Methods and compositions for combinatorial barcoding

Patent metadata

Field	Value
Publication number	US-11535882-B2
Application number	US-201615084307-A
Country	US
Kind code	B2
Filing date	Mar 29, 2016
Priority date	Mar 30, 2015
Publication date	Dec 27, 2022
Grant date	Dec 27, 2022

Abstract

The present disclosure provide compositions, methods and kits for generating a set of combinatorial barcodes, and uses thereof for barcoding samples such as single cells or genomic DNA fragments. Some embodiments disclosed herein provide compositions comprising a set of component barcodes for producing a set of combinatorial barcodes. The set of component barcodes can comprise, for example, n×m unique component barcodes, wherein n and m are integers, each of the component barcodes comprises: one of n unique barcode subunit sequences; and one or two linker sequences or the complements thereof, wherein the component barcodes are configured to connect to each other through the one or two linker sequences or the complements thereof to produce a set of combinatorial barcodes.

First claim

What is claimed is:

1. A composition comprising a set of component barcodes for producing a set of combinatorial barcodes, comprising:
n×m unique component barcodes, wherein n and m are positive integers, each of the component barcodes comprises:
one of n unique barcode subunit sequences; and
one or two linker sequences or the complements thereof,

wherein the component barcodes are configured to connect to each other through the one or two linker sequences or the complements thereof to produce a set of combinatorial barcodes; and
a plurality of solid supports,
wherein a plurality of the component barcodes are attached to a solid support, wherein each of the component barcodes attached to the solid support comprise a first label, wherein component barcodes attached to the same solid support comprise the same first label, and wherein component barcodes attached to different solid supports comprise different first labels, and
wherein each one of the set of combinatorial barcodes comprises:
a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide is attached to a solid support, wherein the second oligonucleotide is not attached to a solid support, wherein each of the first oligonucleotide and the second oligonucleotide comprises a target-specific region and at least one of n unique barcode subunit sequences, wherein the target-specific region of the first oligonucleotide is configured to bind the target, wherein the target-specific region of the second oligonucleotide is configured to bind a reverse complement of the target, and wherein only the target-specific region of the first oligonucleotide is oligo-dT, wherein the target comprises messenger RNA (mRNA).

2. The composition of claim 1 , wherein each one of the set of n unique barcode subunit sequences comprises a molecular label, a cellular label, a dimensional label, a universal label, or any combination thereof.

3. The composition of claim 1 , wherein the total number of different linker sequences is m−1 or m−2.

4. The composition of claim 1 , wherein each of the component barcodes comprises:
a) barcode subunit sequence-linker;
b) complement of linker-barcode subunit sequence-linker; or
c) complement of linker-barcode subunit sequence.

5. The composition of claim 1 , wherein:
the first oligonucleotide comprises the formula: [first barcode subunit sequence]-[first linker]-[second barcode subunit sequence]-[second linker]- . . . -[third barcode subunit sequence]; and
the second oligonucleotide comprises the formula: [fourth barcode subunit sequence]- . . . -[third linker]-[fifth barcode subunit sequence]-[fourth linker]-[sixth barcode subunit sequence].

6. The composition of claim 1 , wherein each one of the one or two linker sequences is different.

7. The composition of claim 1 , wherein the set of combinatorial barcodes has at least 100,000 unique combinatorial barcodes.

8. The composition of claim 1 , wherein the set of combinatorial barcodes has at least 1,000,000 unique combinatorial barcodes.

9. The composition of claim 1 , wherein the solid support comprises a bead.

10. The composition of claim 9 , wherein the bead is a sepharose bead, a streptavidin bead, an agarose bead, a magnetic bead, a conjugated bead, a hydrogel bead, a protein A conjugated bead, a protein G conjugated bead, a protein A/G conjugated bead a protein L conjugated bead an oligo(dT) conjugated bead a silica bead a silica-like bead, an anti-biotin microbead, an anti-fluorochrome microbead, or any combination thereof.

11. The composition of claim 1 , wherein each one of the set of n unique barcode subunit sequences comprises a molecular label sequence, and the set of combinatorial barcodes comprise at least 10,000 unique molecular label sequences.

12. The composition of claim 1 , wherein m is an integer from 2 to 10.

13. The composition of claim 1 , wherein n is an integer from 4 to 100.

14. The composition of claim 1 , wherein the n unique barcode subunit sequences are the same length.

15. The composition of claim 1 , wherein at least two of the n unique barcode subunit sequences have different lengths.

16. The composition of claim 1 , wherein the linker sequences are the same length.

17. The composition of claim 1 , wherein the linker sequences have different lengths.

18. The composition of claim 1 , wherein the set of combinatorial barcodes has equal to or less than nm unique combinatorial barcodes.

Assignees

• Cellular Res Inc
• Becton Dickinson Co

Inventors

• Fu Glenn
• Fodor Stephen P A

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6837

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C12Q2563/185

  Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title