Intermittent detection during analytical reactions

• US 8,628,940 B2
• Filed: 12/30/2010
• Issued: 01/14/2014
• Est. Priority Date: 09/24/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Sorenson, Jon, Bashir, Ali, Christians, Fred, Turner, Stephen, Olivares, Eric C., Underwood, Jason
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US12/982,029
• Publication Number:
  US 20110195406A1
• Time in Patent Office: 1,111 Days
• Field of Search: 435/91.2, 435/6.1
• US Class Current: 435/91.2
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68
• 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
• CPC Class Codes: G16B 30/20 Sequence assembly

Abstract

Methods, devices, and systems for performing intermittent detection during analytical reactions are provided. Such methods facilitate collection of reaction data from disparate reaction times. Further, such methods are useful for reducing photo-induced damage of one or more reactants in an illuminated analytical reaction at a given reaction time. In preferred embodiments, the reaction mixture is subjected to at least one illuminated and non-illuminated period and allowed to proceed such that the time in which the reaction mixture is illuminated is less than a photo-induced damage threshold period.

Claims

1. A method of performing paired-end sequencing on a single template molecule, the method comprising:

a) providing a double-stranded nucleic acid molecule comprising a first terminal portion, an intermediate portion, and a second terminal portion;

b) ligating a first linker to the first terminal portion of the nucleic acid molecule, wherein the first linker connects the 3′

terminus at the first terminal portion of the sense strand with the 5′

terminus at the first terminal portion of the antisense strand;

c) ligating a second linker to the second terminal portion of the nucleic acid molecule, wherein the second linker connects the 3′

terminus at the second terminal portion of the antisense strand with the 5′

terminus at the second terminal portion of the sense strand, and further wherein the nucleic acid molecule having both the first linker and the second linker ligated thereto forms a single-stranded circular template molecule that contains (i) the first linker between the 3′

terminus of the sense strand and the 5′

terminus of the antisense strand, and (ii) the second linker between the 3′

terminus of the antisense strand and the 5′

terminus of the sense strand; and

d) subjecting the template molecule to a sequencing process in which sequence reads are generated, for the first terminal portion and the second terminal portion, but sequence reads are not generated for the intermediate portion, thereby performing paired-end sequencing on the template molecule.

2. The method of claim 1, wherein the first linker and the second linker are identical to one another.

3. The method of claim 1, wherein the first linker and the second linker are not identical to one another.

4. The method of claim 1, wherein the first linker and the second linker are hybridized together prior to the ligating of steps b and c.

5. The method of claim 4, wherein the first linker and the second linker are separated prior to the sequencing process of step d.

6. The method of claim 4, wherein the first linker and the second linker are separated during the sequencing process of step d.

7. The method of claim 1, wherein the sequencing process comprises at least one detection period and at least one non-detection period such that the intermediate portion of the template molecule is subjected to the sequencing process during the non-detection period.

8. The method of claim 1, further comprising subjecting the template molecule to fragmentation and ligation prior to step d, wherein the fragmentation removes the intermediate portion.

9. The method of claim 1, wherein the sequencing process generates redundant sequence data from one or both of the first terminal portion and the second terminal portion.

10. The method of claim 1, wherein the sequencing process further generates sequence data from an additional portion of the template molecule that is noncontiguous with the first terminal portion and the second terminal portion.

11. The method of claim 1, wherein the sequencing process involves circularizing the template molecule by separating complementary strands of the template molecule and using the complementary strands in template-directed nascent strand synthesis catalyzed by a single polymerase enzyme.

12. The method of claim 1, wherein the intermediate portion is processed by a polymerase enzyme during the sequencing process.

13. The method of claim 1, wherein the template molecule comprises a primer binding site, a registration sequence, and/or a synthesis blocking moiety.

14. The method of claim 13, wherein the primer binding site, a registration sequence, or synthesis blocking moiety is present in the first linker, but absent from the second linker.

15. The method of claim 13, wherein the synthesis blocking moiety is selected from the group consisting of an abasic site, a nick, a synthetic linker, a non-native nucleotide or analog thereof, a primer, a large photolabile group, a strand-binding moiety, a damaged base, and a modified base.

16. The method of claim 15, wherein the abasic site is introduced by treatment with a DNA-glycosylase or by ligating a linker or adapter sequence comprising the abasic site.

17. The method of claim 16, wherein the abasic site is introduced by treatment with a uracil-DNA glycosylase subsequent to steps a-c, and further wherein steps a-c are performed in the presence of an inhibitor of a uracil-DNA glycosylase.

18. The method of claim 13, wherein the synthesis blocking moiety permanently blocks progression of a polymerase enzyme on the template during the sequencing process.

19. The method of claim 13, wherein the synthesis blocking moiety temporarily blocks progression of a polymerase enzyme on the template during the sequencing process.