Delaying real-time sequencing

• US 10,081,836 B2
• Filed: 07/15/2016
• Issued: 09/25/2018
• Est. Priority Date: 10/24/2013
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Clark, Tyson A., Korlach, Jonas, Heiner, Cheryl, Travers, Kevin, Miller, Erik
• Primary Examiner(s): Kim, Young J
• Application Number:
  US15/211,890
• Publication Number:
  US 20170009289A1
• Time in Patent Office: 802 Days
• Field of Search: None
• 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 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2527/113 Time

Abstract

Methods, compositions, and systems are provided that allow for reliable sequencing of the initial sequence region of a sequence of interest. The methods of the invention allow for more reliable barcoding of subpopulations of nucleic acids to be sequenced.

Claims

1. A method for delaying the sequencing of a sequence of interest in a single-molecule, real-time sequencing reaction comprising:

simultaneously performing a plurality of single-molecule, real-time sequencing reactions on a plurality of nucleic acid templates, wherein each of the nucleic acid templates comprises an adaptor region and an insert region, wherein the adaptor region comprises an initiation site and a runway region and the insert region comprises a sequence of interest, wherein the runway region is between the initiation site and the insert region, wherein the runway region comprises at least 200 nucleotides.

2. The method of claim 1, wherein each initiation site in each of the nucleic acid templates comprises a substantially identical sequence.

3. The method of claim 1, wherein each runway region in each of the nucleic acid templates comprises a substantially identical sequence.

4. The method of claim 1, wherein each of the nucleic acid templates comprises a double-stranded region.

5. The method of claim 4, wherein the double-stranded region comprises the runway region and the insert region.

6. The method of claim 5, wherein the adaptor region is a hairpin adaptor region.

7. The method of claim 6, wherein each of the nucleic acid templates comprises hairpins at both ends of the double-stranded region to form a topologically circular molecule.

8. The method of claim 5, wherein the template is a linear template comprising a single strand overhang on at least one end, wherein the single strand overhang comprises the initiation site.

9. The method of claim 1, wherein the single-molecule, real-time sequencing reaction comprises nanopore sequencing.

10. The method of claim 1, wherein the length of the runway region is 500 nucleotides or greater.

11. The method of claim 1, wherein the length of the runway region is between 200 and 2,000 nucleotides.

12. The method of claim 1, wherein the runway region comprises at least one modified base that is absent from the insert region.

13. The method of claim 1, further comprising a barcode region between the runway region and the insert region.