Identification of nucleic acid template-linked barcodes comprising nucleic acid modifications

• US 10,294,523 B2
• Filed: 09/23/2015
• Issued: 05/21/2019
• Est. Priority Date: 12/11/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Flusberg, Benjamin, Korlach, Jonas, Wegener, Jeffrey, Clark, Tyson A., Vilfan, Igor Drasko, Kislyuk, Andrey, Turner, Stephen, Sorenson, Jon, Travers, Kevin, Heiner, Cheryl, Tomaney, Austin B., Marks, Patrick, Lee, Jessica, Jia, Lei, Webster, Dale, Lyle, John, Hanes, Jeremiah
• Primary Examiner(s): Bertagna, Angela M.
• Application Number:
  US14/863,133
• Publication Number:
  US 20160153038A1
• Time in Patent Office: 1,336 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• 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 1/689 for bacteria
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2600/154 Methylation markers

Abstract

Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.

Claims

1. A method of identifying a barcode, the method comprising:

a) providing a sequencing template comprising a nucleic acid linked to a barcode, wherein the barcode consists of a nucleotide sequence comprising a base modification;

b) subjecting the sequencing template to a single-molecule sequencing reaction;

c) collecting reaction data in real time during the single-molecule sequencing reaction, wherein said reaction data comprises a sequence read for the sequencing template, and wherein the reaction data further comprises a kinetic change in the single-molecule sequencing reaction, wherein the kinetic change is indicative of the base modification in the nucleotide sequence of the barcode; and

d) analyzing both the sequence read and the kinetic change in the reaction data to detect the nucleotide sequence of the barcode, thereby identifying the barcode in the sequencing template.

2. The method of claim 1, wherein the sequencing template comprises a first polynucleotide region and a second polynucleotide region complementary to the first polynucleotide region, where the first polynucleotide region and the second polynucleotide region are on a single strand of the sequencing template.

3. The method of claim 1, wherein the sequencing template is subjected to a treatment to alter the modified base prior to the single-molecule sequencing reaction.

4. The method of claim 1, wherein the single-molecule sequencing reaction comprises a polymerase enzyme.

5. The method of claim 1, wherein the single-molecule sequencing reaction comprises template-directed synthesis of a nascent strand that is complementary to the sequencing template.

6. The method of claim 1, wherein the single-molecule sequencing reaction comprises single nucleotides that are differentially labeled to be distinguishable from one another during the single-molecule sequencing reaction.

7. The method of claim 1, wherein the kinetic change is detected as an alteration in interpulse duration during the single-molecule sequencing reaction.

8. The method of claim 1, wherein the kinetic change is detected as an alteration in pulse width during the single-molecule sequencing reaction.

9. The method of claim 1, wherein the sequencing template forms a complex with a polymerase, and further wherein the complex is immobilized at a reaction site on a substrate during the single-molecule sequencing reaction.

10. The method of claim 1, wherein the kinetic change in the reaction data occurs at the base modification.

11. The method of claim 1, wherein the kinetic change in the reaction data occurs at one or more positions upstream or downstream of the base modification.

12. The method of claim 1, wherein the kinetic change in the reaction data occurs at different locations selected from at the base modification, at one or more positions upstream of the modification, and at one or more positions downstream of the base modification.

13. The method of claim 1, wherein the base modification is selected from the group consisting of a methylated base, a 5-hydroxymethylcytosine, a pseudouridine base, a 7,8-dihydro-8-oxoguanine base, a 2′

-O-methyl derivative base, N7-methylguanosine, and a bulky base adduct.

14. The method of claim 1, wherein the providing comprises introducing the modified base into the barcode, and linking the barcode comprising the modified base to the nucleic acid, thereby providing the sequencing template.

15. The method of claim 1, wherein the barcode identifies the source of the nucleic acid.

16. The method of claim 1, wherein two sequencing templates, each comprising a different barcode, are subjected to the single-molecule sequencing reaction and the reaction data comprising the sequence reads and kinetic changes for each sequencing template is used to distinguish between the barcodes.