WO2025207588A2 - Séquençage unicellulaire à l'aide de multiples adaptateurs de transposase - Google Patents

Séquençage unicellulaire à l'aide de multiples adaptateurs de transposase

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Publication number
WO2025207588A2
WO2025207588A2 PCT/US2025/021282 US2025021282W WO2025207588A2 WO 2025207588 A2 WO2025207588 A2 WO 2025207588A2 US 2025021282 W US2025021282 W US 2025021282W WO 2025207588 A2 WO2025207588 A2 WO 2025207588A2
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sequence
antisense
sequences
strand
oligonucleotides
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WO2025207588A3 (fr
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Qiang Zhang
Man CHENG
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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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • transcript isoforms can create phenotypic variations within a population by modulating gene function.
  • the method comprises, providing fixed and permeabilized cells; diffusing reverse transcriptase and nucleotides and a primer into the fixed and permeabilized cells and performing reverse transcription of RNA in the cells to form RNA/first strand cDNA hybrids; diffusing into the fixed and permeabilized cells a set of at least three (e.g., 3-20, at least 3, 4, 5, 6, 7, 8, 9, 10, etc.) transposases, each of the at least three transposases carrying different adaptor oligonucleotides, that introduce breaks in the RNA/first strand cDNA hybrids to form RNA/first strand cDNA hybrid fragments and inserts homoadaptor oligonucleotides at the breaks, wherein the adaptor oligonucleotides comprise a first adaptor strand comprising, 5' to 3’: an Rn sequence, wherein Rn sequences differ for the adaptor oligonucleotides of each of the at least three transposases, optionally, one
  • the method further comprises displacing the second adaptor strand and (i) extending 3’ ends of the first strand cDNAs with a polymerase using the RNA and first adaptor strand linked thereto as a template to form first strand cDNA fragments linked to a 5’ first Rn sequence (Rl) and a 3’ antisense second Rn sequence (R2) or (ii) extending 3’ ends of the RNAs with a polymerase that initiates from RNA using the first strand cDNAs and first adaptor strand linked thereto as a template to form RNA fragments linked to a 5’ first Rn sequence (Rl) and a 3’ antisense second Rn sequence (R2) and then performing a second reverse transcription to form the first strand cDNA fragments linked to a 5‘ first Rn sequence (Rl) and a 3’ antisense second Rn sequence (R2);
  • the method further comprises forming partitions comprising (i) single permeabihzed cells comprising the first strand cDNA fragments and (ii) a bead linked to a plurality of clonal barcoding oligonucleotides having free 3’ ends, the barcoding oligonucleotides comprising a first 5’ PCR handle sequence, a 3’ capture sequence and a barcode sequence unique to the bead to which the barcode oligonucleotide is linked, wherein the plurality of clonal barcoding oligonucleotides have different 3 ?
  • the method further comprises, in the partitions, annealing a second of the clonal barcoding oligonucleotide 3’ capture sequences to the antisense of the first Rn sequence on the adaptor-linked intermediate second strand cDNAs and extending the second of the clonal barcoding oligonucleotides using the adaptor-linked intermediate second strand cDNAs as a template with the DNA-dependent DNA polymerase to form doublebarcoded first strand cDNA fragments that comprise 5 ’-3’: the first 5’ PCR handle sequence, the barcode sequence unique to the bead, the first Rn sequence, optionally the one or more optional barcode sequences, the ME sequence, the first strand cDNA fragment, and an antisense of the ME sequence, an antisense of the first 5’ PCR handle sequence, optionally an antisense of the one or more optional barcode sequences, and the antisense of the second Rn sequence;
  • the method further comprises extending the adaptor-linked intermediate second strand cDNAs using the double-barcoded first strand cDNAs as a template to form double-stranded double-barcoded cDNA fragments.
  • the method comprises, providing fixed and permeabilized cells; diffusing reverse transcriptase and nucleotides and a pnmer into the fixed and permeabilized cells and performing reverse transcription of RNA in the cells to form RNA/first strand cDNA hybrids; diffusing into the fixed and permeabilized cells a set of at least three (e.g., 3-20, at least 3, 4, 5, 6, 7, 8, 9, 10.
  • transposases each of the at least three transposases carrying different adaptor oligonucleotides, that introduce breaks in the RNA/first strand cDNA hybrids to form RNA/first strand cDNA hybrid fragments and inserts homoadaptor oligonucleotides at the breaks, wherein the adaptor oligonucleotides comprise a first adaptor strand comprising, 5’ to 3': an Rn sequence, wherein Rn sequences differ for the adaptor oligonucleotides of each of the at least three transposases, optionally, one or more barcode sequences and a mosaic end (ME) sequence, and a second adaptor strand comprising an antisense ME sequence, wherein one of the transposases covalently links 3 ?
  • the adaptor oligonucleotides comprise a first adaptor strand comprising, 5’ to 3': an Rn sequence, wherein Rn sequences differ for the adaptor oligonucleotides of each of the at least
  • the method comprises, providing fixed and permeabilized cells; diffusing reverse transcriptase and nucleotides and a primer into the fixed and permeabilized cells and performing reverse transcription of RNA in the cells to form RNA/first strand cDNA hybrids; diffusing into the fixed and permeabilized cells a set of at least three (e.g., 3-20, at least 3, 4.
  • each of the at least three transposases carrying different adaptor oligonucleotides, that introduce breaks in the RNA/first strand cDNA hybrids to form RNA/first strand cDNA hybrid fragments and inserts homoadaptor oligonucleotides at the breaks, wherein the adaptor oligonucleotides comprise a first adaptor strand comprising, 5’ to 3’: an Rn sequence, wherein Rn sequences differ for the adaptor oligonucleotides of each of the at least three transposases, optionally, one or more barcode sequences and a mosaic end (ME) sequence, and a second adaptor strand comprising an antisense ME sequence, wherein one of the transposases covalently links 3 ?
  • the adaptor oligonucleotides comprise a first adaptor strand comprising, 5’ to 3’: an Rn sequence, wherein Rn sequences differ for the adaptor oligonucleotides of each of the at least three transposa
  • first strand cDNAs with a polymerase using the RNA and first adaptor strand linked thereto as a template to form first strand cDNA fragments linked to a 5’ first Rn sequence (Rl) and a 3’ antisense second Rn sequence (R2) or (ii) extending 3’ ends of the RNAs with a polymerase that initiates from RNA using the first strand cDNAs and first adaptor strand linked thereto as a template to form RNA fragments linked to a 5’ first Rn sequence (Rl) and a 3’ antisense second Rn sequence (R2) and then performing a second reverse transcription to form the first strand cDNA fragments linked to a 5’ first Rn sequence (Rl) and a 3’ antisense second Rn sequence (R2); forming partitions comprising (i) single permeabilized cells comprising the first strand cDNA fragments and (ii) a bead linked to a plurality of clonal barcoding oli
  • the plurality of different bridging oligonucleotides include end sequences that anneal to or comprise all of the different Rn sequences on the transposases; optionally releasing the clonal barcoding oligonucleotides in the partitions; and in the partitions, linking barcoding oligonucleotides to both ends of the first strand cDNA fragments linked to a 5’ first Rn sequence (Rl) and a 3’ antisense second Rn sequence (R2) via annealing and polymerase extension using the bridging oligonucleotides to form doublestranded double-barcoded cDNA fragments that comprise 5 ’-3’: the first 5’ PCR handle sequence, the barcode sequence unique to the bead, the first Rn sequence, optionally the one or more optional barcode sequences, the ME sequence, the first strand cDNA fragment, and an antisense of the ME sequence, an antisense of the first 5’
  • the displacing and extending is performed with a blend of polymerases, wherein the blend has RNA- and DNA-dependent DNA polymerase activity.
  • the adaptor oligonucleotides comprise one or more barcodes.
  • the one or more barcodes comprises a unique molecular identifier (UMI) barcode.
  • the one or more barcodes comprises a sample barcode.
  • the partitions are droplets, impermeable capsules or semi- permeable capsules or microwells.
  • partitions comprise,
  • the method comprises, providing fixed and permeabilized cells; performing reverse transcription in the cells to form RNA/first strand cDNA hybrids; diffusing into the fixed and permeabilized cells a set of at least three different transposases each of the at least three transposases earning adaptor oligonucleotides that comprise a mosaic end (ME) sequence and different Rn sequences to form RNA/first strand cDNA hybrid fragments having 5’ overhangs; extending 3’ ends of the first strand cDNAs with a polymerase using the RNA and first adaptor strand linked thereto as a template to form first strand cDNA fragments linked to a 5 ’ first Rn sequence and a 3 ?
  • MME mosaic end
  • partitions comprising (i) single permeabilized cells comprising the first strand cDNA fragments and (ii) a bead linked to a plurality of clonal barcoding oligonucleotides having free 3’ ends, the barcoding oligonucleotides comprising a first 5’ PCR handle sequence, a 3’ capture sequence and a barcode sequence unique to the bead to which the barcode oligonucleotide is linked, wherein the plurality of clonal barcoding oligonucleotides have different 3’ capture sequences such that the plurality includes all of the different Rn sequences on the transposases; optionally releasing the clonal barcoding oligonucleotides in the partitions; in the partitions forming double-stranded double-barcoded cDNA fragments that comprise 5'- 3‘: the first 5‘ PCR handle sequence, the barcode sequence unique to the bead, a first Rn sequence,
  • the polymerase is Bst3.0, Bst2.0, Superscript II, or Superscript III.
  • the displacing and extending is performed with a blend of polymerases, wherein the blend has RNA- and DNA-dependent DNA polymerase activity.
  • the extending with the DNA-dependent DNA polymerase occurs at a higher temperature than the displacing and annealing.
  • the adaptor oligonucleotides comprise one or more barcodes.
  • the one or more barcodes comprises a unique molecular identifier (UMI) barcode.
  • the one or more barcodes comprises a sample barcode.
  • permeabilized cells are provided.
  • the cells can be permeabilized to allow for entry of reagents while the cells themselves remain substantially intact.
  • Permeabilization can remove cellular membrane lipids to allow molecules such as enzymes to enter the cell while substantially retaining RNA (e.g., >100 or 500 nucleotides long).
  • a detergent is used for permeabilization.
  • Exemplary detergents can include, for example, Tween-20, Triton X-100, and NP-40 are used for permeabilization (for example, at 0. 1-0.5% (v/v, in PBS).
  • Suitable reverse transcriptases can include but are not limited to Maxima RNAse+ (Thermo), Maxima RNAse- (Thermo), murine leukemia virus (MLV) reverse transcriptase (Gerard and Grandgenett, Journal of Virology 15:785-797, 1975; Verma, Journal of Virology’ 15:843-854, 1975) or feline leukemia virus (FLV) reverse transcriptase (Rho and Gallo, Cancer Lett., 10:207-221, 1980 or SEQ ID NO: 1, bovine leukemia virus (BLV) (Demirhan et al., Anticancer Res., 16:2501-5, 1996; Drescher et al., Arch Geschwulstforsch., 49:569-79, 1979), Avian Myeloblastosis Virus (AMV) reverse transcriptase, Respiratory Syncytial Virus (RSV) reverse transcriptase, Equine Infectious Anemia Virus (AMV)
  • tagmentation results in fragmentation of polynucleotides and addition of oligonucleotides on the ends of the resulting fragments.
  • a transposase, carrying two oligonucleotides, that introduces breaks in cDNA/RNA hybrids and introduces oligonucleotides into the break sites is referred to as a “tagmentase” and the action of the tagmentase is referred to as “tagmentation” and can involve introduction of different adaptor sequences on different sides of a DNA breakage point or the adaptor sequences added by a transposases can be identical.
  • RNA/DNA hybrids are described in, e.g., Bo LuLiting et al., eLife 9:e54919 (2020).
  • 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.
  • tagmentases As described herein, at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10, e.g., 3-10, 3-20, etc.) different tagmentases are used to cleave the cDNA/RNA hybrids in the permeabilized cells. See, e.g., FIG. 1.
  • “Different tagmentases” means for the purposes of this disclosure that the tagmentases (i. e. , copies of the same enzyme) carry different oligonucleotides differing at least by their 5" end Rn sequences. This is shown schematically in the top right of FIG. 1, showing the result of random fragmentation with different tagmentases carrying 9 different adaptor oligonucleotides.
  • Adaptor oligonucleotides delivered by the tagmentases can have the following sequence 5'-3’: an Rn sequence, wherein Rn sequences differ for the adaptor oligonucleotides of each of the at least transposases, optionally a spacer sequence, optionally one or more barcode sequences and a mosaic end (ME) sequence (e.g., 5'- CTGTCTCTTATACACATCT-3').
  • Rn sequence will be used as an adaptor sequence for annealing in dow nstream step(s) after the Rn sequence is added as part of the adaptor oligonucleotide to DNA or RNA fragments by the tagmentase.
  • the length and complexity of the Rn sequence can be selected based on various criteria, e.g., such as the number of Rn sequences used.
  • the Rn sequence will in some embodiments be at least 14 nucleotides long, e.g., 14-30 nucleotides long.
  • the adaptor oligonucleotides loaded on the tagmentase will including a doublestranded portion comprising the ME sequence, and thus a short antisense ME sequence is present and is delivered to the fragment but is not covalently linked to the fragment, and instead remains solely by base pairing with the ME sequence, which has been covalently linked to the fragment. See, e.g., FIG. 2.
  • a 9 base pair sequence at the site of cleavage is replaced such that both fragments formed by the tagmentase comprise the same 9 bp sequence. As discussed later, this can be used in sequencing to splice sequencing reads into longer sequences.
  • the adaptor oligonucleotides delivered by the tagmentases will comprise one or more barcode, i.e., between the Rn and ME sequences. See, e.g., FIG.
  • the adaptor oligonucleotides cany' a unique molecular identifier (UMI) barcode sequence allowing for a unique sequence for different oligonucleotides, which as used herein can be used to identify specific molecules to which the UMI are linked.
  • UMI is labeled as “Ui” in the figures.
  • the adaptor oligonucleotides carry a sample barcode sequence allowing for identification of which sample was reverse transcribed and allowing for cDNA fragments from different samples to be combined downstream while allowing for their association with a particular sample or patient.
  • the sample barcode is labeled as “Ti” in the figures.
  • the methods comprise generating an RNA/cDNA hybrid in permeabilized cells and then contacting the RNA/cDNA hybrids with different transposases earn ing three or more different adaptor oligonucleotides, allowing for the fragments to be accessible for any variety' of downstream workflows for ultimate sequencing of the resulting fragments. Exemplary doyvnstream workflows are described below.
  • the tagmented products and the permeabilized cells that contain them can be partitioned to form partitions containing single cells and one or more bead comprising a plurality of clonal barcoding oligonucleotides having free 3’ ends and used subsequently to add a barcode sequence specific for the bead, allowing for partition-specific barcoding.
  • the permeabilized cells can be partitioned such that individual cells are the only cell within a particular partition.
  • Exemplary partitions can include but are not limited to droplets within an emulsion, microwells, capsules, including but not limited to semi-permeable capsules (SPCs). Methods and compositions for partitioning are described, for example, in published patent applications WO 2010/036352, US 2010/0173394, US 2011/0092373, and US 2011/0092376.
  • 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 droplets described herein are relatively stable and have minimal coalescence between two or more droplets.
  • 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.
  • An SPC is a capsule having a semi-permeable shell that allows for small molecules to pass through the shell while substantially retaining larger molecules, such as DNA (e.g., having at least 100 or at least 500 nucleotides), mRNA and optionally some proteins.
  • DNA e.g., having at least 100 or at least 500 nucleotides
  • mRNA e.g., mRNA
  • WO-2023117364 describes SPCs with a semipermeable shells comprising a gel formed from a polyampholyte and/or a polyelectrolyte, wherein the polyampholyte and/or the polyelectrolyte in the gel is covalently cross-linked.
  • plurality of copies of barcoding oligonucleotides linked to a head can be delivered to the partitions (including for example forming the partitions with the beads and the cells), wherein different partitions receive different beads and accompanying barcoding oligonucleotides.
  • the bead can be attached to multiple copies of the same oligonucleotide, for example, at least about 10, 50, 100, 500, 1000, 5000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 10 8 , 10 9 , 10 10 or more copies of the same or substantially identical oligonucleotide can be attached to one (e g., the same) bead.
  • the barcoding oligonucleotides will comprise at least a bead-specific barcode sequence and a 3’ capture sequence for annealing to a target sequence (e.g., an Rn sequence) on the adaptor oligonucleotides or alternatively to a bridging oligonucleotide as described below.
  • the barcoding oligonucleotides further comprise a 5’ PCR handle sequence, allowing for a common 5’ sequence between sequences comprising different cell-specific barcodes, allowing them all to be amplified with a universal primer that anneals to the PCR handle sequence.
  • the oligonucleotides can be cleaved from the bead prior to linking the oligonucleotides to the DNA fragments.
  • the 3’ capture sequence on the barcoding oligonucleotides anneal to a reverse complement (i.e.. antisense) of a Rn sequence as it occurs linked to a first strand cDNA fragment. See, e.g., FIG. 3.
  • the 3’ capture sequence on the barcoding oligonucleotides anneals to a sense Rn sequence.
  • the barcoding oligonucleotides on the bead will comprise a mixture of oligonucleotides having the same barcode sequence, but varying in that different 3‘ capture sequence sequences, that can anneal to different antisense Rn sequences, are present.
  • a uridine incorporated site can be cleaved, for example, using a uracil glycosylase enzyme (e.g., a uracil N-glycosylase enzyme or uracil DNA glycosylase (UDG) enzyme).
  • a uracil glycosylase enzyme e.g., a uracil N-glycosylase enzyme or uracil DNA glycosylase (UDG) enzyme
  • the cleavable linker comprises a photocleavable nucleotide.
  • Photocleavable nucleotides include, for example, photocleavable fluorescent nucleotides and photocleavable biotinylated nucleotides. See, e.g., Li et al., PNAS, 2003, 100:414-419; Luo et al.. Methods Enzymol, 2014, 549: 115-131.
  • 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.
  • CPG controlled pore glass
  • oxalyl-controlled pore glass See, e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527
  • the bead material is a polystyrene resin or poly(methyl methacrylate) (PMMA).
  • PMMA poly(methyl methacrylate)
  • 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.
  • the bridging oligonucleotides can be blocked on their 3' ends such that the bridging oligonucleotides themselves are not extended. See, e.g., FIGs, 8-9.
  • one or both ends of the bridging oligonucleotides, or reverse complements thereof formed by primer extension are ligated to the tagmented and gap-filled cDNA fragments and the barcoding oligonucleotides, optionally before or after a primer extension step.
  • a product can be generated in which a barcoding oligonucleotide has been linked to both ends of the tagmented cDNA fragment.
  • a further amplification can occur for example to add a first PCR handle sequence. See e.g., bottom of FIG. 3. An example of this is shown at the bottom of FIG. 3.
  • first 5’ PCR handle sequence the barcode sequence unique to the bead
  • a first Rn sequence optionally one or more optional barcode sequences e.g., a UMI and/or sample barcode
  • the ME sequence the first strand cDNA fragment
  • an antisense of the ME sequence an antisense of the first 5 ? PCR handle sequence
  • an antisense of the one or more optional barcode sequences optionally an antisense sequence of a second Rn sequence (which is different from the first Rn sequence). See, e.g., bottom of FIG. 3.
  • the contents of partitions can be combined (e.g.. different microwell contents can be mixed, droplets or SPCs can be disrupted, etc.) such that the double-stranded double-barcoded cDNA fragments from different partitions are together in a bulk solution.
  • Reactions can then be performed in the bulk solution as desired to generate sequences that can be used in sequencing reactions, e.g.. next generation sequencing reactions, which can include for example sequencing by synthesis of other methods.
  • primer extension or PCR reactions are performed to add one or more PCR handle sequences to one or both ends of the double-barcoded cDNA fragments.
  • This reaction can form strands comprising 5 ’-3’ the second PCR handle sequence and the ME sequence, a first strand cDNA or second strand cDNA fragment, the antisense of the ME sequence, the antisense of the first 5’ PCR handle sequence, optionally the antisense of the one or more optional barcode sequences, optionally the antisense of the spacer sequence, the antisense of the first or second Rn sequence, and the antisense of the barcode sequence unique to the bead.
  • the primers comprising 5’-3’ a second PCR handle sequence and the ME sequence are used in a higher concentration than that of the underlying template, for example to facilitate annealing of the primers compared to potential competition from self- annealing of the template.
  • Subsequent introduction of a 3’-5 ? exonuclease that digests 3’ singlestranded overhangs but not double-stranded DNA, can be used to remove sequences 3’ of the antisense ME sequence on the double-stranded cDNAs.
  • Exemplary 3’-5’ endonucleases can include, for example, Macosko et al. , RESOURCE ⁇ Vol. 161, Issue 5, P1202-1214, MAY 21, 2015.
  • the result is a singly -barcoded cDNA fragment.
  • different PCR handle sequences e.g., Illumina P5 and P7 sequences
  • primers that are used to amplify the singly-barcoded fragment.
  • Resulting products will be double-stranded DNA molecules comprising the cDNA fragments, a beadspecific barcode and different Rn sequences at either end of the cDNA fragment sequence.
  • the original tagmentase reaction comprises N different Rn sequences
  • the products will be mixtures of double stranded DNA molecules that include different combinations of Rn sequences flanking different cDNA fragment sequences. Two such exemplary sequences are depicted in FIG. 4, but is should be appreciated that many more combinations of Rn sequences can occur as more Rn sequences are used in the tagmentation.
  • Sequencing of the double-stranded DNA molecules can be performed as desired.
  • Methods for high throughput sequencing and genotyping are known in the art.
  • sequencing technologies include, but are not limited to, pyrosequencing, sequencing-by- ligation, single molecule sequencing, sequence-by -synthesis (SBS), massive parallel clonal, massive parallel single molecule SBS, massive parallel single molecule real-time, massive parallel single molecule real-time nanopore technology, etc.
  • SBS sequence-by -synthesis
  • Morozova and Marra provide a review of some such technologies in Genomics, 92: 255 (2008), herein incorporated by reference in its entirety.
  • Exemplary' DNA sequencing techniques include fluorescence-based sequencing methodologies (See, e.g..
  • the present technology 7 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. 5,750,341 ; and 6,306,597, both of which are herein incorporated by reference in their entireties).
  • sequencing techniques include the Church polony technology (Mitra et al., 2003, Analytical Biochemistry 320, 55-65; Shendure et al., 2005 Science 309, 1728-1732; and U.S. Pat. Nos. 6.432,360; 6,485.944; 6,511.803; herein incorporated by reference in their entireties), the 454 picotiter pyrosequencing technology (Margulies et al., 2005 Nature 437, 376-380; U.S. Publication No. 2005/0130173; herein incorporated by reference in their entireties), the Solexa single base addition technology 7 (Bennett et al., 2005, Pharmacogenomics, 6, 373-382; U.S. Pat.
  • Amplification-requiring methods include pyrosequencing commercialized by Roche as the 454 technology 7 platforms (e.g., GS 20 and GS FLX), the Solexa platform commercialized by Illumina, and the Supported Oligonucleotide Ligation and Detection (SOLiD) platform commercialized by Applied Biosystems.
  • Non-amphfication approaches also known as single-molecule sequencing, are exemplified by the HeliScope platform commercialized by Heli cos BioSciences, and platforms commercialized by VisiGen, Oxford Nanopore Technologies Ltd., Life Technologies/Ion Torrent, and Pacific Biosciences, respectively.
  • sequencing reads are generated that comprise the Rn sequences, any barcodes, and the cDNA fragment
  • the different sequences can be aligned as desired to a reference genome or cDNA sequence. This will allow for example one to identify a likely identity of which cDNA the fragment originated from. Fragments from the cDNA should align with the same reference sequence.
  • fragments from the precise original cDNA i.e., the original RNA molecule, can be identified and subsequently stitched together. An example of stitching is shown e.g.. in FIG. 7.
  • Various criteria can be used including one or some or all of:
  • sequence reads on the reference genome The ends should map to the same place in the reference genome if they are from the same cDNA molecule.
  • cDNA fragments linked to the same bead barcode sequence can be expected to have originated from the same cell.
  • cDNA fragments linked to different bead barcodes may be from different cells or may come from the same cell that w s in a partition with more than one bead.
  • cDNA end sequences map less than 10 nucleotides apart on the reference genome. Tagmentation results in insert of a 9 bp repeat on either site of the cleavage site of the tagmentase. resulting in cDNA fragments with the same 9 bp end sequence (one on the 5 ’ end and one on the 3’ end of the two fragments).
  • kits that can be used for performing part or all of the methods described herein.
  • the kit will comprise one or more container for holding various reagents, and optionally instructions.
  • Exemplary components of a kit can include, for example, one or more of cell fixatives, cell permeation reagents, adaptor-loaded transposases (optionally more than one), one or more polymerase, which can include for example a reverse transcriptase and a DNA polymerase, one or more exonuclease, primers for primer extension and/or amplification as described herein, reagents for performing droplet-based amplification (dPCR) and optionally a reaction mixture for performing a second PCR amplification.
  • dPCR droplet-based amplification
  • Other reagents as described herein or as useful for performing the steps described herein can also be included in the kit.
  • mRNA was reverse-transcribed to cDNA. forming cDNA/RNA hybrid structure.
  • the cDNA/RNA hybrid structure was further tagmented by transposases carrying 19 different adaptor oligonucleotides (FIG. 2).
  • the tagmented product was gap-filled by Bst 3.0 (FIG. 2), followed by the 1st PCR using these 19 different adaptors as primers (as shown in FIG. 3 but in a bulk reaction and without CBC).
  • the 1st PCR product was denatured, annealed with primer R2, and extended with Bst 3.0 (steps shown in FIG. 4). Leftover primers and single stranded 3’ overhangs were digested by exonuclease (FIG. 4).
  • Full-length sequencing adapters were added during the 2nd PCR (steps show n in FIG. 5).

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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés et des compositions pour générer des lectures d'ADNc à partir de fragments d'ADNc comprenant une tagmentation avec au moins trois transposases portant différents oligonucléotides homoadaptateurs avec différentes séquences d'extrémité.
PCT/US2025/021282 2024-03-27 2025-03-25 Séquençage unicellulaire à l'aide de multiples adaptateurs de transposase Pending WO2025207588A2 (fr)

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CN120624607A (zh) * 2018-05-08 2025-09-12 深圳华大智造科技股份有限公司 用于准确且经济高效的测序、单体型分型和组装的基于单管珠粒的dna共条形码化
US20210222163A1 (en) * 2018-07-11 2021-07-22 X Gen Us Co. Transposome enabled dna/rna-sequencing (ted rna-seq)
EP4413158A4 (fr) * 2021-10-08 2025-07-09 Bio Rad Laboratories Inc Traitement atacseq à base de billes
CN116949133A (zh) * 2023-06-14 2023-10-27 人科(北京)生物技术有限公司 单细胞转录组建库和测序的方法
WO2025024703A1 (fr) * 2023-07-26 2025-01-30 Bio-Rad Laboratories, Inc. Dnaseq unicellulaire à double tagmentation

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