Granted Patent B2

US 12,241,059 · App. 17/376,709 · Granted Mar 4, 2025

Inventors: Robert Meltzer (Belmont, MA); Kristina Fontanez (Arlington, MA); Yi Xue (Shrewsbury, MA)

Assignee: Illumina, Inc.

C12N15/1065C12Q1/6853

Abstract

Claims (34)

1. A method for creating a target capture reagent, the method comprising:

dividing a plurality of beads linked to initial oligos into a first set of partitions;

ligating partition-specific first duplexes comprising first barcodes to the initial oligos to form ligation products;

pooling the ligation products into a pool;

splitting the pool into a second set of partitions;

ligating partition-specific second duplexes comprising second barcodes to the ligation products to form multi-part oligonucleotides each comprising (i) one of the initial oligos, (ii) one of the first barcodes, and (iii) one of the second barcodes;

repeating the pooling, splitting, and ligating steps at least twice with additional sets of partition-specific duplexes and barcodes, wherein each successive repetition comprises a number of partition-specific duplexes and barcodes that is one greater than the immediate prior round of pooling, splitting, and ligating, and wherein a penultimate repeating step comprises a partition-specific duplex having two overhangs on the same strand.

2. The method of claim 1 , wherein the beads comprise hydrogels.

3. The method of claim 2 , wherein the beads comprise agar, glass, polyacrylamide, polystyrene, or polyethylene.

4. The method of claim 2 , wherein the initial oligos are linked to the beads by acrydite linkages, click chemistry, biotin/streptavidin, silane linkage, or amide linkages.

5. The method of claim 2 , wherein the method provides a plurality of beads, each linked to a plurality of copies of one of the multi-part oligonucleotides, wherein the multi-part oligonucleotides have been covalently synthesized on the beads using ligase and without using polymerase.

6. The method of claim 1 , wherein the first set of partitions are wells within a multi-well plate.

7. The method of claim 6 , wherein the second set of partitions are wells in a second well plate that each include copies of one of the first duplexes that hybridize to ends of the initial oligos.

8. The method of claim 1 , wherein the first set of partitions and the second set of partitions are each independently selected from the group consisting of droplets of an emulsion and wells in one or more multi-well plates.

9. The method of claim 1 , wherein the first set of partitions comprise wells in a multi-well plate and the second set of partitions comprise droplets of an emulsion and the splitting step comprises forming the emulsion in the wells.

10. The method of claim 1 , wherein the multi-part oligonucleotides further include one or more of:

a sequencing adaptor;

an amplification primer binding site;

a restriction enzyme recognition site;

a G/C clamp;

a unique molecular identifier; and

a priming sequence that hybridizes to RNA.

11. The method of claim 1 , wherein the initial oligos are linked to beads and the beads include one or more reagents for cell lysis or reverse transcription.

12. The method of claim 1 , wherein the multi-part oligonucleotides are at least 50 to at least 1,000 bases in length.

13. The method of claim 12 , wherein the at least 50 to at least 1,000 bases of length of the multi-part oligonucleotides have been synthesized without polymerase.

14. The method of claim 1 , wherein each ligating step includes annealing a length of about 4 to about 8 bases of a single strand sticky end of a duplex comprising a barcode.

15. The method of claim 1 , wherein the multi-part oligonucleotides have a barcode space between about a few thousand and about several hundreds of millions.

16. The method of claim 1 , further comprising emulsifying the multi-part oligonucleotides with single cells in partitions and labeling cells and molecules from the cells with combinations of at least two barcodes.

17. The method of claim 1 , further comprising emulsifying the multi-part oligonucleotides with single cells in partitions and labeling molecules from the cells with combinations of at least two barcodes that provide cellular barcode labels unique for each cell and UMI barcode labels unique for each molecule.

18. The method of claim 1 further comprising using the plurality of beads and the linked multi-part oligonucleotides for single cell RNA sequencing (scRNASeq).

19. A method of forming oligonucleotides on beads, the method comprising:

performing a plurality of rounds of addition of oligos comprising two 5′ overhangs onto beads covalently linked to initial oligos to form first ligation products;

ligating an oligo comprising two overhangs on the same strand to the first ligation products to form second ligation products;

ligating an oligo comprising a unique molecular identifier (UMI) and a poly-T primer site to the second ligation products to form oligonucleotides on beads.

Assignments (2)

Continuity (2)

References Cited (218)