Reagents, Methods, and Libraries for Bead-Based Sequencing
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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.
109 Citations
379 Claims
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1-354. -354. (canceled)
- 355. A collection of at least two distinguishably labeled oligonucleotide probe families, wherein probes in each probe family comprise a constrained portion and an unconstrained portion, each position in the constrained portion is at least 2-fold degenerate, and probes in each family comprise a scissile internucleoside linkage.
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363. A collection of at least two distinguishably labeled oligonucleotide probe families, wherein the oligonucleotide probes in each probe family have the structure 5′
- -(X)j(N)kNB-3′
or 3′
-(X)j(N)kNB-5′
, wherein N represents any nucleoside or an abasic residue, NB represents a moiety that is not extendable by ligase, (X)j is a constrained portion of the probe in which each X represents a nucleoside or abasic residue, with the proviso that Xl represents a nucleotide, and nucleosides in (X)j are identical or different but are not independently selected, each X is at least 2-fold degenerate, j is between 2 and 5, k is between 1 and 100, inclusive, each probe comprises a detectable moiety at a position other than the nucleoside in (X)j which is at the probe terminus, and wherein probes in each probe family comprise the same label and probes in different probe families comprise different distinguishable labels. - View Dependent Claims (364, 365, 366, 367, 368, 369)
- -(X)j(N)kNB-3′
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370. A collection of at least two distinguishably labeled oligonucleotide probe families, wherein the oligonucleotide probes in each probe family have the structure 5′
- -(X)j(N)kNB-3′
or 3′
-(X)j(N)kNB-5′
, wherein N represents any nucleoside or an abasic residue, NB represents a moiety that is not extendable by ligase, (X)j is a constrained portion of the probe in which each X represents a nucleoside or abasic residue, with the proviso that Xl represents a nucleotide, and nucleosides in (X)j are identical or different but are not independently selected, each X is at least 2-fold degenerate, j is between 2 and 5, k is between 1 and 100, inclusive, each probe comprises a detectable moiety at a position other than the nucleoside in (X)j which is at the probe terminus, and wherein probes in each probe family comprise the same label and probes in different probe families comprise different distinguishable labels. - View Dependent Claims (371, 372, 373, 374, 375, 377)
- -(X)j(N)kNB-3′
- 378. A kit comprising a collection of at least two distinguishably labeled oligonucleotide probe families, wherein the probes comprise a phosphorothiolate linkage.
Specification