Digital Counting of Individual Molecules by Stochastic Attachment of Diverse Labels
First Claim
1. A method of determining a presence of alleles in a genomic sample, the method comprising:
- a) attaching a plurality of oligonucleotides that comprise a label-tag comprising nucleotides selected from purine bases, pyrimidine bases, natural nucleotide bases, chemically modified nucleotide bases, biochemically modified nucleotide bases, non-natural nucleotide bases and derivatized nucleotide bases to nucleic acid molecules of a genomic sample that contain a polymorphic site, thereby producing a population of target-label-tag molecules in which each of the target-label-tag molecules that comprises the polymorphic site is attached to a label-tag of the plurality of oligonucleotides,b) amplifying the population of target-label-tag molecules, thereby producing amplified target-label-tag molecules that contain the polymorphic site;
c) detecting some of the amplified target-label-tag molecules by sequencing, thereby producing a plurality of readouts,wherein the plurality of readouts comprise (i) the nucleotide sequence of at least a portion of the polymorphic site and (ii) the nucleotide sequence of an associated label-tag to which the polymorphic site is attached;
d) determining, for one allele of the polymorphic site, a number of different label-tags that are associated with the allele;
e) repeating step d) for additional alleles of the polymorphic site, thereby determining a presence of the alleles in the genomic sample based on the number of different label-tags associated with the one allele and the additional alleles.
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Accused Products
Abstract
Compositions, methods and kits are disclosed for high-sensitivity single molecule digital counting by the stochastic labeling of a collection of identical molecules by attachment of a diverse set of labels. Each copy of a molecule randomly chooses from a non-depleting reservoir of diverse labels. Detection may be by a variety of methods including hybridization based or sequencing. Molecules that would otherwise be identical in information content can be labeled to create a separately detectable product that is unique or approximately unique in a collection. This stochastic transformation relaxes the problem of counting molecules from one of locating and identifying identical molecules to a series of binary digital questions detecting whether preprogrammed labels are present. The methods may be used, for example, to estimate the number of separate molecules of a given type or types within a sample.
64 Citations
38 Claims
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1. A method of determining a presence of alleles in a genomic sample, the method comprising:
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a) attaching a plurality of oligonucleotides that comprise a label-tag comprising nucleotides selected from purine bases, pyrimidine bases, natural nucleotide bases, chemically modified nucleotide bases, biochemically modified nucleotide bases, non-natural nucleotide bases and derivatized nucleotide bases to nucleic acid molecules of a genomic sample that contain a polymorphic site, thereby producing a population of target-label-tag molecules in which each of the target-label-tag molecules that comprises the polymorphic site is attached to a label-tag of the plurality of oligonucleotides, b) amplifying the population of target-label-tag molecules, thereby producing amplified target-label-tag molecules that contain the polymorphic site; c) detecting some of the amplified target-label-tag molecules by sequencing, thereby producing a plurality of readouts, wherein the plurality of readouts comprise (i) the nucleotide sequence of at least a portion of the polymorphic site and (ii) the nucleotide sequence of an associated label-tag to which the polymorphic site is attached; d) determining, for one allele of the polymorphic site, a number of different label-tags that are associated with the allele; e) repeating step d) for additional alleles of the polymorphic site, thereby determining a presence of the alleles in the genomic sample based on the number of different label-tags associated with the one allele and the additional alleles. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A method for enhancing a determination of the presence of alleles in a genomic sample, said method comprising:
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a) attaching a set of oligonucleotides that comprises a degenerate base region (DBR) comprising at least one nucleotide base selected from;
R, Y, S, W, K, M, B, D, H, V, N and modified versions thereof to the nucleic acid molecules of a genomic sample that contains a polymorphic target region, thereby producing a population of adapter-attached polynucleotides in which each of said adaptor-attached polynucleotides that comprises said polymorphic target region is attached to a DBR sequence of an oligonucleotide of said set of oligonucleotides, wherein said oligonucleotides further comprise a unique multiplex identifier (MID) sequence that identifies a source for each of the nucleic acid molecules to which it is attached;b) amplifying the adapter-attached polynucleotides, thereby producing amplified adapter-attached polynucleotides that contain the polymorphic target region; c) sequencing some of the amplified adapter-attached polynucleotides that contain the polymorphic target region, thereby producing a plurality of sequences, wherein the sequencing step provides, for each of the amplified adaptor-attached polynucleotides that contain the polymorphic target region and are sequenced; (i) the nucleotide sequence of at least a portion of said polymorphic target region and (ii) the DBR sequence of the oligonucleotide to which said polymorphic target region is attached; d) determining, for one allele of the polymorphic target region, the number of different DBR sequences in the oligonucleotides that are associated with said allele; and e) repeating step d) for additional alleles of said polymorphic target region, thereby enhancing said determination of the presence of said alleles in said genomic sample based on the numbers of different DBR sequences in the oligonucleotides that are associated with said allele and said additional alleles. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38)
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Specification