Methods for DNA sequencing and analysis using multiple tiers of aliquots
First Claim
1. A method of processing genomic DNA, comprising:
- preparing a first tier of aliquots, at least some of which contain one or more polynucleotides that include a nucleotide sequence from a portion of a genome tagged with an oligonucleotide such that polynucleotides present in different aliquots have different tag sequences, and polynucleotides present in the same aliquot of the first tier have the same tag sequence;
preparing a second tier of aliquots;
at least some of which contain one or more of the polynucleotides from the first tier of aliquots; and
producing multiple fragments from the polynucleotides in the second tier of aliquots, wherein the fragments include a nucleotide sequence from a portion of the genome.
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Accused Products
Abstract
The invention provides methods and kits for ordering sequence information derived from one or more target polynucleotides. In one aspect, one or more tiers or levels of fragmentation and aliquoting are generated, after which sequence information is obtained from fragments in a final level or tier. Each fragment in such final tier is from a particular aliquot, which, in turn, is from a particular aliquot of a prior tier, and so on. For every fragment of an aliquot in the final tier, the aliquots from which it was derived at every prior tier is known, or can be discerned. Thus, identical sequences from overlapping fragments from different aliquots can be distinguished and grouped as being derived from the same or different fragments from prior tiers. When the fragments in the final tier are sequenced, overlapping sequence regions of fragments in different aliquots are used to register the fragments so that non-overlapping regions are ordered. In one aspect, this process is carried out in a hierarchical fashion until the one or more target polynucleotides are characterized, e.g. by their nucleic acid sequences, or by an ordering of sequence segments, or by an ordering of single nucleotide polymorphisms (SNPs), or the like.
181 Citations
20 Claims
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1. A method of processing genomic DNA, comprising:
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preparing a first tier of aliquots, at least some of which contain one or more polynucleotides that include a nucleotide sequence from a portion of a genome tagged with an oligonucleotide such that polynucleotides present in different aliquots have different tag sequences, and polynucleotides present in the same aliquot of the first tier have the same tag sequence; preparing a second tier of aliquots;
at least some of which contain one or more of the polynucleotides from the first tier of aliquots; andproducing multiple fragments from the polynucleotides in the second tier of aliquots, wherein the fragments include a nucleotide sequence from a portion of the genome. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method of processing genomic DNA, comprising:
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preparing a first tier of aliquots, at least some of which contain one or more polynucleotides that include a nucleotide sequence from a portion of a genome; preparing a second tier of aliquots;
at least some of which contain one or more of the polynucleotides from the first tier of aliquots;producing multiple fragments from the polynucleotides in the second tier of aliquots, wherein the fragments include a nucleotide sequence from a portion of the genome; obtaining sequence reads from each of a plurality of the fragments from the second tier of aliquots; and
thencharacterizing the genome by a process that comprises grouping sequence reads from portions of the genome represented in the same aliquot in the first tier, and grouping sequence reads from portions of the genome represented in the same aliquot in the second tier. - View Dependent Claims (11, 12, 13, 14, 15, 16)
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17. A method of processing a target polynucleotide, comprising:
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preparing a first tier of aliquots, at least some of which contain one or more polynucleotides that include a nucleotide sequence from a portion of a target polynucleotide tagged with an oligonucleotide such that polynucleotides present in different aliquots have different tag sequences, and polynucleotides present in the same aliquot of the first tier have the same tag sequence; preparing a second tier of aliquots;
at least some of which contain one or more of the polynucleotides from the first tier of aliquots; andproducing multiple fragments from the polynucleotides in the second tier of aliquots, wherein the fragments include a nucleotide sequence from a portion of the target polynucleotide.
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18. A method of processing a target polynucleotide, comprising:
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preparing a first tier of aliquots, at least some of which contain one or more polynucleotides that include a nucleotide sequence from a portion of a target polynucleotide; preparing a second tier of aliquots;
at least some of which contain one or more of the polynucleotides from the first tier of aliquots;producing multiple fragments from the polynucleotides in the second tier of aliquots, wherein the fragments include a nucleotide sequence from a portion of the target polynucleotide; obtaining sequence reads from each of a plurality of the fragments from the second tier of aliquots; and
thencharacterizing the target polynucleotide by a process that comprises grouping sequence reads from portions of the target polynucleotide represented in the same aliquot in the first tier, and grouping sequence reads from portions of the target polynucleotide represented in the same aliquot in the second tier. - View Dependent Claims (19, 20)
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Specification