Real-time sequencing methods and systems

• US 8,940,507 B2
• Filed: 06/21/2013
• Issued: 01/27/2015
• Est. Priority Date: 04/27/2009
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Korlach, Jonas, Turner, Stephen, Schadt, Eric
• Primary Examiner(s): Martinell, James
• Application Number:
  US13/924,130
• Publication Number:
  US 20140017680A1
• Time in Patent Office: 585 Days
• US Class Current: 435/91.51
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/702 for retroviruses
• CPC Class Codes: C12Q 2547/101 by confinement to a single tube/container
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The present invention is generally directed to compositions, methods, and systems for performing single-molecule, real-time analysis of a variety of different biological reactions. The ability to analyze such reactions provides an opportunity to study those reactions as well as to potentially identify factors and/or approaches for impacting such reactions, e.g., to either enhance or inhibit such reactions. In certain preferred embodiments, RNA templates are used in single-molecule real-time sequencing reactions.

Claims

1. A method for identifying a nucleic acid modification in a single molecule RNA template, the method comprising:

(a) providing a complex comprising a circularized form of the RNA template and a sequencing engine, wherein the complex is immobilized in an optical confinement;

(b) introducing a set of labeled nucleotides to the optical confinement;

(c) processing the circularized RNA template with the sequencing engine;

(d) detecting an energetic profile of the sequencing engine during the processing step (c), thereby identifying a nucleic acid modification in the RNA template.

2. The method of claim 1, wherein the nucleic acid modification comprises a base modification or a secondary structure.

3. The method of claim 2, wherein the nucleic acid modification comprises a base modification.

4. The method of claim 3, wherein the base modification is selected from the group consisting of a pseudouridine residue and a methylated base.

5. The method of claim 4, wherein the methylated base is an N2-methylguanosine residue.

6. The method of claim 1, wherein the sequencing engine is an RNA-dependent polymerase or a reverse transcriptase.

7. The method of claim 1, wherein the energetic profile comprises a member selected from:

a pause in the processing by the sequencing engine, a change in incorporation duration, a change in interpulse duration, and a change in missed or extra pulses.

8. The method of claim 1, wherein the processing step (c) comprises performing a sequencing-by-synthesis reaction.

9. The method of claim 8, wherein the processing step (c) comprises cognate sampling.

10. The method of claim 9, wherein the set of labeled nucleotides comprises unincorporatable nucleotides, and wherein the introducing step (b) further comprises introducing a set of unlabeled incorporatable nucleotides to the optical confinement.

11. The method of claim 1, wherein the sequencing engine is bound, directly or indirectly, to a surface of the optical confinement.

12. The method of claim 1, wherein the RNA template is bound, directly or indirectly, to a surface of the optical confinement.

13. The method of claim 12, wherein the RNA template is hybridized to an oligonucleotide bound to a surface of the optical confinement.

14. The method of claim 13, wherein the RNA template is hybridized to a sequence-specific region or a poly-T region of the oligonucleotide.

15. The method of claim 1, wherein the processing step (c) occurs multiple times over all or a portion of the RNA template.