Single cell nucleic acid detection and analysis

Patent metadata

Field	Value
Publication number	US-11834712-B2
Application number	US-202318177496-A
Country	US
Kind code	B2
Filing date	Mar 2, 2023
Priority date	Mar 24, 2011
Publication date	Dec 5, 2023
Grant date	Dec 5, 2023

Abstract

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

First claim

What is claimed is:

1. 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 while the cells are in the separate reaction vessels, the RNA comprising a plurality of distinct RNA species,
generating tagged cDNA from the released RNA, wherein the tagged cDNA comprises
(i) a sample barcode sequence corresponding to the single cell and
(ii) a copy number barcode sequence, and

pooling the tagged cDNA from the separate reaction vessels.

2. 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.

3. The method of claim 2 , wherein the copy number barcode sequences are 3 to 75 base pairs in length.

4. The method of claim 3 , wherein the copy number barcode sequences are 6 to 30 base pairs in length.

5. The method of claim 3 , wherein the copy number barcode sequences are of identical length.

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

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

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

9. The method of claim 3 , wherein the cDNA synthesis primers can hybridize to a polyA tail of the released mRNA molecules.

10. The method of claim 9 , wherein the cDNA synthesis primer comprise a polyT sequence for hybridizing to a polyA tail of the released mRNA molecules.

11. 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 sample barcode sequences corresponding to the isolated single cells and a plurality of different copy number barcode sequences.

12. The method of claim 11 , wherein the sample barcode sequences corresponding to the same single cell are identical to each other.

13. The method of claim 1 further preparing a sequencing library configured for sequencing on a massively parallel DNA sequencer wherein the sequencing library comprises the polled tagged cDNA molecules or copies of the pooled tagged cDNA molecules.

14. The method of claim 11 further preparing a sequencing library configured for sequencing on a massively parallel DNA sequencer wherein the sequencing library comprises the polled tagged cDNA molecules or copies of the pooled tagged cDNA molecules.

15. 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, wherein the released RNA is physically separated from RNA released from other 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 single cell and
(ii) a copy number barcode sequence, and

pooling the tagged cDNA derived from the different isolated single cells.

16. The method of claim 15 , 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.

17. The method of claim 16 , wherein the copy number barcode sequences are 3 to 75 base pairs in length.

18. The method of claim 17 , wherein the copy number barcode sequences are 6 to 30 base pairs in length.

19. The method of claim 17 , wherein the copy number barcode sequences are of identical length.

20. The method of claim 16 , wherein the copy number barcode sequences are random in sequence.

21. The method of claim 17 , wherein the copy number barcode sequences are separated by an error distance of at least 1.

22. The method of claim 21 , wherein the copy number barcode sequences are separated by an error distance in the range of 1 to 9.

23. The method of claim 16 , wherein the cDNA synthesis primers can hybridize to a polyA tail of an mRNA molecule.

24. The method of claim 23 , wherein the cDNA synthesis primer comprise a polyT sequence for hybridizing to a polyA tail of the released mRNA molecules.

25. The method of claim 15 , wherein the step of priming the synthesis of cDNA comprises contacting the released RNA with a set of cDNA synthesis primers that comprise sample barcode sequences corresponding to the isolated single cells and a plurality of different copy number barcode sequences.

26. The method of claim 25 , wherein the sample barcode sequences corresponding to the same single cell are identical to each other.

27. The method of claim 15 further preparing a sequencing library configured for sequencing on a massively parallel DNA sequencer wherein the sequencing library comprises the pooled tagged cDNA molecules or copies of the pooled tagged cDNA molecules.

28. The method of claim 25 further preparing a sequencing library configured for sequencing on a massively parallel DNA sequencer wherein the sequencing library comprises the pooled tagged cDNA molecules or copies of the pooled tagged cDNA molecules.

Assignees

• Harvard College

Inventors

• Xie Xiaoliang Sunney
• Shiroguchi Katsuyuki
• Sims Peter A
• Jia Tony Z

Classifications

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12N15/1065

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/6853 Primary

  using modified primers or templates · CPC title

• C12N2320/10

  in screening processes · CPC title

• C12Q2525/131

  incorporating a restriction site · CPC title