Methods for obtaining a single molecule consensus sequence

• US 9,582,640 B2
• Filed: 04/01/2016
• Issued: 02/28/2017
• Est. Priority Date: 03/28/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Travers, Kevin, Otto, Geoff, Turner, Stephen, Heiner, Cheryl, Ma, Congcong
• Primary Examiner(s): Lu, Frank
• Application Number:
  US15/089,071
• Publication Number:
  US 20160237485A1
• Time in Patent Office: 333 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.1, 435/91.2, 435/91.51, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• 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 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

Methods obtaining a single molecule consensus sequence for a single template molecule, and for obtaining a plurality of single molecule consensus sequences for a plurality of single template molecules is provided. Template molecules having two complementary regions connected with a linker are sequenced. A single read from each template molecule can be obtained, the read containing sequence information for each of the complementary regions. Single molecule consensus sequences can be determined from these reads by comparing the sequence information of the two complementary regions.

Claims

1. A method of obtaining a single molecule consensus sequence of a single template nucleic acid molecule, comprising:

performing a single-molecule sequencing process on a single template nucleic acid molecule, wherein the single template nucleic acid molecule comprises;

i) a first strand having a 3′

end and a 5′

end, ii) a second strand, complementary to the first strand, and having a 3′

end and a 5′

end, and iii) a linker that links the 3′

end of the first strand to the 5′

end of the second strand, wherein the linker comprises a linking oligonucleotide,

thereby generating a sequence read from said single template nucleic acid molecule, the sequence read comprising a first sequence corresponding to the sequence of the first strand, and a second sequence corresponding to the sequence of the second strand; and

comparing the first sequence and the second sequence from said sequence read, thereby obtaining a single molecule consensus sequence of the single template nucleic acid molecule.

2. The method of claim 1, wherein said sequence read comprises a sequence of the linking oligonucleotide.

3. The method of claim 1, wherein said single-molecule sequencing process is performing using a sequencing by synthesis technology.

4. The method of claim 3, wherein said sequencing by synthesis technology comprises detecting incorporation of each nucleotide incorporated by a polymerase mediated, template dependent sequencing process.

5. The method of claim 1, wherein said single-molecule sequencing process is performing using a nanopore sensor.

6. The method of claim 1, wherein said sequence read comprises the sequence of at least 1000 bases.

7. The method of claim 1, wherein said linking oligonucleotide comprises a registration sequence.

8. The method of claim 1, wherein the linking oligonucleotide comprises a barcode sequence.

9. The method of claim 1, wherein the first strand, the second strand, or both the first strand and the second strand comprise genomic DNA.

10. The method of claim 1, wherein the first strand, the second strand, or both the first strand and the second strand comprise an amplification product.

11. The method of claim 1, wherein the first strand, the second strand, or both the first strand and the second strand comprise a PCR amplification product.

12. The method of claim 1, wherein the single template nucleic acid molecule also comprises a linker oligonucleotide that links the 5′

end of the first strand to the 3′

end of the second strand to form a completely contiguous molecule.

13. A method of obtaining a single molecule consensus sequence of a plurality of single template nucleic acid molecules, comprising:

performing a plurality of single-molecule sequencing processes on a plurality of single template nucleic acid molecules, wherein each of said plurality of single template nucleic acid molecules comprises i) a first strand having a 3′

end and a 5′

end, ii) a second strand, complementary to the first strand, having a 3′

end and a 5′

end, and iii) a linker that links the 3′

end of the first strand to the 5′

end of the second strand, wherein the linker comprises a linking oligonucleotide,

thereby generating a sequence read from each of said plurality of single template nucleic acid molecules, the sequence read from each of said plurality of single template nucleic acid molecules comprising a first sequence corresponding to the sequence of the first strand, and a second sequence corresponding to the sequence of the second strand; and

comparing the first sequence and the second sequence from each of said plurality of single template nucleic acid molecules, thereby obtaining a single molecule consensus sequence of said plurality of single template nucleic acid molecules.

14. The method of claim 13, wherein said plurality of single-molecule sequencing processes include sequencing the linking oligonucleotide.

15. The method of claim 13, wherein said single-molecule sequencing processes are performed using a sequencing by synthesis technology.

16. The method of claim 15, wherein said sequencing by synthesis technology comprises detecting incorporation of each nucleotide incorporated by a polymerase mediated, template dependent sequencing process.

17. The method of claim 13, wherein said single molecule sequencing processes are performed using a nanopore sensor.

18. The method of claim 13, wherein said read comprises the sequence of at least 1000 base pairs.

19. The method of claim 13, wherein said linking oligonucleotide comprises a registration sequence.

20. The method of claim 13, wherein the linking oligonucleotide comprises a barcode sequence.

21. The method of claim 13, wherein the first strand, the second strand, or both the first strand and the second strand of each of said plurality of single template nucleic acid molecules comprise genomic DNA.

22. The method of claim 13, wherein the first strand, the second strand, or both the first strand and the second strand of each of said plurality of single template nucleic acid molecules comprise an amplification product.

23. The method of claim 13, wherein the first strand, the second strand, or both the first strand and the second strand of each of said plurality of single template nucleic acid molecules comprise a PCR amplification product.

24. The method of claim 13, wherein each of said plurality of single template nucleic acid molecules also comprises a linker oligonucleotide that links the 5′

end of the first strand to the 3′

end of the second strand to form a completely contiguous molecule.