Massively Multiplexed RNA Sequencing

• US 20160024572A1
• Filed: 03/14/2014
• Published: 01/28/2016
• Est. Priority Date: 03/14/2013
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: SHISHKIN, Alexander A., GUTTMAN, Mitchell, SURKA, Christine F., LANDER, Eric S.
• Granted Patent: US 10,612,088 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

A method for parallel sequencing target RNA from samples from multiple sources while maintaining source identification is provided. The method includes providing samples of RNA comprising target RNA from two or more sources; labeling, at the 3′ end, the RNA from the two or more sources with a first nucleic acid adaptor that comprises a nucleic acid sequence that differentiates between the RNA from the two or more sources; reverse transcribing the two or more sources to create a single stranded DNA comprising the nucleic acid sequence that differentiates between the RNA from the two or more sources; amplifying the single stranded DNA to create DNA amplification products that comprise the nucleic acid sequence that differentiates between the RNA from the two or more sources; sequencing the DNA amplification products thereby parallel sequencing target RNA from samples from multiple sources while maintaining source identification.

Claims

1. A method for parallel sequencing target RNA from samples from multiple sources while maintaining source identification, comprising:

providing samples of RNA comprising target RNA from two or more sources;

labeling, at the 3′

end, the RNA from the two or more sources with a first nucleic acid adaptor that comprises a nucleic acid sequence that differentiates between the RNA from the two or more sources;

optionally pooling the 3′

labeled RNA;

reverse transcribing the RNA from the two or more sources to create a single stranded DNA comprising the nucleic acid sequence that differentiates between the RNA from the two or more sources;

amplifying the single stranded DNA to create DNA amplification products that comprise the nucleic acid sequence that differentiates between the RNA from the two or more sources;

sequencing the DNA amplification products;

thereby parallel sequencing target RNA from samples from multiple sources while maintaining source identification.

3. The method of claim 1, further comprising labeling, at the 3′

end, the single stranded DNA with a second nucleic acid adaptor that comprises a site for binding of a PCR primer wherein the second nucleic acid adaptor optionally comprises a sequencing adaptor and/or a second nucleic acid sequence that can differentiate between single stranded DNA from two or more sources.

6. The method of claim 1, wherein the first nucleic acid adaptor comprises a sequencing adaptor.

9. The method of claim 1, further comprising depleting the samples of non-target RNA.

10. The method of claim 9, wherein depleting the samples of non-target RNA comprises using one or more probes that specifically hybridize to the non-target RNA, wherein the one or more probes comprise a label that facilitates removal of the probe from the sample.

13. The method of claim 1, wherein

the first nucleic acid adaptor is 3′

end blocked and/or wherein the first nucleic acid adaptor comprises RNA, DNA or a RNA-DNA hybrid.

15. The method of claim 3, wherein the second nucleic acid adaptor is 3′

end blocked and/or wherein the second nucleic acid adaptor comprises RNA, DNA or a RNA-DNA hybrid.

17. The method of claim 1, wherein the nucleic acid sequence that differentiates between the RNA from the two or more sources is at least two nucleotides in length and/or wherein the nucleic acid sequence that differentiates between the single stranded DNA from the two or more sources is at least two nucleotides in length.

19. The method of claim 1, further comprising removing and/or degrading the reverse transcription primers and/or removing and/or degrading the RNA from the sample.

21. The method of claim 1, wherein the samples of different origins comprise samples of the same cell type and/or tissue type exposed to different environmental conditions.

22. The method of claim 21, where the different environmental conditions are contacts with different test agents.

23. The method of claim 1, where the target RNA is mRNA, and differences in expression of the mRNA are measured across samples from different sources.

24. The method of claim 1, further comprising quantifying the sequenced target RNAs of samples from multiple sources.

25. The method of claim 1, comprising sorting the sequenced target RNAs to their respective sources.

26. The method of claim 1, further comprising labeling, at the 5′

end, the RNA from the two or more sources with a first nucleic acid adaptor that comprises a nucleic acid sequence that differentiates between the RNA from the two or more sources.

27. The method of claim 26, further comprising labeling, at the 5′

end, the single stranded DNA with a second nucleic acid adaptor that comprises a site for binding of a PCR primer and/or a sequencing adaptor.

2. (canceled)

4. (canceled)

5. (canceled)

8. The method of claim 5, further comprising pooling the single stranded DNA with single stranded DNA from one or more additional sources, wherein the single stranded DNA from one or more additional sources is similarly labeled.

7. (canceled)

11. (canceled)

12. (canceled)

14. (canceled)

16. (canceled)

18. (canceled)

20. (canceled)

28. (canceled)

29. A kit for parallel sequencing target RNA from samples from multiple sources while maintaining source identification, comprising:

a first nucleic acid adaptor for labeling, at the 3′

end, the RNA from the two or more sources that comprises a nucleic acid sequence that differentiates between the RNA from the two or more sources and optionally wherein the first nucleic acid adaptor comprises a sequencing adapter; and

a second nucleic acid adaptor for end labeling the 3′

end of single stranded DNA, wherein the second nucleic acid adaptor comprises a site for binding of a PCR primer.

30. The kit of claim 29, wherein the second nucleic acid adaptor comprises a sequencing adapter and/or a second nucleic acid sequence that can differentiate between single stranded DNA from two or more sources and/or a site for binding of a PCR primer.

34. The kit of claim 29, further comprising a first nucleic acid adaptor for labeling, at the 5′

end, the RNA from the two or more sources that comprises a nucleic acid sequence that differentiates between the RNA from the two or more sources

35. The kit of claim 34, further comprising a second nucleic acid adaptor for end labeling the 5′

end of single stranded DNA, wherein the second nucleic acid adaptor comprises a site for binding of a PCR primer and/or wherein the second nucleic acid adaptor comprises a sequencing adaptor.

37. The kit of claim 29, further comprising one or more probes that specifically hybridize to non-target RNA, wherein the one or more probes comprise a label that facilitates removal of the probe from the sample.

40. The kit of claim 29, wherein the first nucleic acid adaptor is 3′

end blocked and/or wherein the first nucleic acid adaptor comprises RNA, DNA or a RNA-DNA hybrid.

42. The kit of claim 29, wherein the second nucleic acid adapter is 3′

end blocked and/or wherein the second nucleic acid adapter comprises RNA, DNA or a RNA-DNA hybrid.

44. The kit of claim 29, wherein the nucleic acid sequence that differentiates between the RNA from the two or more sources is at least two nucleotides in length and/or wherein the nucleic acid sequence that differentiates between the single stranded DNA from the two or more sources is at least two nucleotides in length.

31. (canceled)

32. (canceled)

33. (canceled)

36. (canceled)

38. (canceled)

39. (canceled)

41. (canceled)

43. (canceled)

45. (canceled)