IP Library › Granted Patent US 12,448,649
Granted Patent B2
US 12,448,649 · App. 18/527,896 · Granted Oct 21, 2025

Single cell nucleic acid detection and analysis

Inventors: Xiaoliang Sunney Xie (Lexington, MA); Katsuyuki Shiroguchi (Arlington, MA); Peter A. Sims (Cambridge, MA); Tony Z. Jia (Cambridge, MA)
Assignee: President and Fellows of Harvard College
C12Q1/6874C12N15/1065C12Q1/6853C12N2320/10
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Quick Facts
Patent No.
US 12,448,649
App. No.
18/527,896
Granted
Oct 21, 2025
Kind
B2
Abstract

Methods and compositions for digital profiling of nucleic acid sequences present in a sample are provided.

Claims (42)

1. A method of quantitatively measuring a plurality of different RNA species in a plurality of isolated single cells, comprising

physically isolating a plurality of single cells to produce isolated single cells,

releasing RNA from the isolated single cells, the RNA comprising a plurality of distinct RNA species,

forming tagged cDNA from the released RNA, wherein the tagged cDNA comprises

(i) a sample barcode sequence corresponding to the isolated single cells, wherein the sample barcode sequence identifies the tagged cDNA molecules derived from the RNA released from the same isolated single cell, and

(ii) a copy number barcode sequence, wherein the copy number barcode sequences are of identical length,

pooling the tagged cDNA derived from the isolated single cells, and

sequencing the pooled tagged cDNA molecules so as to produce DNA sequence information sufficient to identify (i) the RNA species, (ii) the sample barcode sequences, and (iii) the copy number barcode sequences.

2. The method of 1 , further comprising counting the quantity of distinct copy number barcode sequences present in tagged cDNA molecules derived from the same RNA species and having the same sample barcode sequence.

3. The method of claim 1 , wherein the step of generating tagged cDNA comprises priming synthesis of cDNA from the released RNA with a set of cDNA synthesis primers that comprise a plurality of different copy number barcode sequences.

4. The method of claim 3 , wherein the copy number barcode sequences are 3 to 75 base pairs in length.

5. The method of claim 4 , wherein the copy number barcode sequences are 6 to 30 base pairs in length.

6. The method of claim 3 , wherein the copy number barcode sequences are separated by a barcode distance of at least 1.

7. The method of claim 3 , wherein the copy number barcode sequences are separated by a barcode distance in the range of 1 to 9.

8. The method of claim 3 , wherein the cDNA synthesis primers can hybridize to a polyA tail of an mRNA molecule, wherein the RNA being analyzed is mRNA.

9. The method of claim 3 , wherein the step of priming the synthesis of cDNA comprises contacting the released RNA with a set of cDNA synthesis primers that comprise a plurality of different copy number barcode sequences and different sample barcode sequences, wherein the priming occurs before the cDNA pooling step.

10. The method of claim 1 , wherein the copy number barcode sequences are random in sequence.

11. The method of 1 , wherein the sequencing comprises massively parallel sequencing.

12. A method of quantitatively measuring a plurality of different RNA species in a cell, comprising

isolating a plurality of single cells, each cell being isolated in a separate reaction vessel,

releasing RNA from the isolated single cells, the RNA comprising a plurality of distinct RNA species,

generating tagged cDNA molecules from the released RNA, wherein the tagged cDNA molecules comprise

(i) a sample barcode sequence corresponding to the isolated single cells, wherein the sample barcode sequence identifies the tagged cDNA molecules derived from the RNA released from the same isolated single cell, and

(ii) a copy number barcode sequence, and

sequencing the tagged cDNA molecules so as to produce DNA sequence information sufficient to identify for each sequenced cDNA (i) the RNA species, (ii) the sample barcode sequences, and (iii) the copy number barcode sequences.

13. The method of claim 12 , further comprising counting the quantity of distinct copy number barcode sequences present in tagged cDNA molecules derived from the same RNA species and having the same sample barcode sequence.

14. The method of claim 12 , wherein the step of generating tagged cDNA comprises priming synthesis of cDNA from the released RNA with a set of cDNA synthesis primers that comprise a plurality of different copy number barcode sequences.

15. The method of claim 14 , wherein the copy number barcode sequences are 3 to 75 base pairs in length.

16. The method of claim 14 , wherein the copy number barcode sequences are separated by a barcode distance of at least 1.

17. The method of claim 14 , wherein the copy number barcode sequences are separated by a barcode distance in the range of 1 to 9.

18. A method of quantitatively measuring a plurality of different RNA species in a cell, comprising

isolating a plurality of single cells, each cell being isolated in a separate reaction vessel,

releasing RNA from the isolated single cells, the RNA comprising a plurality of distinct RNA species,

generating tagged cDNA molecules from the released RNA, wherein the tagged cDNA molecules comprise

(i) a sample barcode sequence corresponding to the isolated single cells, wherein the sample barcode sequence identifies the tagged cDNA molecules derived from the RNA released from the same isolated single cell, and

(ii) a copy number barcode sequence, and

sequencing the tagged cDNA molecules from a pool comprising the tagged cDNA molecules derived from a plurality of the isolated single cells so as to produce DNA sequence information sufficient to identify (i) the RNA species, (ii) the sample barcode sequences, and (iii) the copy number barcode sequences.

19. The method of claim 18 , further comprising counting the quantity of distinct copy number barcode sequences present in tagged cDNA molecules derived from the same RNA species and having the same sample barcode sequence.

20. The method of claim 18 , wherein the step of generating tagged cDNA comprises priming synthesis of cDNA from the released RNA with a set of cDNA synthesis primers that comprise a plurality of different copy number barcode sequences.

21. The method of claim 20 , wherein the copy number barcode sequences are 3 to 75 base pairs in length.

22. The method of claim 20 , wherein the copy number barcode sequences are separated by a barcode distance of at least 1.

23. The method of claim 20 , wherein the copy number barcode sequences are separated by a barcode distance in the range of 1 to 9.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 11, 2025
From: JIA, TONY Z.; SHIROGUCHI, KATSUYUKI; SIMS, PETER A.; XIE, XIAOLIANG SUNNEY
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 070469/0556 →
Continuity (11)
Continuation 18177496 · Mar 2, 2023
Continuation 17719800 · Apr 13, 2022
Continuation 17363053 · Jun 30, 2021
Continuation 16774104 · Jan 28, 2020
Continuation 16364947 · Mar 26, 2019
Continuation 15730157 · Oct 11, 2017
Continuation 14990286 · Jan 7, 2016
Continuation 14006971
Provisional Application 61583787 · Jan 6, 2012
Provisional Application 61467037 · Mar 24, 2011
Related Publication 20240117427A1 · Apr 11, 2024
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