Single cell nucleic acid detection and analysis

• US 9,260,753 B2
• Filed: 03/22/2012
• Issued: 02/16/2016
• Est. Priority Date: 03/24/2011
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Xie, Xiaoliang Sunney, Shiroguchi, Katsuyuki, Sims, Peter A., Jia, Tony Z.
• Primary Examiner(s): Kim, Young J
• Application Number:
  US14/006,971
• Publication Number:
  US 20140155274A1
• Time in Patent Office: 1,426 Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 2320/10 in screening processes
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

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

Claims

1. A method of determining copy number of nucleic acid molecule in a sample including a plurality of nucleic acid molecules comprising:

attaching a unique barcode sequence to substantially each of the plurality of nucleic acid molecules in the sample to produce a plurality of differently barcoded nucleic acid molecules,

amplifying the plurality of differently barcoded nucleic acid molecules in the sample to produce amplicons of the plurality of differently barcoded nucleic acid molecules,

sequencing each amplicon to identify an associated nucleic acid sequence and an associated barcode sequence,

selecting a first target nucleic acid sequence and determining the number of unique associated barcode sequence for the first target nucleic acid sequence, wherein the number of unique associated barcode sequences is the copy number of the first target nucleic acid sequence.

2. The method of claim 1 wherein the nucleic acid molecules are DNA or RNA.

3. The method of claim 1, wherein the plurality of differently barcoded nucleic acid molecules is a plurality of differently barcoded RNA molecules and further including the steps of reverse transcribing the plurality of differently barcoded RNA molecules to produce differently barcoded cDNA molecules and amplifying the differently barcoded cDNA molecules to produce amplicons of the differently barcoded cDNA molecules.

4. The method of claim 3 comprising repeatedly reverse transcribing the plurality of differently barcoded RNA molecules to produce linear pre-amplified differently barcoded cDNA molecules and amplifying the linear pre-amplified differently barcoded cDNA molecules to produce amplicons of the linear pre-amplified differently barcoded cDNA molecules.

5. The method of claim 4, wherein the step of repeatedly reverse transcribing the plurality of differently barcoded RNA molecules includes using reverse transcriptase and a nicking enzyme.

6. The method of claim 1, wherein the plurality of differently barcoded nucleic acid molecules is a plurality of differently barcoded DNA molecules and further including the steps of repeated replication of the plurality of differently barcoded DNA molecules to produce a plurality of pre-amplified differently barcoded DNA molecules and amplifying the plurality of pre-amplified differently barcoded DNA molecules to produce amplicons of the plurality of pre-amplified differently barcoded DNA molecules.

7. The method of claim 6, wherein the step of repeated replication of the plurality of differently barcoded DNA molecules includes using DNA polymerase and a nicking enzyme.

8. the method of claim 1, wherein the sample is obtained from one or more cells of a first cell type and wherein amplification includes use of a primer derived from genomic DNA of the first cell type.

9. A method of counting nucleic acid molecules in a sample including a plurality of nucleic acid molecules comprising:

attaching a unique barcode sequence to substantially each of the plurality of nucleic acid molecules in the sample to produce a plurality of differently barcoded nucleic acid molecules,

amplifying the plurality of differently barcoded nucleic acid molecules in the sample to produce amplicons of the plurality of differently barcoded nucleic acid molecules,

sequencing each amplicon to identify an associated barcode sequence, and

counting the number of unique associated barcode sequences as a measure of the number of nucleic acid molecules in the sample.

10. The method of claim 9 wherein the nucleic acid molecules are DNA or RNA.

11. The method of claim 9, wherein the plurality of differently barcoded nucleic acid molecules is a plurality of differently barcoded RNA molecules and further including the steps of reverse transcribing the plurality of differently barcoded RNA molecules to produce differently barcoded cDNA molecules and amplifying the differently barcoded cDNA molecules to produce amplicons of the differently barcoded cDNA molecules.

12. The method of claim 11 comprising repeatedly reverse transcribing the plurality of differently barcoded RNA molecules to produce linear pre-amplified differently barcoded cDNA molecules and amplifying the linear pre-amplified differently barcoded cDNA molecules to produce amplicons of the linear pre-amplified differently barcoded cDNA molecules.

13. The method of claim 12, wherein the step of repeatedly reverse transcribing the plurality of differently barcoded RNA molecules includes using reverse transcriptase and a nicking enzyme.

14. The method of claim 9, wherein the plurality of differently barcoded nucleic acid molecules is a plurality of differently barcoded DNA molecules and further including the steps of repeated replication of the plurality of differently barcoded DNA molecules to produce a plurality of pre-amplified differently barcoded DNA molecules and amplifying the plurality of pre-amplified differently barcoded DNA molecules to produce amplicons of the plurality of pre-amplified differently barcoded DNA molecules.

15. The method of claim 14, wherein the step of repeated replication of the plurality of differently barcoded DNA molecules includes using DNA polymerase and a nicking enzyme.

16. The method of claim 9, wherein the sample is obtained from one or more cells of a first cell type and wherein amplification includes use of a primer derived from genomic DNA of the first cell type.

17. A method of determining copy numbers of nucleic acid molecules in a sample comprising:

attaching a unique barcode sequence to substantially each of the nucleic acid molecules in the sample to produce a plurality of differently barcoded nucleic acid molecules,

amplifying the plurality of differently barcoded nucleic acid molecules in the sample to produce amplicons of the plurality of differently barcoded nucleic acid molecules,

sequencing the amplicons of the plurality of differently barcoded nucleic acid molecules to identify for each amplicon an associated nucleic acid sequence and an associated barcode sequence, and

determining the number of unique associated barcode sequences for each nucleic acid sequence in the sample.

18. The method of claim 17 wherein the nucleic acid molecules are DNA or RNA.

19. The method of claim 17, wherein the plurality of differently barcoded nucleic acid molecules is a plurality of differently barcoded RNA molecules and further including the steps of reverse transcribing the plurality of differently barcoded RNA molecules to produce differently barcoded cDNA molecules and amplifying the differently barcoded cDNA molecules to produce amplicons of the differently barcoded cDNA molecules.

20. The method of claim 19 comprising repeatedly reverse transcribing the plurality of differently barcoded RNA molecules to produce linear pre-amplified differently barcoded cDNA molecules and amplifying the linear pre-amplified differently barcoded cDNA molecules to produce amplicons of the linear pre-amplified differently barcoded cDNA molecules.

21. The method of claim 20, wherein the step of repeatedly reverse transcribing the plurality of differently barcoded RNA molecules includes using reverse transcriptase and a nicking enzyme.

22. The method of claim 17, wherein the plurality of differently barcoded nucleic acid molecules is a plurality of differently barcoded DNA molecules and further including the steps of repeated replication of the plurality of differently barcoded DNA molecules to produce a plurality of pre-amplified differently barcoded DNA molecules and amplifying the plurality of pre-amplified differently barcoded DNA molecules to produce amplicons of the plurality of pre-amplified differently barcoded DNA molecules.

23. The method of claim 22, wherein the step of repeated replication of the plurality of differently barcoded DNA molecules includes using DNA polymerase and a nicking enzyme.

24. The method of claim 17, wherein the sample is obtained from one or more cells of a first cell type and wherein amplification includes use of a primer derived from genomic DNA of the first cell type.