Reagents, methods, and libraries for bead-based sequencing
First Claim
1. 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, wherein:
- (a) the constrained portion comprises at least two constrained residues where each position in the constrained portion is at least 2-fold degenerate and less than all possible nucleotide combinations for the constrained portion are present within a single probe family, and where;
(i) at least two probe families include different subsets of all possible nucleotide combinations for the constrained portion, or(ii) all possible nucleotide combinations for the constrained portion are present in the collection, and(b) optionally, probes in each family comprise a scissile internucleoside linkage that can be cleaved under conditions that will not substantially cleave phosphodiester bonds.
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Accused Products
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. 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 immobilized beads. The invention further provides sets of labeled probes containing phosphorothiolate linkages. 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.
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Citations
57 Claims
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1. 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, wherein:
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(a) the constrained portion comprises at least two constrained residues where each position in the constrained portion is at least 2-fold degenerate and less than all possible nucleotide combinations for the constrained portion are present within a single probe family, and where; (i) at least two probe families include different subsets of all possible nucleotide combinations for the constrained portion, or (ii) all possible nucleotide combinations for the constrained portion are present in the collection, and (b) optionally, probes in each family comprise a scissile internucleoside linkage that can be cleaved under conditions that will not substantially cleave phosphodiester bonds. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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36. A collection of at least two distinguishably labeled oligonucleotide probe families, wherein each probe family comprises a plurality of oligonucleotide probes having identical labels but different sequences that comprise:
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(a) a constrained portion of least two constrained residues, wherein the constrained residues; (i) are at least 2-fold degenerate among the probes in the probe family, (ii) need not be located contiguously, and (iii) are not selected independently of each other, such that (a) knowing the label of a probe eliminates one or more combinations of nucleotides as possibilities for the sequence of the constrained portion, and (b) knowing the identity of a constrained residue in that probe further eliminates at least one possible identity for another constrained residue in the probe, and (b) optionally, an unconstrained portion comprising at least k nucleosides N, wherein k is at least 2, and wherein nucleosides N are independently selected and need not be located contiguously. - View Dependent Claims (37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57)
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Specification