comprise a bead bead-specific barcode sequence and the first universal sequence.
  • the random breaks are induced by a transposase that inserts the adaptor oligonucleotides.
  • the dsDNA detection oligonucleotide further comprises a second universal adaptor sequence 5’ of the partition barcode sequence and wherein the double-stranded detection cDNAs comprise the first universal adaptor sequence and the second universal adaptor sequence
  • the method further comprises amplifying the double- stranded detection cDNAs and the cellular cDNA fragments that comprise the first universal adaptor sequence and the second universal adaptor sequence with a DNA polymerase and a first primer that anneal to the first universal adaptor sequence and a second primer that anneals to the second universal primer sequence.
  • the promoter is a T7 or SP6 promoter and the RNA polymerase is T7 or SP6 RNA polymerase, respectively.
  • the partitions are droplets in an emulsion or microwells.
  • the cell is a mammalian cell.
  • the bead is a hydrogel bead.
  • generating the plurality of detection RNAs occurs at a temperature of between 35-50 degrees Celsius; performing the reverse transcription occurs at a temperature of between 37-55 degrees Celsius; and/or performing the second strand synthesis occurs at a temperature of between 60-70 degrees Celsius.
  • partitions comprise (i) a different dsDNA detection oligonucleotide, wherein the detection oligonucleotide comprises 5’-3’: a promoter, a partition barcode sequence specific for the partition in which the detection oligonucleotide resides, and a poly A 3’ sequence; (ii) an RNA polymerase that recognizes the promoter; (iii) a reverse transcriptase; (iv) a lysed cell and cellular RNA; and (v) a bead linked to multiple copies of a barcoding oligonucleotide comprising 5’-3’: a first universal adapter sequence, a bead-specific barcode sequence and a poly T 3’ end sequence; wherein at least one partition contains (a) a first bead linked to a first barcoding oligonucleotide comprising a first bead-
  • the different partitions comprise two or more different dsDNA detection oligonucleotides having different partition barcode sequences specific for the partition in which the detection oligonucleotide resides.
  • the detection oligonucleotide comprises a sequencing adapter sequence between the promoter and the barcode sequence.
  • the promoter is a T7 or SP6 promoter and the RNA polymerase is T7 or SP6 RNA polymerase, respectively.
  • the partitions are droplets in an emulsion or microwells.
  • the cell is a mammalian cell.
  • the bead is a hydrogel bead.
  • FIG.1 depicts barcode deconvolution as described herein.
  • CBC refers to bead- specific barcode sequences.
  • DO ID refers to detection oligonucleotide ID, also referred herein as the partition barcode.
  • R1 and R2 refer to universal sequences that can be used for example as “PCR handles” to amplify the nucleic acids resulting from the steps depicted.
  • cDNAs formed by extending the bead-specific oligonucleotide using the detection RNAs as a template are depicted at the bottom of the Figure and show how various combinations can occur and be interpretated to deconvolute later sequencing reads.
  • the bottom of the figure depicts hypothetical products from the method described herein.
  • Bottom right products represent cellular cDNAs formed in partitions from cellular RNA and a barcoding oligonucleotide that was delivered to the partition linked to a bead.
  • Different cellular cDNAs have different bead-specific barcodes (CBC1, CBC2, CBC3, CBC4, and CBC5 referring to bead-specific barcodes having different sequences).
  • FIG.2A and 2D depict an exemplary workflow for performing the methods described herein.
  • FIG.2B-2C depicts reactions within partitions. In an initial step the partitions are exposed to conditions to allow for in vitro transcription to form detection RNAs from detection oligonucleotides in the partitions. Optionally simultaneously, cellular RNAs are released into the partition, for example by cell lysis.
  • Barcoding oligonucleotides linked to beads in the partitions can also be released from the beads.
  • reverse transcription is performed to generate first strand cDNAs from (i) the detection RNAs primed from the barcoding oligonucleotides and (ii) the cellular RNAs primed from the barcoding oligonucleotides.
  • second strand synthesis is performed.
  • FIG.2D depicts optional sample preparation reactions following combination of contents of the partitions into a “bulk” reaction, allowing for subsequent generation of sequencing reads.
  • detection cDNAs can be separated from cellular cDNAs, for example based on nucleic acid length as the detection cDNAs will in generally be shorter than cellular cDNAs, and already of a length useful for sequencing.
  • cellular cDNAs are contacted with a tagmentase that fragments the cellular cDNAs to generate fragments of appropriate size for sequencing.
  • fragmentation results in fragments comprising the ends of cellular cDNAs that include the bead-specific barcode and universal or other sequences introduced in the RT reaction by the bead-specific barcode.
  • FIG.3 depicts exemplary alternative options for configurations of the detection oligonucleotide. As can be seen in the figure, in some embodiments, the barcode sequence is discontinuous, separated by constant sequences.
  • FIG.4A-B depicts data regarding bead merging as discussed in the Example.
  • FIG.5 depicts data for bead distribution as discussed in the Example.
  • amplification reaction refers to any in vitro method for multiplying the copies of a target sequence of nucleic acid in a linear or exponential manner.
  • RNA transcription-based amplification reactions e.g., amplification that involves T7, T3, or SP6 primed RNA polymerization
  • TAS transcription amplification system
  • NASBA nucleic acid sequence based amplification
  • 3SR self-sustained sequence replication
  • SPIA single-primer isothermal amplification
  • LAMP loop mediated isothermal amplification
  • SDA strand displacement amplification
  • MDA multiple displacement amplification
  • RCA rolling circle amplification
  • “Amplifying” refers to a step of submitting a solution to conditions sufficient to allow for amplification of a polynucleotide if all of the components of the reaction are intact.
  • Components of an amplification reaction include, e.g., primers, a polynucleotide template, polymerase, nucleotides, and the like.
  • the term “amplifying” typically refers to an “exponential” increase in target nucleic acid.
  • PCR Polymerase chain reaction
  • PCR refers to a method whereby a specific segment or subsequence of a target double-stranded DNA, is amplified in a geometric progression. PCR is well known to those of skill in the art; see, e.g., U.S. Pat. Nos.4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds, 1990. Exemplary PCR reaction conditions typically comprise either two or three step cycles.
  • a “primer” refers to a polynucleotide sequence that hybridizes to a sequence on a target nucleic acid and optionally serves as a point of initiation of nucleic acid synthesis. Primers can be of a variety of lengths. In some embodiments, a primer is less than 100 or 50 nucleotides in length, e.g., from about 10 to about 900, from about 15 to about 80, or from about 30-85 to about 30 nucleotides in length.
  • primers for use in an amplification reaction can be designed based on principles known to those of skill in the art; see, e.g., PCR Protocols: A Guide to Methods and Applications, Innis et al., eds, 1990.
  • the primer can include or be completely formed from DNA, RNA or non-natural nucleotides.
  • a primer comprises one or more modified and/or non- natural nucleotide bases.
  • a primer comprises a label (e.g., a detectable label).
  • a nucleic acid, or portion thereof “hybridizes” to another nucleic acid under conditions such that non-specific hybridization is minimal at a defined temperature in a physiological buffer.
  • a nucleic acid, or portion thereof hybridizes to a conserved sequence shared among a group of target nucleic acids.
  • a primer, or portion thereof can hybridize to a primer binding site if there are at least about 6, 8, 10, 12, 14, 16, or 18 contiguous complementary nucleotides, including “universal” nucleotides that are complementary to more than one nucleotide partner.
  • a primer, or portion thereof can hybridize to a primer binding site if there are fewer than 1 or 2 complementarity mismatches over at least about 12, 14, 16, or 18 contiguous complementary nucleotides.
  • the defined temperature at which specific hybridization occurs is room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is higher than room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is at least about 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, or 80°C.
  • nucleic acid refers to DNA, RNA, single-stranded, double- stranded, or more highly aggregated hybridization motifs, and any chemical modifications thereof.
  • Modifications include, but are not limited to, those providing chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, points of attachment and functionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole.
  • Such modifications include, but are not limited to, peptide nucleic acids (PNAs), phosphodiester group modifications (e.g., phosphorothioates, methylphosphonates), 2’-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases, isocytidine and isoguanidine and the like.
  • Nucleic acids can also include non-natural bases, such as, for example, nitroindole.
  • Modifications can also include 3’ and 5’ modifications including but not limited to capping with a fluorophore (e.g., quantum dot) or another moiety.
  • partitioning or “partitioned” refers to separating a sample into a plurality of portions, or “partitions.” Partitions are generally physical, such that a sample in one partition does not, or does not substantially, mix with a sample in an adjacent partition. Partitions can be solid or fluid. In some embodiments, a partition is a solid partition, e.g., a microchannel or microwell. In some embodiments, a partition is a fluid partition, e.g., a droplet.
  • a fluid partition (e.g., a droplet) is a mixture of immiscible fluids (e.g., water and oil).
  • a fluid partition (e.g., a droplet) is an aqueous droplet that is surrounded by an immiscible carrier fluid (e.g., oil).
  • an immiscible carrier fluid e.g., oil.
  • the array of wells can be configured to facilitate bead capture in at least one of a single-solid support format or optionally in small groups of solid supports.
  • Exemplary microwell arrays and methods of delivery of beads to the microwells and analysis thereof is described in, e.g., PCT/US2021/034152.
  • the term “bead” refers to any solid support that can be in a partition, e.g., a small particle or other solid support.
  • Exemplary beads can include hydrogel beads. In some cases, the hydrogel is in sol form. In some cases, the hydrogel is in gel form. An exemplary hydrogel is an agarose hydrogel. Other hydrogels include, but are not limited to, those described in, e.g., U.S.
  • a “barcode” is a short nucleotide sequence (e.g., at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30 or more nucleotides long) that identifies a molecule to which it is conjugated. In some embodiments, a barcode is used to identify molecules in a partition.
  • partition-specific barcode (or multiple different partition-specific barcodes) can be unique for that partition as compared to barcodes present in other partitions.
  • partitions containing target RNA from single cells can be subjected to reverse transcription conditions using primers that contain a different partition-specific barcode sequence in different (e.g., each) partition, incorporating a copy of a unique “cellular barcode” into the reverse transcribed nucleic acids of each partition.
  • nucleic acid from each cell can be distinguished from nucleic acid of other cells due to the unique “cellular barcode.”
  • a barcode is present on oligonucleotides conjugated to a particle, wherein the “particle barcode” or “bead barcode” is shared by (e.g., identical or substantially identical amongst) all, or substantially all, of the oligonucleotides conjugated to that particle or bead.
  • the barcode is discontinuous. See, e.g., FIG.3.
  • “Clonal” copies of a polynucleotide means the copies are identical in sequence.
  • oligonucleotides there are at least 100, 1000, 10 4 or more clonal copies of oligonucleotides in linked to a bead.
  • deconvolution refers to the assignment of 2 or more barcodes (and optionally the beads to which they were attached) as being from the same partition or originally occupying the same partition. Deconvolution can be determined as described herein by the detection of the same detection oligonucleotide barcode associated with two different bead-specific barcodes during sequencing. DETAILED DESCRIPTION OF THE INVENTION [0045] The inventors have discovered that in-partition transcription and reverse transcription can be used to generate a plurality of copies of a partition-specific barcode.
  • Linkage of the partition-specific barcodes with bead-specific barcodes delivered to partitions linked to beads can be detected using sequencing to determine whether and which bead- specific barcodes originated in the same partition. Because bead-specific barcodes are also linked to sample nucleic acids in partitions, one can use whether bead-specific barcodes where linked to partition-specific barcodes to tell whether multiple bead-specific barcodes were present in a single partition, and thus sample nucleic acid sequences having such bead- specific barcodes (linked to the same partition-specific barcode) are identified as being from the same partition in spite of a portion of the sequences being linked to different bead- specific barcodes.
  • the presence of more than one differently-bead barcoded oligonucleotide primer can interfere with sequence analysis and quantification because different barcodes are assumed to be from different partitions when in fact some fraction of the barcodes occur together (for example as a function of a Poisson distribution).
  • deconvolute i.e., determine that multiple bead barcodes are from the same partition and account for that in the sequencing analysis
  • disregard e.g., discard
  • partitions comprising different double-stranded (ds) DNA detection oligonucleotides.
  • partitions can be provided such that different partitions contain different dsDNA detection oligonucleotides that differ at least by having different partition barcode sequences.
  • the dsDNA detection oligonucleotides comprise 5’-3’: a promoter operably linked to a sequence comprising a partition barcode sequence specific for the partition in which the detection oligonucleotide resides and optionally a poly A 3’ sequence.
  • the partitions further comprise an RNA polymerase that can recognizes the promoter and that generates RNA transcripts comprising reverse complements of the partition barcode sequences.
  • the partitions can also contain a reverse transcriptase and one or more bead linked to a barcoding oligonucleotide as a primer comprising a bead-specific barcode sequence such that the RNA transcripts can be converted to cDNAs comprising the partition barcode sequences and the bead-specific barcode sequence, thereby providing a plurality of copies of the partition barcode sequences in the partition itself.
  • cellular RNA from a biological sample
  • a barcoding oligonucleotide as a primer
  • the resulting cDNAs comprise a bead-specific barcode sequence.
  • the various reagents described, as provided in partitions can be present when the partitions are formed or can be added to partitions after the partitions are formed. While one can inject reagents into droplets for example, it may be more convenient when the partitions are droplets to have the reagents at a desired concentration in a bulk solution and then form droplets from the bulk solution.
  • reagents such as buffers, nucleotides, and enzymes should be provided so most or all partitions receive a sufficient concentration to allow for the reactions described herein, e.g., transcription and reverse transcription.
  • the number or concentration of DNA detection oligonucleotides can be selected such that a majority (e.g., at least 60, 70, 80, 90% or more) of the partitions contain at least one DNA detection oligonucleotide. Due to Poisson distribution, the average number of detection oligonucleotides per partition will be more than one copy per partition to avoid an excess of partitions lacking a detection oligonucleotide.
  • the number or concentration of detection oligonucleotides is selected such that the average number of detection oligonucleotides in a partition is for example between 1-100, e.g., 5-50, 10-40, 10- 30, e.g., 25 or a different range or value therein.
  • Each of the different detection oligonucleotides in a partition can have a different sequence, for example differing by their barcode sequence.
  • the barcode sequence for each will be unique or substantially unique to the partition in which the detection oligonucleotide resides, but there can be different barcodes within a particular partition wherein each is different from each other and unique to the partition, i.e., the barcode sequence(s) do not occur in other partitions.
  • the detection oligonucleotides are composed of DNA (e.g., dsDNA) or a nucleotide analog that can be recognized by the RNA polymerase to form RNA transcripts.
  • the detection oligonucleotide will at least have a promoter operably linked a sequence comprising a partition barcode sequence.
  • a sequencing adapter sequence can occur between the promoter and the barcode sequence, allowing for the user to better identify the start position of the barcode sequence.
  • the sequencing adapter sequence comprises a phase-shift region, for example as described in PCT/US2017/013205.
  • the partition barcode sequence length and complexity can be selected based on the number of unique sequences desired. For example, for a given number of partitions desired, one could select an excess number (e.g., 2X, 5X, 10X, 20X, etc.) of unique detection oligonucleotides.
  • the barcode sequence is 15-30, e.g., 18-22, e.g., 19, 20 or 21 nucleotides long and can be continuous or discontinuous (e.g., composed of two or more segments).
  • the detection oligonucleotides are between 150-200 nucleotides long.
  • the barcode sequence can be discontinuous, for example two or more sub-sequences separated by a constant sequence.
  • the detection oligonucleotides also comprise a poly thymine (poly-T) sequence at the 3’ end of the detection oligonucleotides.
  • Poly-T sequence are single stranded sequences of deoxythymine (dT).
  • the length of the poly-T sequence can vary, for example from 6 bases to 30 bases and may be a mixture of poly-T sequences with different lengths.
  • the promoter in the detection oligonucleotides can be any promoter recognized by the RNA polymerase to be used in the partitions.
  • RNA polymerases can include but are not limited to the T7 polymerase (see, e.g., Cheetham, G. M., et al. Nature 399, 80–83 (1999) and the SP6 polymerase (see, e.g., Stump et al., Nucleic Acids Res.21(23): 5480– 5484 (1993).
  • Promoter sequences recognized by the T7 polymerase include but not limited to a sequence comprising TAATACGACTCACTATAG.
  • Promoter sequences recognized by the SP6 polymerase include but not limited to a sequence comprising ATTTAGGTGACACTATAGAAGNG.
  • In vitro transcription can be initiated in the partitions with the RNA polymerase using the detection oligonucleotides as a template(s).
  • Any desired in vitro transcription conditions can be selected that are compatible with the stability of the partitions and allow for activity by the RNA polymerase.
  • Exemplary conditions can include, for example, performing in vitro transcription at 37 degrees C or for example between 35-39 degrees C with the appropriate free ribonucleotides to form RNA transcripts.
  • the in vitro transcription generates detection RNAs in the partitions comprising a reverse complement of the partition barcode sequence and a poly A tail sequence.
  • partitions may contain more than one detection oligonucleotide (indeed, in some embodiments, the partitions on average contain more than one detection oligonucleotide as discussed herein), detection oligonucleotides between partitions will differ thereby allowing the partition barcode(s) in a droplet to be unique for that partition and thus the resulting detection RNAs are also unique for the partition in which they are produced. [0053] After, or simultaneous with, the production of the detection RNAs, reverse transcription can be performed in the partitions.
  • reverse transcription can occur in the partition to generate first strand cDNAs based on the (i) detection RNAs and (ii) sample mRNAs as templates.
  • the reverse transcriptase has an optimal temperature that is higher than the RNA polymerase.
  • the partitions are incubated at a lower temperature (e.g., 37 degrees C) to generate the detection RNAs and then the partitions are incubated at a higher temperature (e.g., 50 degrees C) to generate first strand cDNAs from the detection RNAs and sample mRNAs as templates.
  • Reverse transcription is an amplification method that copies RNA into DNA.
  • a variety of reagents can be included in the partitions for the reverse transcription reaction. RT reactions can be performed with reaction mixtures as desired. Components and conditions for RT reactions are generally known.
  • Reverse transcriptases (RTs) can be selected based on the conditions of the reaction and user preference.
  • the reverse transcriptase is an MMLV RT or a variant thereof (for example but not limited to variants with improved thermostability compared to native MMLV RT). See, e.g., WO2018200867, Arezi et al., Nucleic Acids Res.37(2): 473–481 (2009); Baba et al., Protein Engineering, Design and Selection, Volume 30, Issue 8, August 2017, Pages 551–557.
  • Partitions will comprise cellular RNA from single cells or isolated nuclei.
  • the partitions are formed with cells or nuclei are a sufficiently low concentration such that a majority of the partitions containing a cell or nuclei contain only one (a single) cell or nuclei.
  • the cells are fixed cells.
  • the cells are formalin-fixed, paraffin-embedded (FFPE) samples.
  • the cells are not fixed.
  • the cells are permeabilized to allow for entry of reagents while the cells themselves remain substantially intact.
  • the cells or nuclei are lysed to release cellular RNA in cells or nuclei into the partitions, allowing for the RNA to take part in the reverse transcription reaction.
  • the cells or nuclei in the partitions have been permeabilized such that the RNA in the permeabilized cell or nuclei can diffuse from the cells or nuclei into the remaining inside portion of the partitions.
  • Permeabilization can remove cellular membrane lipids to allow large molecules such as enzymes to enter the cell.
  • a detergent is used for permeabilization.
  • Exemplary detergents can include, for example, Triton X-100 and NP-40 are used for permeabilization (for example, at 0.1–0.5% (v/v, in PBS).
  • a steroidal saponin or saraponin
  • An exemplary saraponin is Digitonin.
  • the appropriate permeabilization reagent can be selected to be compatible with the integrity of the partition used.
  • the cells or isolated nuclei are lysed so that their contents, including RNA, are released into the partition in which the cell or nucleus is present.
  • Any type of cells can be used according to the methods and compositions described herein.
  • the cells are mammalian, for example human cells.
  • the cells are from a biological sample. Biological samples can be obtained from any biological organism, e.g., an animal, plant, fungus, pathogen (e.g., bacteria or virus), or any other organism.
  • the biological sample is from an animal, e.g., a mammal (e.g., a human or a non-human primate, a cow, horse, pig, sheep, cat, dog, mouse, or rat), a bird (e.g., chicken), or a fish.
  • a mammal e.g., a human or a non-human primate, a cow, horse, pig, sheep, cat, dog, mouse, or rat
  • a bird e.g., chicken
  • a biological sample can be any tissue or bodily fluid obtained from the biological organism, e.g., blood, a blood fraction, or a blood product (e.g., serum, plasma, platelets, red blood cells, and the like), sputum or saliva, tissue (e.g., kidney, lung, liver, heart, brain, nervous tissue, thyroid, eye, skeletal muscle, cartilage, or bone tissue); cultured cells, e.g., primary cultures, explants, and transformed cells, stem cells, or cells found in stool, urine, etc.
  • tissue e.g., kidney, lung, liver, heart, brain, nervous tissue, thyroid, eye, skeletal muscle, cartilage, or bone tissue
  • cultured cells e.g., primary cultures, explants, and transformed cells, stem cells, or cells found in stool, urine, etc.
  • the plurality of partitions can be, for example, a plurality of emulsion droplets, or a plurality of microwells, etc.
  • one or more reagents are added during droplet formation or to the droplets after the droplets are formed.
  • Methods and compositions for delivering reagents to one or more partitions include microfluidic methods as known in the art; droplet or microcapsule combining, coalescing, fusing, bursting, or degrading (e.g., as described in U.S.2015/0027,892; US 2014/0227,684; WO 2012/149,042; and WO 2014/028,537); droplet injection methods (e.g., as described in WO 2010/151,776); and combinations thereof.
  • the partitions can be picowells, nanowells, or microwells.
  • the partitions can be pico-, nano-, or micro- reaction chambers, such as pico, nano, or microcapsules.
  • the partitions can be pico-, nano-, or micro- channels.
  • the partitions can be droplets, e.g., emulsion droplets.
  • a droplet comprises an emulsion composition, i.e., a mixture of immiscible fluids (e.g., water and oil).
  • a droplet is an aqueous droplet that is surrounded by an immiscible carrier fluid (e.g., oil).
  • a droplet is an oil droplet that is surrounded by an immiscible carrier fluid (e.g., an aqueous solution).
  • the droplets described herein are relatively stable and have minimal coalescence between two or more droplets.
  • emulsions can also have limited flocculation, a process by which the dispersed phase comes out of suspension in flakes. In some cases, such stability or minimal coalescence is maintained for up to 4, 6, 8, 10, 12, 24, or 48 hours or more (e.g., at room temperature, or at about 0, 2, 4, 6, 8, 10, or 12 °C).
  • the droplet is formed by flowing an oil phase through an aqueous sample or reagents.
  • the oil phase of an emulsion can comprise a fluorinated base oil which can additionally be stabilized by combination with a fluorinated surfactant such as a perfluorinated polyether.
  • a fluorinated surfactant such as a perfluorinated polyether.
  • the sample is partitioned into, or into at least, 500 partitions, 1000 partitions, 2000 partitions, 3000 partitions, 4000 partitions, 5000 partitions, 6000 partitions, 7000 partitions, 8000 partitions, 10,000 partitions, 15,000 partitions, 20,000 partitions, 30,000 partitions, 40,000 partitions, 50,000 partitions, 60,000 partitions, 70,000 partitions, 80,000 partitions, 90,000 partitions, 100,000 partitions, 200,000 partitions, 300,000 partitions, 400,000 partitions, 500,000 partitions, 600,000 partitions, 700,000 partitions, 800,000 partitions, 900,000 partitions, 1,000,000 partitions, 2,000,000 partitions, 3,000,000 partitions, 4,000,000 partitions, 5,000,000 partitions, 10,000,000 partitions, 20,000,000 partitions, 30,000,000 partitions, 40,000,000 partitions, 50,000,000 partitions, 60,000,000 partitions, 70,000,000 partitions, 80,000,000 partitions, 90,000,000 partitions, 100,000,000 partitions, 150,000,000 partitions, or 200,000,000 partitions.
  • the droplets that are generated are substantially uniform in shape and/or size.
  • the droplets are substantially uniform in average diameter.
  • the droplets that are generated have an average diameter of about 0.001 microns, about 0.005 microns, about 0.01 microns, about 0.05 microns, about 0.1 microns, about 0.5 microns, about 1 microns, about 5 microns, about 10 microns, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns, about 70 microns, about 80 microns, about 90 microns, about 100 microns, about 150 microns, about 200 microns, about 300 microns, about 400 microns, about 500 microns, about 600 microns, about 700 microns, about 800 microns, about 900 microns, or about 1000 microns.
  • the droplets that are generated have an average diameter of less than about 1000 microns, less than about 900 microns, less than about 800 microns, less than about 700 microns, less than about 600 microns, less than about 500 microns, less than about 400 microns, less than about 300 microns, less than about 200 microns, less than about 100 microns, less than about 50 microns, or less than about 25 microns.
  • the droplets that are generated are non-uniform in shape and/or size. [0063] In some embodiments, the droplets that are generated are substantially uniform in volume.
  • the standard deviation of droplet volume can be less than about 1 picoliter, 5 picoliters, 10 picoliters, 100 picoliters, 1 nL, or less than about 10 nL. In some cases, the standard deviation of droplet volume can be less than about 10-25% of the average droplet volume.
  • the droplets that are generated have an average volume of about 0.001 nL, about 0.005 nL, about 0.01 nL, about 0.02 nL, about 0.03 nL, about 0.04 nL, about 0.05 nL, about 0.06 nL, about 0.07 nL, about 0.08 nL, about 0.09 nL, about 0.1 nL, about 0.2 nL, about 0.3 nL, about 0.4 nL, about 0.5 nL, about 0.6 nL, about 0.7 nL, about 0.8 nL, about 0.9 nL, about 1 nL, about 1.5 nL, about 2 nL, about 2.5 nL, about 3 nL, about 3.5 nL, about 4 nL, about 4.5 nL, about 5 nL, about 5.5 nL, about 6 nL, about 6.5 nL, about 7 nL, about 7.5 nL, about 8 nL, about
  • one or more beads linked to clonal copies of a barcoding oligonucleotides are present when the partitions are formed or are added to the partitions.
  • the barcoding oligonucleotides can comprise, for example, 5’-3’: a first universal adapter sequence, a bead-specific barcode sequence and a poly T 3’ end sequence.
  • the barcoding oligonucleotides are used as RT primers by annealing to RNA in a partition to form cDNAs that include the bead-specific barcode sequence.
  • UMIs unique molecule identifiers
  • sample-specific barcodes can also be included in the oligonucleotide sequence.
  • Any bead of useful size and composition for delivery to partitions can be used.
  • the particle or bead can be any particle or bead having a solid support surface.
  • Solid supports suitable for particles include controlled pore glass (CPG)(available from Glen Research, Sterling, Va.), oxalyl-controlled pore glass (See, e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527), TentaGel Support—an aminopolyethyleneglycol derivatized support (See, e.g., Wright, et al., Tetrahedron Letters 1993, 34, 3373), polystyrene, Poros (a copolymer of polystyrene/divinylbenzene), or reversibly cross-linked acrylamide. Many other solid supports are commercially availa—e and amenable to the methods described herein.
  • CPG controlled pore glass
  • oxalyl-controlled pore glass See, e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527
  • TentaGel Support an aminopolyethyleneglycol derivatized support (
  • the bead material is a polystyrene resin or poly(methyl methacrylate) (PMMA).
  • the bead material can be metal.
  • the particle or bead comprises hydrogel or another similar composition.
  • the hydrogel is in sol form.
  • the hydrogel is in gel form.
  • An exemplary hydrogel is an agarose hydrogel.
  • Other hydrogels include, but are not limited to, those described in, e.g., U.S. Patent Nos.4,438,258; 6,534,083; 8,008,476; 8,329,763; U.S. Patent Appl. Nos.20020009591; 20130022569; 20130034592; and International Patent Publication Nos.
  • compositions and methods for making and using hydrogels include those described in, e.g., Klein et al., Cell, 2015 May 21;161(5):1187-201.
  • the solid support surface of the bead can be modified to include a linker for attaching barcode oligonucleotides.
  • the linkers may comprise a cleavable moiety, which may be cleaved before reverse transcription is performed in the partitions.
  • Non-limiting examples of cleavable moieties include a disulfide bond, a dioxyuridine moiety, and a restriction enzyme recognition site.
  • the cleavable sequence can be any cleavable sequence that can be targeted enzymatically or otherwise while leaving the rest of the nucleic sequences in the mixture intact.
  • the cleavable sequence comprises one or more uracils.
  • the cleavable sequence can include 1, 2, 3, 4 or more uracils, which can be contiguous. Uracils can be selectively removed and the backbone cleaved (nicked) by contacting with uracil DNA glycosylase and endonuclease VIII, which excises the one or more uracil.
  • the cleavable sequence comprises one or more ribonucleotide(s).
  • the cleavable sequence can include 1, 2, 3, 4 or more ribonucleotides, which can be contiguous. This allows one to use an enzyme that selectively cleaves ribonucleotides and does not substantially cleave deoxribonucleotides.
  • RNAseH is used to specifically cleave at a ribonucleotide in the cleavable sequence.
  • a restriction enzyme is selected such that its recognition and/or cleavage site only occurs in the cleavable sequence and does not occur elsewhere in the oligonucleotides in the mixture.
  • Oligonucleotides can be linked to beads as desired. Methods of linking oligonucleotides to beads are described in, e.g., WO 2015/200541.
  • the oligonucleotide configured to link a hydrogel bead to the barcode is covalently linked to the hydrogel. Numerous methods for covalently linking an oligonucleotide to one or more hydrogel matrices are known in the art.
  • aldehyde derivatized agarose can be covalently linked to a 5’-amine group of a synthetic oligonucleotide.
  • First strand cDNA synthesis is performed as described herein followed by second strand synthesis in the partitions to form double-stranded cDNAs, i.e., using first strand detection cDNAs and first strand cellular cDNAs, respectively, as templates, thereby forming double-stranded detection cDNAs and double-stranded cellular cDNAs.
  • the enzymes in the bulk solution can be inactivated after second strand synthesis, for example by applying heat to the partitions.
  • the inactivating comprises incubating the partitions at 75-90 degrees Celsius.
  • some partitions will not contain any beads, some partitions will contain one bead, some two beads, and some more than two beads.
  • partitions that contain two or more beads and associated barcoding oligonucleotides will comprise: (i)(a) a first cDNA in the partition comprises the first bead-specific barcode sequence and a specific partition barcode sequence (formed by extending a first barcoding oligonucleotide from a first bead in the partition with the reverse transcriptase using the detection RNAs as a template) and (i)(b) a second cDNA in the partition comprises the second bead-specific barcode sequence and the specific partition barcode sequence (formed by extending a second barcoding oligonucleotide from a second bead in the partition with the reverse transcriptase using the detection RNAs as a template), and (ii) cellular cDNAs (formed by extending the first and/or second barcoding oligonucleotides with the reverse transcriptase using the cellular RNAs as a template).
  • the above pattern of bead-specific and partition barcode sequences can then be used to identify those partitions having more than one bead-specific barcode. While the example above provides for two bead-specific barcodes in a partition, the same approach can be used for a higher number of beads in the partition (e.g., 3, 4, 5, or more), for example using different bead-specific barcodes associated with the same partition barcode sequence to determine that those different bead-specific barcodes resided in the same partition. See, e.g., FIG.1. [0072] Once the cDNAs are linked to bead-specific barcode sequences, the contents of the partitions can be combined into a bulk solution.
  • the droplets are burst such that their contents are combined in form a bulk aqueous solution.
  • the fragments in the resulting bulk solution can be nucleotide sequenced as desired. In some embodiments, a certain size of fragments is desired for the sequencing reaction.
  • the detection cDNAs are sufficiently short to be processed for sequencing directly, whereas it can be desirable to fragment the cellular cDNAs, which are longer than the detection cDNAs.
  • SPRI solid-phase reversible immobilization
  • FIG.2D depicts embodiments in which tagmentation is used to fragment the cellular cDNAs and add adapter sequences to the ends of the fragments. As shown in FIG.2D, some of the fragments will include the end of the cellular cDNA having at one end the bead-specific barcode and universal sequence introduced by the barcoding oligonucleotide and an adapter sequence on the fragment’s other end introduced by the tagmentase.
  • the universal sequence introduced from the barcoding oligonucleotide and the adapter sequence introduced by the tagmentase is subsequently used to amplify the desired fragments. See, e.g., FIG.2D, bottom panel.
  • the number of cycles of amplification can be determined by the user depending on the amount of amplified product desired for sequencing.
  • the action of some transposases sometimes referred to as “tagmentation” and can involve introduction of different adapter sequences on different sides of a DNA breakage point or the adapter sequences added can be identical. In either case, the adapter sequences are common adapter sequences in that the adapter sequences are the same across a diversity of DNA fragments.
  • a tagmentase is an enzyme that is capable of forming a functional complex with a transposon end-containing composition and catalyzing insertion or transposition of the transposon end-containing composition into the double-stranded target DNA with which it is incubated in an in vitro transposition reaction.
  • Exemplary transposases include but are not limited to modified Tn5 transposases that are hyperactive compared to wildtype Tn5, for example can have one or more mutations selected from E54K, M56A, or L372P.
  • Wild-type Tn5 transposon is a composite transposon in which two near-identical insertion sequences (IS50L and IS50R) are flanking three antibiotic resistance genes (Reznikoff WS. Annu Rev Genet 42: 269–286 (2008)).
  • Each IS50 contains two inverted 19-bp end sequences (ESs), an outside end (OE) and an inside end (IE).
  • ESs inverted 19-bp end sequences
  • OE outside end
  • IE inside end
  • wild-type ESs have a relatively low activity and were replaced in vitro by hyperactive mosaic end (ME) sequences.
  • a complex of the transposase with the 19-bp ME is thus all that is necessary for transposition to occur, provided that the intervening DNA is long enough to bring two of these sequences close together to form an active Tn5 transposase homodimer (Reznikoff WS., Mol Microbiol 47: 1199–1206 (2003)).
  • Transposition is a very infrequent event in vivo, and hyperactive mutants were historically derived by introducing three missense mutations in the 476 residues of the Tn5 protein (E54K, M56A, L372P), which is encoded by IS50R (Goryshin IY, Reznikoff WS.1998. J Biol Chem 273: 7367–7374 (1998)).
  • Transposition works through a “cut-and- paste” mechanism, where the Tn5 excises itself from the donor DNA and inserts into a target sequence, creating a 9-bp duplication of the target (Schaller H. Cold Spring Harb Symp Quant Biol 43: 401–408 (1979); Reznikoff WS., Annu Rev Genet 42: 269–286 (2008)).
  • current commercial solutions NexteraTM DNA kits, Illumina
  • free synthetic ME adapters are end-joined to the 5′-end of the target DNA by the transposase (tagmentase).
  • Sequencing platforms can be selected as desired.
  • IlluminaTM-supported sequencing methods are employed. See, e.g., U.S.
  • Exemplary DNA sequencing techniques include fluorescence-based sequencing methodologies (See, e.g., Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y.; herein incorporated by reference in its entirety).
  • automated sequencing techniques understood in that art are utilized.
  • the present technology provides parallel sequencing of partitioned amplicons (PCT Publication No. WO 2006/0841,32, herein incorporated by reference in its entirety).
  • DNA sequencing is achieved by parallel oligonucleotide extension (See, e.g., U.S. Pat. Nos.
  • EXAMPLE [0078] This example describes the production of barcode oligonucleotide chimeras in single-cell 3’ RNA library preparation workflow.
  • Cell preparation HEK293 cells (ATCC, CRL-1573) and NIH3T3 cells (ATCC, CRL-1658) were used for ddSEQ reagents setup. Cells were prepared according to Illumina SureCell WTA 3’ Reference Guide. HEK293 and NIH3T3 cells were mixed in a 1:1 ratio for the experiment. Droplet generation and incubation.
  • the Bead suspension mix includes Tris buffer, salts, detergents, density gradient media, T7 RNA polymerase, and 5950 barcoded beads/ ⁇ l.
  • the Cell suspension mix contains Tris buffer, salts, detergent, density gradient media, NTPs, dNTPs, enzyme mix, detection oligos, and 400 cells/ ⁇ l mixed HEK293/NIH3T3 cells. Assembled bead and cell suspension mixes were loaded and run in the ddSEQ Single-Cell Isolator as instructed in Illumina SureCell WTA 3’ Reference Guide.
  • the droplet emulsions were transferred to a Bio-Rad’s ddPCR 96-well plate, sealed with an 8-cap strip, and incubated as follows: 37°C for 60 minutes, 50°C for 15 minutes, 70°C for 45 minutes, 80°C for 5 minutes, and hold at 4°C. Library preparation. Droplet contents were released by adding 40 ⁇ l droplet disruptor to each sample. Cell cDNA was cleaned up and separated from detection cDNA was by performing two rounds of 0.68X ampure XP. Detection cDNA was extracted from the supernatant of the first ampure round and purified by double-sided size selection. The purified cell cDNA was then tagmented according to protocol in the Illumina SureCell WTA 3’ Reference Guide.
  • the index PCR reaction consisted of Kapa HiFi HotStart Ready Mix (Roche, PN 9420398001), custom P5-Read 1 primer, and Nextera P7 index primer.
  • the reactions were thermal cycled in Bio-Rad C1000 thermal cycler using separate protocols for cell cDNA and detection cDNA.
  • Thermal profile for cell cDNA 95°C for 3 min, 13 cycles of 98°C for 20 s, 60°C for 45 s, 72°C for 60 s, and 72°C for 5 min in the last cycle, hold at 4°C.
  • Thermal profile for detection cDNA 95°C for 3 min, 15 cycles of 98°C for 20 s, 60°C for 45 s, 72°C for 30 s, and 72°C for 5 min in the last cycle, hold at 4°C.
  • Final DNA libraries were cleaned up using two rounds of 0.6X ampure XP for the cell library and two rounds of 0.8X ampure XP for the DO library. Purified final DNA libraries were QC’ed on an Agilent Bioanalyzer before sequencing. Sequencing and analysis. The DNA libraries were sequenced in the Illumina Nextseq 500/550 system using the high output 150 cycles kit. Sequencing was set up for paired-end reads with 54 cycles for Read 1, 75 cycles for Read 2, and 8 cycles for index 1.
  • FIG.4A showed 4242 cells recovered from an initial 8000 cell input (400 cells/ ⁇ l x 20 ⁇ l). The 53% yield is consistent with the expected 64% cell recovery based on double poisson calculation of bead 1.19 ⁇ and cell 0.08 ⁇ . The lower observed yield can be explained by loss in dead volume and during droplet transfer.
  • FIG.4B displayed 4.01% crosstalk between HEK293 and NIH3T3 cells and matched with the 4.1% expected crosstalk at 8000 cell input.

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Abstract

L'invention concerne des procédés et des compositions pour déconvoluer des cloisons comprenant de multiples codes-barres spécifiques de billes. Les procédés peuvent impliquer par exemple la transcription in vitro d'oligonucléotides de détection à l'intérieur de cloisons, qui peuvent être liées à des codes-barres spécifiques de billes et séquencées.
PCT/US2024/038585 2023-07-19 2024-07-18 Procédés et compositions pour résoudre des cloisons à billes multiples Pending WO2025019701A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015200541A1 (fr) * 2014-06-24 2015-12-30 Bio-Rad Laboratories, Inc. "barcoding" par pcr numérique
WO2018148700A1 (fr) * 2017-02-13 2018-08-16 Yale University Polyomique monocellulaire à haut débit
WO2020247950A1 (fr) * 2019-06-07 2020-12-10 Bio-Rad Laboratories, Inc. Résolution de multiples particules par gouttelette

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015200541A1 (fr) * 2014-06-24 2015-12-30 Bio-Rad Laboratories, Inc. "barcoding" par pcr numérique
WO2018148700A1 (fr) * 2017-02-13 2018-08-16 Yale University Polyomique monocellulaire à haut débit
WO2020247950A1 (fr) * 2019-06-07 2020-12-10 Bio-Rad Laboratories, Inc. Résolution de multiples particules par gouttelette

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