Digital Counting of Individual Molecules by Stochastic Attachment of Diverse Labels

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.

That which is claimed is: 
     
         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.   
     
     
         2 . The method of  claim 1 , further comprising:
 f) using a probability analysis to determine the presence of each of the alleles in the genomic sample using the numbers of the different label-tags associated with the allele and the additional alleles; and   g) determining a genotype for the genomic sample based on the probability analysis in step f).   
     
     
         3 . The method of  claim 1 , wherein the oligonucleotides comprise a label-tag comprising at least 2 nucleotide bases, wherein each of the at least 2 nucleotide bases is 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. 
     
     
         4 . The method of  claim 3 , wherein the label-tag comprises from 2 to 20 nucleotide bases, wherein each of the 2 to 20 nucleotide bases is selected purine bases, pyrimidine bases, natural nucleotide bases, chemically modified nucleotide bases, biochemically modified nucleotide bases, non-natural nucleotide bases and derivatized nucleotide bases. 
     
     
         5 . The method of  claim 1 , wherein the oligonucleotides further comprises a universal primer sequence. 
     
     
         6 . The method of  claim 1 , wherein detecting some of the amplified target-label-tag molecules by sequencing comprises sequencing some of the amplified target-label-tag molecules on a next-generation sequencing platform. 
     
     
         7 . The method of  claim 1 , wherein the genomic sample containing the polymorphic site is enriched prior to the attaching step. 
     
     
         8 . The method of  claim 1 , wherein the genomic sample containing the polymorphic site is enriched prior to detecting the amplified target-label-tag molecules by sequencing. 
     
     
         9 . The method of  claim 1 , wherein the genomic sample comprises nucleic acid molecules from tumor cells. 
     
     
         10 . The method of  claim 1 , wherein the genomic sample comprises nucleic acid molecules from bacteria. 
     
     
         11 . The method of  claim 1 , wherein the attaching step comprises ligating the plurality of oligonucleotides to the nucleic acid molecules. 
     
     
         12 . The method of  claim 1 , wherein the plurality of oligonucleotides is primers and the attaching step is done by primer extension. 
     
     
         13 . The method of  claim 1 , wherein the genomic sample comprises a plurality of genomic samples. 
     
     
         14 . The method of  claim 13 , wherein the plurality of genomic samples is from a human subject. 
     
     
         15 . The method of  claim 13 , wherein the plurality of genomic samples comprises nucleic acids from tumor cells. 
     
     
         16 . The method of  claim 1 , wherein the genomic sample comprises polynucleotides encoding viral epitopes. 
     
     
         17 . The method of  claim 1 , wherein the oligonucleotides further comprise a unique sequence tag that is used to distinguish mixed genomes in the genomic sample. 
     
     
         18 . The method of  claim 17 , wherein the method further comprises combining the population of target-label-tag molecules with other populations of target-label-tag molecules, and wherein each of the populations of target-label-tag molecules is associated with the unique sequence tag. 
     
     
         19 . A method for enhancing a determination of the presence of alleles in a genomic sample, said method comprising:
 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.   
     
     
         20 . The method of  claim 19 , further comprising: f) independently calculating the likelihood that each of the alleles is present in said genomic sample using the numbers of different DBR sequences in the oligonucleotides that are associated with said allele and said additional alleles; and g) calling a genotype for said genomic sample based on the likelihood calculated in step f). 
     
     
         21 . The method of  claim 19 , wherein said oligonucleotides comprise a degenerate base region comprises at least 2 nucleotide bases, wherein each of the at least 2 nucleotide bases are selected from: R, Y, S, W, K, M, B, D, H, V, N, and modified versions thereof. 
     
     
         22 . The method of  claim 21 , wherein the degenerate base region comprises from 3 to 20 nucleotide bases, wherein each of the 3 to 10 nucleotide bases is selected from: R, Y, S, W, K, M, B, D, H, V, N, and modified versions thereof. 
     
     
         23 . The method of  claim 19 , wherein the oligonucleotides further comprises a sequencing primer site. 
     
     
         24 . The method of  claim 19 , wherein said method further comprises combining said population of adapter-attached polynucleotides with other populations of adaptor attached polynucleotides, and wherein each of said populations of adaptor attached polynucleotides is associated with said unique MID sequence. 
     
     
         25 . The method of  claim 19 , wherein said sequencing step comprises sequencing said some of the amplified adapter-attached polynucleotides on a next-generation sequencing platform. 
     
     
         26 . The method of  claim 19 , wherein the genomic sample containing said polymorphic target region is enriched prior to the attaching step. 
     
     
         27 . The method of  claim 19 , wherein the genomic sample containing said polymorphic target region is enriched prior to the sequencing step. 
     
     
         28 . The method of  claim 19 , wherein the genomic sample comprises polynucleotides made from a tumor tissue. 
     
     
         29 . The method of  claim 19 , wherein the genomic sample comprises polynucleotides made from microorganisms and/or viruses. 
     
     
         30 . The method of  claim 19 , wherein said attaching step comprises ligating said set of oligonucleotides to the nucleic acid molecules of said genomic sample. 
     
     
         31 . The method of  claim 19 , wherein said set of oligonucleotides are primers and the attaching step is done by primer extension. 
     
     
         32 . The method of  claim 19 , wherein said genomic sample comprises multiple genomic samples derived from different sources. 
     
     
         33 . The method of  claim 32 , wherein each of the multiple genomic samples is derived from a different subject. 
     
     
         34 . The method of  claim 32 , wherein each of the multiple genomic samples is derived from a different human subject. 
     
     
         35 . The method of  claim 32 , wherein the multiple genomic samples are derived from different sections of a tumor. 
     
     
         36 . The method of  claim 32 , wherein the multiple genomic samples are derived from different tumors from a subject. 
     
     
         37 . The method of  claim 32 , wherein the multiple genomic samples are derived from a subject over time. 
     
     
         38 . The method of  claim 37 , wherein the polymorphic target region is in a viral sample.