Centroid markers for image analysis of high denisty clusters in complex polynucleotide sequencing
First Claim
1. A method for producing at least one nucleic acid cluster having an identifiable center, comprising:
- (I) generating, on a solid support, at least one immobilized nucleic acid complement of at least one nucleic acid template, wherein the at least one nucleic acid template or the at least one nucleic acid complement comprises a detectable label; and
(II) after step (I) amplifying the at least one nucleic acid template and the at least one nucleic acid complement to obtain on the solid support at least one nucleic acid cluster, wherein each cluster comprises (a) a substantially central location comprising the at least one nucleic acid template and the at least one nucleic acid complement, and (b) a surrounding region comprising one or more immobilized copies of the at least one nucleic acid template and of the at least one nucleic acid complement, and thereby producing the at least one nucleic acid cluster having an identifiable center.
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Accused Products
Abstract
Improved compositions, methods, apparatus, and kits for high-throughput nucleic acid amplification, detection and sequencing are disclosed. A nucleic acid cluster having an identifiable center is produced by generating on a solid support an immobilized nucleic acid complement from a template, one of which comprises a detectable label; and amplifying the complement and the template to obtain a nucleic acid cluster on the support, the cluster having a substantially central location marked by the detectable label and a surrounding region comprising immobilized copies. Also disclosed are nucleotide sequence determination in nucleic acid clusters so produced, center position annotation in the clusters, assignment of sequence information to overlapping clusters, and related compositions and methods.
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Citations
26 Claims
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1. A method for producing at least one nucleic acid cluster having an identifiable center, comprising:
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(I) generating, on a solid support, at least one immobilized nucleic acid complement of at least one nucleic acid template, wherein the at least one nucleic acid template or the at least one nucleic acid complement comprises a detectable label; and (II) after step (I) amplifying the at least one nucleic acid template and the at least one nucleic acid complement to obtain on the solid support at least one nucleic acid cluster, wherein each cluster comprises (a) a substantially central location comprising the at least one nucleic acid template and the at least one nucleic acid complement, and (b) a surrounding region comprising one or more immobilized copies of the at least one nucleic acid template and of the at least one nucleic acid complement, and thereby producing the at least one nucleic acid cluster having an identifiable center. - 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)
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24. A method for annotating a center position of at least one nucleic acid cluster, comprising:
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(I) generating, on a solid support, at least one immobilized nucleic acid complement of at least one nucleic acid template, wherein the at least one nucleic acid template or the at least one nucleic acid complement comprises a detectable center label; and (II) after step (I) amplifying the at least one nucleic acid template and the at least one nucleic acid complement to obtain on the solid support at least one nucleic acid cluster, wherein each cluster comprises (a) a substantially central location comprising the at least one nucleic acid template and the at least one nucleic acid complement, and (b) a surrounding region comprising one or more immobilized copies of the at least one nucleic acid template and of the at least one nucleic acid complement; and (III) recording a center position in the at least one nucleic acid cluster by detecting the detectable center label to identify the substantially central location in the at least one nucleic acid cluster as the center position, and thereby annotating the center position of the at least one nucleic acid cluster.
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25. A method for assigning nucleotide sequence information to at least one of two or more nucleic acid clusters that overlap on a solid support, comprising:
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(a) providing two or more nucleic acid clusters that detectably overlap on a solid support, each of said clusters comprising (i) a substantially central location comprising an immobilized central nucleic acid having a detectable center label, and (ii) a surrounding region comprising one or more immobilized copies of the immobilized central nucleic acid, wherein the detectable center label distinguishes the substantially central location from the surrounding region; (b) detecting the detectable center label in each of said overlapping nucleic acid clusters and therefrom identifying in each cluster a first position for the substantially central location on the solid support; (c) after step (b) determining a nucleotide sequence in at least one cluster of said overlapping nucleic acid clusters by performing one or a plurality of sequencing steps thereupon, thereby identifying a second position for the at least one cluster on the solid support; and (d) assigning a nucleotide sequence determined in (c) to a discrete nucleic acid cluster by substantially correlating (i) the first position for the substantially central location of (b) with (ii) the second position for the at least one cluster on the solid support of (c), and thereby assigning nucleotide sequence information to at least one of two or more nucleic acid clusters that overlap on the solid support.
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26. A composition comprising:
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(a) a solid support; and (b) one or more nucleic acid clusters each comprising a plurality of nucleic acids having the same sequence and being immobilized to the solid support, wherein each nucleic acid cluster comprises an identifiable center comprising a first subpopulation of the plurality of nucleic acids that is surrounded by a second subpopulation of the nucleic acids, wherein each of the nucleic acids in the first subpopulation comprises one or more detectable center labels that distinguish the nucleic acids in the first subpopulation from the nucleic acids in the second population, and wherein the second subpopulation of the nucleic acids lacks the detectable label.
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Specification