Delaying real-time sequencing

• US 9,416,414 B2
• Filed: 10/22/2014
• Issued: 08/16/2016
• 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:
  US14/521,109
• Publication Number:
  US 20150118685A1
• Time in Patent Office: 664 Days
• Field of Search: None
• US Class Current: 1/1
• 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 initiating a plurality of single-molecule, real-time sequencing reactions on a plurality of polymerase-template complexes, wherein each of the polymerase-template complexes comprises a polymerase enzyme and a nucleic acid template wherein the nucleic acid template comprises, in order, a priming region, a runway region, and an insert region comprising a sequence of interest, said runway region and said insert region each being from a different source, wherein from the time the polymerase incorporates its first nucleotide to the time when the polymerase enzyme sequences a first base of the insert region is greater than about 30 seconds for a majority of the polymerase-template complexes, wherein the runway region has a length based on the polymerization rate of the polymerase enzyme to provide for said about 30 seconds.

2. The method of claim 1 wherein each priming region in each nucleic acid template in each of the polymerase-template complexes comprises an identical sequence.

3. The method of claim 1 wherein each runway region in each nucleic acid template in each of the polymerase-template complexes comprises an identical sequence.

4. The method of claim 1 wherein the template nucleic acid comprises a double-stranded region and a hairpin connecting the strands of the double-stranded region.

5. The method of claim 1 wherein the template nucleic acid comprises a double-stranded region and hairpins on each end of the double-stranded region connecting the two strands.

6. The method of claim 1 wherein the single-molecule, real-time sequencing reactions comprise incorporation of nucleotide residues, wherein the incorporation of nucleotide residues is detected using fluorescence.

7. The method of claim 1 wherein the single-molecule, real-time sequencing reactions comprise incorporation of nucleotide residues, wherein the incorporation of nucleotide residues is detected electronically or magnetically.

8. The method of claim 1 wherein the single-molecule, real-time sequencing reactions comprise incorporation of nucleotide residues, wherein the incorporation of nucleotide residues is detected by electrochemistry, capacitance, conductivity, impedance, or with a field effect transducer.

9. The method of claim 1 wherein the length of the runway region is 200 nucleotides or greater.

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

11. The method of claim 1 wherein the time from the initiation of the sequencing reactions to the time of sequencing the insert region is greater than about 60 seconds for a majority of the polymerase-template complexes.

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

13. The method of claim 1, wherein the single-molecule, real-time sequencing reaction comprises observing labels corresponding to labeled nucleotides during polymerase mediated nucleic acid synthesis.

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

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

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

initiating a single-molecule, real-time sequencing reaction on a polymerase-template complex, wherein the polymerase-template comprises a polymerase enzyme and a nucleic acid template, wherein the nucleic acid template comprises, in order, a priming region, a runway region, and an insert region comprising a sequence of interest, said runway region and said insert region each being from a different source, wherein from the time the polymerase incorporates its first nucleotide to the time when the polymerase enzyme sequences a first base of the insert region is greater than about 30 seconds, wherein the runway region has a length based on the polymerization rate of the polymerase enzyme to provide for said about 30 seconds.

17. The method of claim 16 wherein the template nucleic acid comprises a double-stranded region, and a hairpin connecting the strands of the double-stranded region.

18. The method of claim 16 wherein the template nucleic acid comprises a double-stranded region and hairpins on each end of the double-stranded region connecting the two strands.

19. The method of claim 16 wherein the single-molecule, real-time sequencing reactions comprise incorporation of nucleotide residues, wherein the incorporation of nucleotide residues is detected using fluorescence.

20. The method of claim 16 wherein the single-molecule, real-time sequencing reactions comprise incorporation of nucleotide residues, wherein the incorporation of nucleotide residues is detected electronically or magnetically.

21. The method of claim 16 wherein the single-molecule, real-time sequencing reactions comprise incorporation of nucleotide residues, wherein the incorporation of nucleotide residues is detected by electrochemistry, capacitance, conductivity, impedance, or with a field effect transducer.

22. The method of claim 16 wherein the length of the runway region is 200 nucleotides or greater.

23. The method of claim 16 wherein the length of the runway region is between 200 and 2,000 nucleotides.

24. The method of claim 16 wherein the runway region comprises at least one modified base that is absent from the insert region.

25. The method of claim 16 wherein the time from the initiation of the sequencing reactions to the time of sequencing the insert region is greater than about 60 seconds.

26. The method of claim 16 further comprising a barcode region between the runway region and the insert region.