Methods and compositions for nucleic acid sequence analysis
First Claim
1. A method of determining nucleotide sequences of a population of polynucleotides, the method comprising the steps of. attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide of the population to form tag-polynucleotide conjugates;
- generating a size ladder of polynucleotide fragments for each tag-polynucleotide conjugate by an extension reaction, each polynucleotide fragment of the same size ladder having an end and the same oligonucleotide tag as every other polynucleotide fragment of the size ladder and each polynucleotide fragment for each tag-polynucleotide conjugate differing in length by one or more nucleotides;
separating the polynucleotide fragments to form a plurality of fractions;
copying and labeling the oligonucleotide tag of each polynucleotide fragment in each fraction according to the identity of one or more nucleotides at the end of such polynucleotide fragments;
hybridizing the labeled oligonucleotide tags of each fraction with their respective complements tinder stringent hybridization conditions, the respective complements each being attached to a spatially discrete region on a solid phase support; and
detecting a sequence of signals from the labels of oligonucleotide tags hybridized to the solid phase support to determine the nucleotide sequences of the polynucleotides of the population.
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Abstract
The invention provides methods, kits and materials for determinining simultaneously signature sequences of a population of tagged polynucleotides. Tags comprise at least two parts: a hybridization tag and a correlation tag. Size ladders of polynucleotide fragments are generated from the population of tagged polynucleotides that contain a plurality of size classes. After the size classes are separated, hybridization tags of the separated fragments are copied and labeled according to the identity of one or more bases at the ends of the fragments. In a preferred embodiment, the labeled tags are specifically hybridized to a plurality of random microarrays of tag complements. Signals generated at hybridization sites of different random microarrays are correlated by sequencing of the unique correlation tag. Signature sequences are determined by signals generated at hybridization sites having the same correlation tag on each of the plurality of random microarrays.
172 Citations
16 Claims
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1. A method of determining nucleotide sequences of a population of polynucleotides, the method comprising the steps of.
attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide of the population to form tag-polynucleotide conjugates; -
generating a size ladder of polynucleotide fragments for each tag-polynucleotide conjugate by an extension reaction, each polynucleotide fragment of the same size ladder having an end and the same oligonucleotide tag as every other polynucleotide fragment of the size ladder and each polynucleotide fragment for each tag-polynucleotide conjugate differing in length by one or more nucleotides;
separating the polynucleotide fragments to form a plurality of fractions;
copying and labeling the oligonucleotide tag of each polynucleotide fragment in each fraction according to the identity of one or more nucleotides at the end of such polynucleotide fragments;
hybridizing the labeled oligonucleotide tags of each fraction with their respective complements tinder stringent hybridization conditions, the respective complements each being attached to a spatially discrete region on a solid phase support; and
detecting a sequence of signals from the labels of oligonucleotide tags hybridized to the solid phase support to determine the nucleotide sequences of the polynucleotides of the population. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method of determinining nucleotide sequences of a population of polynucleotides, the method comprising the steps of:
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generating a size ladder of polynucleotide fragments by an extension reaction, each polynucleotide fragment of the same size ladder having an end and an oligonucleotide tag that is the same for every polynucleotide fragment of the size ladder, the oligonucleotide tag being selected from a minimally cross-hybridizing set of oligonucleotides;
separating the polynucleotide fragments to form a plurality of fractions;
copying and labeling the oligonucleotide tag of each polynucleotide fragment in each fraction according to the identity of one or more nucleotides at the end of such polynucleotide fragments;
hybridizing the labeled oligonucleotide tags of each fraction with their respective complements under stringent hybridization conditions, the respective complements each being attached to a spatially discrete region on a solid phase support; and
detecting a sequence of signals from the labels of oligonucleotide tags hybridized to the solid phase support to determine the nucleotide sequences of the polynucleotides of the population. - View Dependent Claims (11, 12, 13, 15)
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14. A method of monitoring a population of polynucleotides in a reaction using oligonucleotide tags, the method comprising the steps of:
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forming tag-polynucleotide conjugates between polynucleotides of the population and oligonucleotide tags of a tag repertoire such that substantially every oligonucleotide tag of the repertoire forms a tag-polynucleotide conjugate with substantially every polynucleotide of the population;
isolating a sample of the tag-polynucleotide conjugates having a size less than or substantially equal to that of the tag repertoire;
conducting a reaction with a plurality of reaction outcomes on the sample, such that each tag-polynucleotide conjugate of the sample has a single reaction outcome;
copying and labeling each oligonucleotide tag of a tag-polynucleotide conjugate according to its reaction outcome such that tag-polynucleotide conjugates having different reaction outcomes have oligonucleotide tags with distinguishable labels;
hybridizing the labeled oligonucleotide tags of each tag-polynucleotide conjugate with their respective complements under stringent hybridization conditions, the respective complements each being attached to a spatially discrete region on a solid phase support; and
detecting signals from the labels of oligonucleotide tags hybridized to the solid phase support to determine reaction outcomes of the polynucleotides of the population.
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16. A method of measuring relative genomic amplification over a genome, the method comprising the steps of:
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providing a partition of a genome, the partition comprising a plurality of fragments uniformly distributed over the genome, each fragment having a genomic location;
generating a signature sequence from each fragment; and
tabulating signature sequences of the fragments at each genomic location; and
determining relative genomic amplification by a relative abundance of each fragment from the tabulated signature sequences.
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Specification