MOLECULAR BARCODING ON OPPOSITE TRANSCRIPT ENDS

• US 20190338278A1
• Filed: 05/01/2019
• Published: 11/07/2019
• Est. Priority Date: 05/03/2018
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Shum, Eleen, Fan, Christina
• Granted Patent: US 11,365,409 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Disclosed herein include systems, methods, compositions, and kits for molecular barcoding on the 5′-end of a nucleic acid target. After barcoding a nucleic acid target using an oligonucleotide barcode comprising a target binding region and a molecular label to generate a barcoded nucleic acid molecule, an oligonucleotide comprising a complement of the target binding region can be added to generate a barcoded nucleic acid molecule comprising the target-binding region and the complement of the target-binding region. A stem loop is formed with intra-molecular hybridization of the barcoded nucleic acid molecule, which can be extended to generate an extended barcoded nucleic acid molecule comprising the molecular label and a complement of the molecular label.

Claims

1. A method for analyzing a nucleic acid target in a sample, comprising:

barcoding copies of the nucleic acid target in a sample using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid molecules each comprising a sequence of the nucleic acid target, a molecular label, and a target-binding region, and wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular label sequences;

attaching an oligonucleotide comprising a complement of the target binding region to the plurality of barcoded nucleic acid molecules to generate a plurality of barcoded nucleic acid molecules each comprising the target-binding region and the complement of the target-binding region;

hybridizing the target-binding region and the complement of the target-binding region within each of the plurality of barcoded nucleic acid molecules to form a stem loop; and

extending 3′

-ends of the plurality of barcoded nucleic acid molecules to extend the stem loop to generate a plurality of extended barcoded nucleic acid molecules each comprising the molecular label and a complement of the molecular label.

2. The method of claim 1, further comprising:

determining the number of the nucleic acid target in the sample based on the number of molecular labels with distinct sequences, complements thereof, or a combination thereof, associated with the plurality of extended barcoded nucleic acid molecules.

3. The method of claim 1, comprising:

determining the number of the nucleic acid target in the sample based on the number of complements of molecular labels with distinct sequences associated with the plurality of extended barcoded nucleic acid molecules.

4. The method of claim 1, wherein barcoding the copies of the plurality of targets comprises:

contacting copies of the nucleic acid target to the plurality of oligonucleotide barcodes, wherein each of the plurality of oligonucleotide barcodes comprises the target-binding region capable of hybridizing to the nucleic acid target; and

extending the copies of the nucleic acid target hybridized to the oligonucleotide barcodes to generate the plurality of barcoded nucleic acid molecules.

5. (canceled)

6. The method of claim 1, comprising

amplifying the plurality of barcoded nucleic acid molecules to generate a plurality of amplified barcoded nucleic acid molecules,

wherein attaching the oligonucleotide comprising the complement of the target-binding region comprises:

attaching the oligonucleotide comprising the complement of the target binding region to the plurality of amplified barcoded nucleic molecules to generate the plurality of barcoded nucleic acid molecules each comprising the target-binding region and a complement of the target-binding region.

7. The method of claim 2,

comprising amplifying the plurality of extended barcoded nucleic acid molecules to generate a plurality of single-labeled nucleic acid molecules each comprising the complement of the molecular label,

wherein determining the number of the nucleic acid target in the sample comprises:

determining the number of the nucleic acid target in the sample based on the number of complements of molecular labels with distinct sequences associated with the plurality of single-labeled nucleic acid molecules.

8. The method of claim 2,

comprising amplifying the plurality of extended barcoded nucleic acid molecules to generate copies of the plurality of extended barcoded nucleic acid molecules,

wherein determining the number of the nucleic acid target in the sample comprises:

determining the number of the nucleic acid target in the sample based on the number of complements of molecular labels with distinct sequences associated with the copies of plurality of extended barcoded nucleic acid molecules.

9. A method for determining the numbers of a nucleic acid target in a sample, comprising:

contacting copies of a nucleic acid target in a sample to a plurality of oligonucleotide barcodes, wherein each of the plurality of oligonucleotide barcodes comprises a molecular label and a target-binding region capable of hybridizing to the nucleic acid target, and wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular label sequences;

extending the copies of the nucleic acid target hybridized to the oligonucleotide barcodes to generate a plurality of nucleic acid molecules each comprising a sequence complementary to at least a portion of the nucleic acid target;

amplifying the plurality of barcoded nucleic acid molecules to generate a plurality of amplified barcoded nucleic acid molecules;

attaching an oligonucleotide comprising the complement of the target-binding region to the plurality of amplified barcoded nucleic acid molecules to generate a plurality of barcoded nucleic acid molecules each comprising the target-binding region and a complement of the target-binding region;

hybridizing the target-binding region and the complement of the target-binding region within each of the plurality of barcoded nucleic acid molecules to form a stem loop;

extending 3′

-ends of the plurality of barcoded nucleic acid molecules to extend the stem loop to generate a plurality of extended barcoded nucleic acid molecules each comprising the molecular label and a complement of the molecular label;

amplifying the plurality of extended barcoded nucleic acid molecules to generate a plurality of single-labeled nucleic acid molecules each comprising the complement of the molecular label; and

determining the number of the nucleic acid target in the sample based on the number of complements of molecular labels with distinct sequences associated with the plurality of single-labeled nucleic acid molecules.

10. The method of claim 9, wherein the molecular label is hybridized to the complement of the molecular label after extending the 3′

-ends of the plurality of barcoded nucleic acid molecules, the method comprising denaturing the plurality of extended barcoded nucleic acid molecules prior to amplifying the plurality of extended barcoded nucleic acid molecules to generate the plurality of single-labeled nucleic acid molecules.

11. The method of claim 9,

wherein contacting copies of the nucleic acid target in the sample comprises contacting copies of a plurality of nucleic acid targets to a plurality of oligonucleotide barcodes,

wherein extending the copies of the nucleic acid target comprises extending the copies of the plurality nucleic acid targets hybridized to the oligonucleotide barcodes to generate a plurality of barcoded nucleic acid molecules each comprising a sequence complementary to at least a portion of one of the plurality of nucleic acid targets, and

wherein determining the number of the nucleic acid target comprises determining the number of each of the plurality of nucleic acid targets in the sample based on the number of the complements of the molecular labels with distinct sequences associated with single-labeled nucleic acid molecules of the plurality of single-labeled nucleic acid molecules comprising a sequence of the each of the plurality of nucleic acid targets

12-15. -15. (canceled)

16. The method of claim 9,

wherein the target-binding region comprises a poly(dT) sequence, and

wherein attaching the oligonucleotide comprising the complement of the target binding region comprises:

adding a plurality of adenosine monophosphates to the plurality of barcoded nucleic acid molecules using a terminal deoxynucleotidyl transferase.

17. The method of claim 9, wherein extending the copies of the nucleic acid target hybridized to the oligonucleotide barcodes comprises (1) reverse transcribing the copies of the nucleic acid target hybridized to the oligonucleotide barcodes to generate a plurality of barcoded complementary deoxyribonucleic acid (cDNA) molecules, (2) extending the copies of the nucleic acid target hybridized to the oligonucleotide barcodes using a DNA polymerase lacking at least one of 5′

to 3′

exonuclease activity and 3′

to 5′

exonuclease activity, or both.

18-19. -19. (canceled)

20. The method of claim 9, comprising obtaining sequence information of the plurality of extended barcoded nucleic acid molecules.

21-29. -29. (canceled)

30. The method of claim 9, wherein the nucleic acid target comprises a cellular component binding reagent, or the nucleic acid molecule is associated with the cellular component binding reagent.

31-33. -33. (canceled)

34. The method of claim 9, wherein the oligonucleotide barcode comprises an identical sample label, and/or an identical cell label.

35-37. -37. (canceled)

38. The method of claim 9, wherein at least one of the plurality of barcoded nucleic acid molecules is associated with a solid support when hybridizing the target-binding region and the complement of the target-binding region within each of the plurality of barcoded nucleic acid molecules to form the stem loop.

39. The method of claim 9, wherein at least one of the plurality of barcoded nucleic acid molecules dissociates from a solid support when hybridizing the target-binding region and the complement of the target-binding region within each of the plurality of barcoded nucleic acid molecules to form the stem loop.

40-43. -43. (canceled)

44. The method of claim 39, wherein the solid support comprises a synthetic particle.

45-49. -49. (canceled)

50. The method of claim 9, wherein the sample comprises a single cell, the method comprising associating a synthetic particle comprising the plurality of the oligonucleotide barcodes with the single cell in the sample.

51-61. -61. (canceled)

62. The method of claim 50, wherein the synthetic particle comprises a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof.

63-65. -65. (canceled)

66. A kit comprising:

a plurality of oligonucleotide barcodes, wherein each of the plurality of oligonucleotide barcodes comprises a molecular label and a target-binding region, and wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular label sequences;

a terminal deoxynucleotidyl transferase or a ligase; and

a DNA polymerase lacking at least one of 5′

to 3′

exonuclease activity and 3′

to 5′

exonuclease activity.

67. The kit of claim 66, further comprising one or more of (1) an plurality of oligonucleotides comprising a complement of the target-binding region, (2) one or more reagents for a reverse transcription reaction, and (3) one or more reagents for an amplification reaction.

68-77. -77. (canceled)

78. The kit of claim 66, wherein at least one of the plurality of oligonucleotide barcodes is immobilized on or enclosed in a synthetic particle.

79-84. -84. (canceled)

85. The kit of claim 66, wherein the synthetic particle comprises a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof.

86-88. -88. (canceled)