Methods and compositions for nucleic acid sequencing
First Claim
1. A method for determining sequences of polynucleotides, comprising(a) performing a sequencing reaction that comprises at least 50 repeated cycles of(i) polymerase catalyzed incorporation of different types of nucleotide conjugates into a plurality of polynucleotides, wherein the different types of nucleotide conjugates comprise reversible terminator moieties, wherein the different types of nucleotide conjugates are simultaneously present and compete for incorporation into the polynucleotides during the polymerase catalyzed incorporation, and(ii) cleaving the reversible terminator moieties from nucleotide conjugates that are incorporated into the polynucleotides;
- (b) detecting signals in the sequencing reaction for the incorporation of three different types of the nucleotide conjugates into the polynucleotides, wherein signals are not detected for a fourth type of the nucleotide conjugates incorporated into the polynucleotides; and
(c) determining the incorporation of the fourth type of the nucleotide conjugates based on the detection pattern of the three different types of the nucleotide conjugates into the polynucleotides,thereby determining sequences comprising at least 50 sequential nucleotides of the polynucleotides, wherein the incorporation of the three different types of the nucleotide conjugates is determined from a signal state and wherein the incorporation of the fourth type of the nucleotide conjugates is determined from a dark state.
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Accused Products
Abstract
The present disclosure provides methods and systems for detecting multiple different nucleotides in a sample. In particular, the disclosure provides for detection of multiple different nucleotides in a sample utilizing fewer detection moieties than the number of nucleotides being detected and/or fewer imaging events than the number of nucleotides being detected.
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Citations
26 Claims
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1. A method for determining sequences of polynucleotides, comprising
(a) performing a sequencing reaction that comprises at least 50 repeated cycles of (i) polymerase catalyzed incorporation of different types of nucleotide conjugates into a plurality of polynucleotides, wherein the different types of nucleotide conjugates comprise reversible terminator moieties, wherein the different types of nucleotide conjugates are simultaneously present and compete for incorporation into the polynucleotides during the polymerase catalyzed incorporation, and (ii) cleaving the reversible terminator moieties from nucleotide conjugates that are incorporated into the polynucleotides; -
(b) detecting signals in the sequencing reaction for the incorporation of three different types of the nucleotide conjugates into the polynucleotides, wherein signals are not detected for a fourth type of the nucleotide conjugates incorporated into the polynucleotides; and (c) determining the incorporation of the fourth type of the nucleotide conjugates based on the detection pattern of the three different types of the nucleotide conjugates into the polynucleotides, thereby determining sequences comprising at least 50 sequential nucleotides of the polynucleotides, wherein the incorporation of the three different types of the nucleotide conjugates is determined from a signal state and wherein the incorporation of the fourth type of the nucleotide conjugates is determined from a dark state. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method for determining sequences of polynucleotides, comprising
(a) performing a sequencing reaction that comprises repeated cycles of (i) polymerase catalyzed incorporation of different types of nucleotide conjugates into a plurality of polynucleotides, wherein the different types of nucleotide conjugates comprise reversible terminator moieties, wherein the different types of nucleotide conjugates are simultaneously present and compete for incorporation into the polynucleotides during the polymerase catalyzed incorporation, and (ii) cleaving the reversible terminator moieties from nucleotide conjugates that are incorporated into the polynucleotides; -
(b) detecting signals in the sequencing reaction for the incorporation of three different types of the nucleotide conjugates into the polynucleotides, wherein the detecting of the signals comprises; (i) detecting a first type of the three different types of the nucleotide conjugates in a first channel; (ii) detecting a second type of the three different types of the nucleotide conjugates in a second channel; and (iii) detecting a third type of the three different types of the nucleotide conjugates in the first channel and in the second channel, wherein the third type of the three different types of the nucleotide conjugates comprises a mixture of different nucleotide conjugates that differ by being attached to a first fluorophore that is detected in the first channel or a second fluorophore that is detected in the second channel, wherein signals are not detected for a fourth type of the nucleotide conjugates incorporated into the polynucleotides; and (c) determining the incorporation of the fourth type of the nucleotide conjugates based on the detection pattern of the three different types of the nucleotide conjugates into the polynucleotides, thereby determining the sequences of the polynucleotides, wherein the incorporation of the three different types of the nucleotide conjugates is determined from a signal state and wherein the incorporation of the fourth type of the nucleotide conjugates is determined from a dark state. - View Dependent Claims (23, 24, 25, 26)
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Specification