Reagents, Methods, and Libraries for Bead-Based Sequencing

• US 20100297626A1
• Filed: 11/30/2009
• Published: 11/25/2010
• Est. Priority Date: 02/01/2005
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: MCKERNAN, Kevin, Kotler, Lev, Costa, Gina, Blanchard, Alan
• Application Number:
  US12/628,209
• Publication Number:
  US 20100297626A1
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• 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 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/137 Chromatographic separation
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles comprise steps of extension, ligation, and, preferably, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. In certain embodiments the methods make use of extension probes containing an abasic residue or a damaged base and employ agents appropriate to cleave linkages between a nucleoside and an abasic residue and/or agents appropriate to remove a damaged base from a nucleic acid. The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to beads, which are immobilized in or on a semi-solid support. The invention further provides sets of labeled extension probes containing phosphorothiolate linkages or trigger residues that are suitable for use in the method. In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides. The invention further provides efficient methods for preparing templates, particularly for performing sequencing multiple different templates in parallel. The invention also provides methods for performing ligation and cleavage. The invention also provides new libraries of nucleic acid fragments containing paired tags, and methods of preparing microparticles having multiple different templates (e.g., containing paired tags) attached thereto and of sequencing the templates individually. The invention also provides automated sequencing systems, flow cells, image processing methods, and computer-readable media that store computer-executable instructions (e.g., to perform the image-processing methods) and/or sequence information. In certain embodiments the sequence information is stored in a database.

Claims

1-48. -48. (canceled)

49. A method for determining information about a template sequence, the method comprising the steps of:

(I) performing a sequencing reaction on a probe-template complex having a duplex portion, wherein the sequencing reaction comprises;

(a) contacting the complex with a collection of oligonucleotide probes, the collection comprising at least two probe families, wherein;

(i) a probe family is a group of one or more different probes that are identically labeled, and

(ii) the probe families are labeled distinguishably from each other;

(b) extending the duplex portion by ligating an extendable terminus of the duplex portion with a labeled probe from the collection that has hybridized to the template;

(c) detecting the label of the ligated probe to identify its probe family , wherein the identity of the probe family eliminates one or more possibilities for the identity of at least one template sequence nucleotide but leaves at least two such possibilities open for that nucleotide; and

(d) repeating steps (a) to (c) for a desired number of cycles to obtain an ordered series of probe family names of successively ligated probes,

(II) optionally performing at least one more sequencing reaction; and

(III) generating an ordered list of probe family names from at least one ordered series of probe family names from a sequencing reaction, wherein the ordered list provides information about the template sequence.

50. The method of claim 49, wherein the method comprises using the ordered list from step (III) to determine the identity of one or more nucleotides in the template sequence,.

51. The method of claim 49, wherein the probe-template complex is initially formed by a process comprising:

(a) rendering the template sequence single-stranded if in double-stranded form, and

(b) hybridizing an initializing oligonucleotide to a single-stranded portion of the template sequence;

and wherein the ordered list of probe family names from step (III) is assembled by combining multiple ordered series of probe family names obtained from a plurality of sequencing reactions using multiple initializing oligonucleotides that initiate from different sites in the template.

53. The method of claim 49, further comprising producing a new extendable terminus on the oligonucleotide probe ligated in step (b) such that the new terminus generated is different from the original terminus of the ligated probe before ligation.

54. The method of claim 49, wherein at least one oligonucleotide probe comprises a phosphorothiolate linkage and the extendable terminus of the duplex is produced by cleaving the phosphorothiolate linkage with a cleavage agent comprising an atom selected from the group consisting of:

Ag, Hg, Cu, Mn, Zn and Cd.

55. The method of claim 49, wherein the collection comprises 2 distinguishably labeled probe families.

56. The method of claim 49, wherein the collection comprises 3 distinguishably labeled probe families.

57. The method of claim 49, wherein the collection comprises 4 distinguishably labeled probe families.

58. The method of claim 49, wherein the collection comprises more than 4 distinguishably labeled probe families.

59. The method of claim 49, wherein at least one probe in a probe family comprises a constrained portion in which nucleotides are not selected independently of each other, in such a manner that knowing the identity of one or more nucleotides in the constrained portion of the probe provides sufficient information to eliminate one or more possible identities for a nucleotide at one of the other positions.

60. The method of claim 49, wherein probes are assigned to four probe families according to a scheme set forth in Table 1.

61. The method of claim 59, wherein at least one nucleotide in the template has a known identity, and wherein the method comprises:

(i) assigning at least one identity to an unknown nucleotide in the template adjacent to the nucleotide of known identity by determining which identity for the unknown nucleotide is consistent with (i) the identity of the known nucleotide and (ii) the possible sequences of the constrained portion of a probe whose proximal nucleotide is ligated opposite the unknown nucleotide on the template adjacent to the nucleotide of known identity;

(ii) assigning at least one identity to a succeeding nucleotide in the template by determining which identity is consistent with possible sequences of the constrained portion of the probe whose proximal nucleotide is ligated opposite the succeeding nucleotide; and

(iii) repeating step (ii) until the sequence information is determined.

62. The method of claim 49, further comprising the step of capping unligated extendable termini on the duplex after ligation.

63. The method of claim 60, wherein the determining step comprises contacting the duplex with a labeled nucleotide in the presence of a polymerase under conditions that allow incorporation of the labeled nucleotide if it is complementary to the template at the position adjacent to the duplex.

64. The method of claim 49, wherein the ordered list is generated from one or more sequencing reactions and method comprises:

generating at least one partial or complete candidate sequence from ordered list of probe family names; and

selecting a candidate sequence as the sequence of nucleotides in the template.

65. The method of claim 64, wherein the generating step comprises generating at least 4 candidate sequences.

66. The method of claim 49, wherein the method comprises:

(i) assuming an identity for a first nucleotide in the sequence of nucleotides;

(ii) assigning an identity for a nucleotide adjacent to the first nucleotide by determining a possible identity for the adjacent nucleotide based on the probe family information for the first nucleotide;

(iii) assigning an identity to a succeeding nucleotide by determining a possible identity for the succeeding nucleotide based on the probe family information for the nucleotide whose identity was most recently assigned;

(iv) repeating step (iii) until a candidate sequence is generated; and

(v) repeating steps (i)-(iv), wherein, in each repetition, a different identity is assumed for the first nucleotide, until a desired number of candidate sequences is generated.

67. The method of claim 64, wherein the selecting step comprises comparing at least one candidate sequence with one or more known sequences and selecting a candidate sequence that exhibits a predetermined degree of identity or is most nearly identical to one or more of the known sequences.

68. The method of claim 67, wherein the template is derived from an organism of interest, and wherein the comparing step comprises comparing at least one candidate sequence with sequences in a database that contains sequences obtained from the organism.

69. The method of claim 67, wherein the comparing step comprises comparing at least one candidate sequence with sequences in a database that contains a plurality of comparison sequences, each of which comprises an alternative possible sequence for the sequence of polynucleotides to be determined.

70. The method of claim 64, wherein the method comprises performing sequencing reactions with a first and second collection of probes, and the selecting step comprises:

(i) obtaining a second ordered list of probe family names from the template using the second collection of distinguishably labeled-probe families, wherein at least one probe family label in the second collection of probe families the probe families corresponds to probe sequences differently from at least one label in the first collection of probe families;

(ii) generating at least one comparison sequence from the second ordered list of probe family names;

(iii) comparing a portion of at least one of the candidate sequences with a portion of at least one of the comparison sequences; and

(iv) selecting a candidate sequence that exhibits a predetermined level of identity or is most nearly identical to a comparison sequence over the portion compared in step (b) as the sequence of nucleotides in the template.

71. The method of claim 70, wherein the portion compared is a single dinucleotide.

72. The method of claim 71, wherein the second ordered list of probe family names contains only a single element.

73. The method of claim 49, wherein the oligonucleotide probes in each probe family have the structure 5′

-(XY)(N)_kN_B*-3′

or 3′

-(XY)(N)_kN_B*-5′

, wherein N represents any nucleoside, N_B represents a moiety that is not extendable by ligase, * represents a detectable moiety, XY is a constrained portion of the probe in which X and Y represent nucleosides that are identical or different but are not independently selected, X and Y are at least 2-fold degenerate, at least one internucleotide linkage is a scissile linkage, and k is between 1 and 100, inclusive, with the proviso that a detectable moiety may be present on Y or on any nucleotide of (N)_k instead of, or in addition to, N_B.

74. The method of claim 73, wherein the scissile linkage is a phorphorothiolate linkage.

75. The method of claim 73, wherein four distinguishably labeled oligonucleotide probe families are used, and wherein oligonucleotide probes having different sequences for the constrained portion of the probe are assigned to first, second, third, and fourth probe families according to one of the 24 schemes set forth in Table 1.

76. The method of claim 49, wherein the detecting step comprises acquiring on average 2 bits of information simultaneously from each of at least 2 nucleotides in the template without acquiring two bits of information from any individual nucleotide.

77. The method of claim 49, wherein the detecting step comprises acquiring less than 2 bits of information simultaneously from each of at least 2 nucleotides in the template.

78. The method of claim 49, wherein the hybridized probe comprises a portion complementary to the portion of the template immediately adjacent to the duplex portion;

and the method comprises optionally generating a new extendable terminus on the extended duplex.

79. The method of claim 78, wherein the detecting step comprises acquiring on average 2 bits of information simultaneously from each of at least 2 nucleotides in the template without acquiring two bits of information from any individual nucleotide.

80. The method of claim 78, wherein the detecting step comprises acquiring less than 2 bits of information simultaneously from each of at least 2 nucleotides in the template.

81. The method of claim 51, wherein the extendable termini of the multiple initializing oligonucleotides are offset by one nucleotide from each other when hybridized to the template.

82. The method of claim 49, wherein the duplex is extended by N nucleotides during extension and the ordered series of probe family names obtained from step (d) provides sequence information for every Nth nucleotide on the template.

83. The method of claim 82, comprising assembling an ordered list in step (III) by combining N different ordered series of probe family names obtained from N sequencing reactions using N different initializing oligonucleotides.

84. The method of claim 83, wherein the extendable termini of the N different initializing oligonucleotides are offset by one nucleotide from each other when hybridized to the template.

85. The method of claim 49, further comprising comparing the ordered list of probe family names to an ordered list corresponding to a reference sequence, wherein a difference of a single isolated probe family between the lists is classified as a sequencing error.

86. The method of claim 49, wherein the template sequence is complementary to a polynucleotide of interest.

52. The method of claim , wherein the oligonucleotide probes in one or more probe families comprise a non-extendable moiety at one terminus.