Reagents, methods, and libraries for bead-based sequencing
First Claim
1. A method for determining a sequence of a nucleic acid template, comprising:
- a) generating a temporally ordered series of signals by interrogating the template, wherei. each signal is derived from and provides sequence information about a corresponding stretch of template comprising two or more consecutive nucleotides and is generated at a different time from any preceding signal; and
ii. each signal'"'"'s corresponding stretch of template overlaps with at least one preceding signal'"'"'s corresponding stretch of template by at least one but not all nucleotides;
b) recording the temporally ordered series of signals on a computer-readable storage medium;
c) obtaining from one or more signals any corresponding sequence information for each nucleotide in the signal'"'"'s corresponding stretch of template; and
d) comparing sequence information provided by a first signal for one or more consecutive nucleotides with sequence information provided by a preceding signal for the same one or more consecutive nucleotides.
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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 extension 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
26 Claims
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1. A method for determining a sequence of a nucleic acid template, comprising:
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a) generating a temporally ordered series of signals by interrogating the template, where i. each signal is derived from and provides sequence information about a corresponding stretch of template comprising two or more consecutive nucleotides and is generated at a different time from any preceding signal; and ii. each signal'"'"'s corresponding stretch of template overlaps with at least one preceding signal'"'"'s corresponding stretch of template by at least one but not all nucleotides; b) recording the temporally ordered series of signals on a computer-readable storage medium; c) obtaining from one or more signals any corresponding sequence information for each nucleotide in the signal'"'"'s corresponding stretch of template; and d) comparing sequence information provided by a first signal for one or more consecutive nucleotides with sequence information provided by a preceding signal for the same one or more consecutive nucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method for determining the sequence of a template nucleic acid, comprising:
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a) recording on a computer-readable storage medium a first signal corresponding to a first stretch of two to five consecutive nucleotides of the template, and a second signal corresponding to a second stretch of the template where the second stretch (i) overlaps with the first stretch but differs by at least one nucleotide from the first stretch, and (ii) has the same number of nucleotides as the first stretch; b) determining the identity of the first signal and the second signal; c) identifying a first subset of one or more possible sequences for the first stretch of the template based solely on the identity of the first signal, and identifying a second subset of one or more possible sequences for the second stretch of the template based solely on the identity of the second signal; and d) comparing the first subset and the second subset. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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Specification