Single cell nucleic acid detection and analysis

Patent metadata

Field	Value
Publication number	US-11608527-B2
Application number	US-202217719800-A
Country	US
Kind code	B2
Filing date	Apr 13, 2022
Priority date	Mar 24, 2011
Publication date	Mar 21, 2023
Grant date	Mar 21, 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,
pooling the tagged cDNA from the separate reaction vessels, 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 claim 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 of identical length.

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

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

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

10. The method of claim 3 , wherein the cDNA synthesis primers can hybridize to a polyA tail of an mRNA molecule.

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 a plurality of different copy number barcode sequences and different sample barcode sequences, wherein the priming occurs before the cDNA pooling step.

12. 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,
pooling the tagged cDNA derived from the different 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.

13. The method of 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 15 , wherein the copy number barcode sequences are 6 to 30 base pairs in length.

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

18. The method of claim 12 , wherein the copy number barcode sequences are random in sequence.

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

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

21. The method of claim 14 , wherein the cDNA synthesis primers can hybridize to a polyA tail of an mRNA molecule.

22. The method of claim 14 , 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.

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

24. The method of 12 , wherein the sequencing comprises massively parallel sequencing.

Assignees

• Harvard College

Inventors

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

Classifications

• 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

• C12Q1/6874 Primary

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

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q2525/301

  Hairpin oligonucleotides · CPC title