Massively parallel contiguity mapping
First Claim
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1. A method for capturing contiguity information comprising:
- (a) producing tagged end fragments of a nucleic acid molecule in situ on a flowcell by;
(i) adding a flowcell compatible end adaptor to each end of a target DNA molecule, wherein each of the end adaptors comprises a first flowcell sequence;
(ii) physically constraining each end of the target DNA molecule at locations on a flowcell by permitting the first flowcell sequences of each end adaptor to hybridize to flowcell surface-bound primers; and
(iii) treating the target DNA molecule physically constrained to the flowcell with at least one transposase loaded with flowcell compatible insertion adaptors resulting in fragmentation of the target DNA molecule and insertion of an insertion adaptor to each resulting fragment end to provide tagged end fragments of the target DNA molecule that remain physically constrained to the flowcell at their respective locations in step (ii), wherein the insertion adaptors comprise a second flowcell sequence;
(b) performing cluster amplification of the tagged end fragments on the flowcell to produce clusters;
(c) sequencing the tagged end fragments; and
(d) capturing contiguity information by associating sequences of the tagged end fragments to provide paired-end reads of the nucleic acid molecule.
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Abstract
Contiguity information is important to achieving high-quality de novo assembly of mammalian genomes and the haplotype-resolved resequencing of human genomes. The methods described herein pursue cost-effective, massively parallel capture of contiguity information at different scales.
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Citations
29 Claims
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1. A method for capturing contiguity information comprising:
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(a) producing tagged end fragments of a nucleic acid molecule in situ on a flowcell by; (i) adding a flowcell compatible end adaptor to each end of a target DNA molecule, wherein each of the end adaptors comprises a first flowcell sequence; (ii) physically constraining each end of the target DNA molecule at locations on a flowcell by permitting the first flowcell sequences of each end adaptor to hybridize to flowcell surface-bound primers; and (iii) treating the target DNA molecule physically constrained to the flowcell with at least one transposase loaded with flowcell compatible insertion adaptors resulting in fragmentation of the target DNA molecule and insertion of an insertion adaptor to each resulting fragment end to provide tagged end fragments of the target DNA molecule that remain physically constrained to the flowcell at their respective locations in step (ii), wherein the insertion adaptors comprise a second flowcell sequence; (b) performing cluster amplification of the tagged end fragments on the flowcell to produce clusters; (c) sequencing the tagged end fragments; and (d) capturing contiguity information by associating sequences of the tagged end fragments to provide paired-end reads of the nucleic acid molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 12, 13, 14, 15, 16, 17, 18)
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9. A method for inferring chromosome conformation comprising:
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a) generating DNA-DNA crosslinks within cells; b) isolating cross-linked DNA from cells; c) fragmenting the cross-linked DNA to provide fragmented, cross-linked DNA molecules; d) end-modifying the fragmented, cross-linked DNA molecules with an end adaptor that comprises a first flowcell sequence that is complementary to or that corresponds to a first surface-bound primer; e) physically constraining each end of the fragmented, cross-linked DNA molecules by hybridizing ends of the fragmented, end-modified target DNA molecules to the first surface-bound primer; f) performing transposition on the hybridized, end-modified target DNA molecules from step e) with non-surface-bound transposase complexes, each non-surface-bound transposase complex comprising a DNA transposase and one or more insertion adaptors that comprise a second flowcell sequence that is complementary to or that corresponds to a second surface-bound primer; g) performing cluster amplification on the hybridized, end-modified target DNA molecules from step f) to produce clusters of clonally derived nucleic acids; h) sequencing the clusters of clonally derived nucleic acids without a step of intramolecular ligation; and i) determining physical interactions between chromosomal positions by paring neighboring clusters together. - View Dependent Claims (10, 11)
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19. A method for capturing contiguity information for non-overlapping, directly adjacent fragments of a target DNA sequence, comprising:
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(a) producing symmetrically tagged non-overlapping fragments of the target DNA sequence by treating the target DNA sequence with a transposase that fragments the target DNA at a site and adds identical or complementary barcode sequences at each flank of the fragmentation site; (b) sequencing the symmetrically tagged non-overlapping fragments to produce independent sequencing reads; and (c) capturing contiguity information by identifying the identical or complementary barcode of each of the symmetrically tagged non-overlapping fragments and assigning a join between independent sequencing reads of the adjacent fragments of a target DNA sequence. - View Dependent Claims (20, 21, 22, 23)
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24. A method for capturing contiguity information for non-overlapping fragments of a target DNA sequence, comprising:
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(a) producing tagged fragments of the target DNA sequence by; (i) treating the target DNA sequence with a transposase resulting in one or more fragmentation events; (ii) during or after step (a)(i), adding or inserting one or more tags to one or more fragments of the target DNA sequence produced in step (a)(i), wherein the tags comprise an identical or complementary barcode sequence; and (iii) prior to or after step (a)(i) but before step (a)(ii), compartmentalizing the target DNA sequence; wherein steps (i), (ii), and (iii) create a plurality of tagged target DNA fragments each comprising an identical or complementary barcode sequence; (b) sequencing the tagged target DNA fragments to produce independent sequencing reads of the target DNA sequence; and (c) capturing contiguity information by identifying the identical or complementary barcode of each of the tagged target DNA fragments and assigning the independent sequencing reads of the target DNA sequence to the same target DNA sequence prior to fragmentation. - View Dependent Claims (25, 26, 27, 28, 29)
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Specification