Sequencing using concatemers of copies of sense and antisense strands

• US 9,910,956 B2
• Filed: 08/19/2016
• Issued: 03/06/2018
• 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:
  US15/242,201
• Publication Number:
  US 20160376647A1
• Time in Patent Office: 564 Days
• Field of Search: 435 61, 435 611, 435 612, 435 911, 435 912, 435 9151, 435183, 436 94, 436501, 536 231, 536 243, 536 2433, 536 253
• 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 and systems for single molecule sequencing using concatemers of copies of sense and antisense strands. Concatemers are provided, for example, by carrying out rolling circle amplification on a circular molecule having sense and antisense regions to produce repeated copies of the sense and antisense regions connected by linking regions. The circular molecules can be produced by ligating hairpin adapters to each end of a double-stranded nucleic acid having a sense and antisense strand. The ligations can be carried out, for example using blunt end ligation. In some cases, a single molecule consensus sequence for a single template molecule is obtained. A single read from each template molecule can be obtained by comparing the sequence information of the sense and antisense regions.

Claims

1. A method of obtaining nucleotide sequence information from nucleic acid segments of a nucleic acid sample, the method comprising:

a) fragmenting a nucleic acid sample, thereby providing at least one double-stranded nucleic acid fragment comprising a double-stranded region comprising a sense strand and a complementary antisense strand of said sense strand;

b) ligating a first hairpin adapter to a first end of said double-stranded nucleic acid fragment and ligating a second hairpin adapter to a second end of double-stranded said nucleic acid fragment, thereby providing a circular nucleic acid;

c) performing a rolling circle replication reaction with said circular nucleic acid as a template, thereby producing a concatemer having segments comprising repeating copies of said sense strand and said antisense strand;

d) performing a single-molecule sequencing process, thereby generating a single sequencing read from said concatemer, said single sequencing read comprising at least one first sequence corresponding to said sense strand and at least one second sequence corresponding to said antisense strand; and

e) comparing or assembling said at least one first sequence and said at least one second sequence from said single sequencing read, thereby obtaining the nucleotide sequence information of said nucleic acid segments of the nucleic acid sample.

2. The method of claim 1, wherein said single-molecule sequencing process is performed using an electrochemical system.

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

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

5. The method of claim 1, wherein said first hairpin adapter and said second hairpin adapter are identical.

6. The method of claim 1, wherein said first hairpin adapter and said second hairpin adapter have blunt ends.

7. The method of claim 1, wherein said first hairpin adapter or said second hairpin adapter, or both said first hairpin adapter and said second hairpin adapter, comprises or comprise a registration sequence.

8. The method of claim 1, wherein the nucleic acid sample comprises genomic DNA.

9. The method of claim 1, wherein the nucleic acid sample comprises amplification products.

10. The method of claim 1, wherein the double-stranded region comprises at least 1000 base pairs.

11. The method of claim 1 further comprises determining a consensus sequence of said nucleic acid segments by said comparing or assembling the at least one first sequence corresponding to the sense region, and at least one second sequence corresponding to the complementary antisense region.

12. The method of claim 11 wherein said consensus sequence of said nucleic acid segments is determined using heuristic methods, Hidden Markov models, Bayesian methods, machine learning approaches, neural networks, or self-organizing maps.

13. A method of obtaining nucleotide sequence information from segments of a single template molecule, the method comprising:

performing a single-molecule sequencing process on a single template molecule, wherein the single template molecule comprises a linear concatemer nucleic acid strand comprising segments comprising repeating, alternating copies of a sense region and a complementary antisense region of said sense region, first linking regions, and second linking regions, and wherein one of said first linking regions are present between 3′

end of one of the sense regions and 5′

end of one of the antisense regions, and wherein one of said second linking regions are present between 3′

end of one of the antisense regions and 5′

end of one of the sense regions,

thereby generating a sequence read from the single template molecule, the read comprising at least one first sequence corresponding to the sense region, and at least one second sequence corresponding to the complementary antisense region; and

comparing or assembling the at least one first sequence and the at least one second sequence from the sequence read, thereby obtaining nucleotide sequence information of the segments of the single template molecule.

14. The method of claim 13, wherein the single-molecule sequencing process comprises a sequencing by synthesis technology.

15. The method of claim 14, wherein the sequencing by synthesis technology comprises detecting incorporation of each nucleotide incorporated by a polymerase mediated, template dependent sequencing process.

16. The method of claim 13, wherein the single-molecule sequencing process is performed using a nanopore sensor.

17. The method of claim 13, wherein the sequence read comprises at least 1000 bases.

18. The method of claim 13, wherein the first linking regions or the second linking regions, or both the first linking regions and the second linking regions, comprise a registration sequence.

19. The method of claim 13, wherein the first linking regions and the second linking regions are identical.

20. The method of claim 13 further comprises determining a consensus sequence of said nucleic acid segments by said comparing or assembling the at least one first sequence corresponding to the sense region, and at least one second sequence corresponding to the complementary antisense region.

21. The method of claim 20 wherein said consensus sequence of said nucleic acid segments is determined using heuristic methods, Hidden Markov models, Bayesian methods, machine learning approaches, neural networks, or self-organizing maps.