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 comprising:
 a) amplifying a population of different target DNA molecules from a tagged genomic sample thereby producing a population of amplified target DNA molecules,   wherein the different target DNA molecules that comprise a polymorphic site are tagged with different label-tags, wherein the label-tags comprise 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; and   wherein each of the population of amplified target DNA molecules comprises the polymorphic site and an associated label-tag of the different label-tags; and   b) detecting the plurality of amplified target DNA molecules by sequencing, thereby producing a plurality of readouts, wherein the plurality of readouts comprises: (i) at least a portion of the target region; and (ii) an associated label-tag of the label-tags.   
     
     
         2 . The method of  claim 1 , further comprising: c) assessing a presence of an allele of the polymorphic site in the tagged genomic sample based on:
 (i) a determination of a number of the different label-tags that are associated with the allele; and   (ii) a determination of a number of readouts that comprise each of the different label-tags that are associated with the allele.   
     
     
         3 . The method of  claim 2 , wherein the assessing step is done by a computer that is programmed to perform the assessing step. 
     
     
         4 . The method of  claim 2 , wherein: the assessing step further comprises independently assessing a presence of an additional allele of the polymorphic site in the tagged genomic sample based on: (i) a determination of a number of the different label-tags that are associated with the additional allele of the polymorphic site; and (ii) a determination of a number of readouts that comprise each of the different label-tags that are associated with the additional allele. 
     
     
         5 . The method of  claim 2 , wherein the assessing of step c) comprises performing a statistical analysis. 
     
     
         6 . The method of  claim 2 , wherein the method further comprises determining an amount of the allele in the tagged genomic sample. 
     
     
         7 . The method of  claim 1 , wherein the population of different target DNA molecules is made by ligating a set of adaptors that comprise the label-tags to an initial nucleic acid sample. 
     
     
         8 . The method of  claim 7 , wherein the initial nucleic acid sample is an amplification product. 
     
     
         9 . The method of  claim 1 , wherein the population of different target DNA molecules is made by extending a set of primers that comprises the label-tags, using an initial nucleic acid sample as a template. 
     
     
         10 . The method of  claim 9 , wherein the initial nucleic acid sample is an amplification product. 
     
     
         11 . The method of  claim 1 , wherein the method comprises, prior to the amplifying step (a), enriching the population of different target DNA molecules from an initial nucleic acid sample. 
     
     
         12 . The method of  claim 1 , wherein the label-tags comprise 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. 
     
     
         13 . The method of  claim 12 , wherein the label-tags comprise at least 8 nucleotide bases, wherein each of the at least 8 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. 
     
     
         14 . The method of  claim 12 , wherein the label-tags comprise from 2 to 20 nucleotide bases, wherein each of the 2 to 20 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. 
     
     
         15 . The method of  claim 1 , wherein the label-tag is used to correct estimation errors. 
     
     
         16 . The method of  claim 1 , wherein the different target DNA molecules that comprise a polymorphic site are tagged with a unique sequence tag that is used to distinguish target DNA molecules from different samples. 
     
     
         17 . The method of  claim 1 , wherein the tagged genomic sample is a mixed sample comprising nucleic acid molecules from different samples, wherein each of the samples is associated with a different unique sequence tag. 
     
     
         18 . The method of  claim 17 , wherein each of the different samples is derived from a human subject. 
     
     
         19 . The method of  claim 17 , wherein each of the different samples comprises nucleic acids from tumor cells. 
     
     
         20 . The method of  claim 1 , wherein the tagged genomic samples comprise nucleic acids from tumor cells. 
     
     
         21 . The method of  claim 1 , wherein the tagged genomic sample comprises polynucleotides from bacteria and/or polynucleotides encoding viral epitopes. 
     
     
         22 . The method of  claim 1 , wherein the tagged genomic sample comprises human genomic DNA and the polymorphic site comprises a single nucleotide polymorphism of a human genome. 
     
     
         23 . The method of  claim 1 , detecting the population of amplified target DNA molecules by sequencing comprises sequencing the plurality of amplified target DNA molecules on a next-generation sequencing platform. 
     
     
         24 . The method of  claim 1 , wherein the amplifying step is done by polymerase chain reaction. 
     
     
         25 . The method of  claim 16 , wherein each of the population of amplified target DNA molecules further comprises the unique sequence tag. 
     
     
         26 . The method of  claim 25 , wherein the plurality of readouts further comprises the unique sequence tag. 
     
     
         27 . A method of sequencing, comprising:
 a) amplifying a population of distinct initial target DNA molecules from a tagged genomic sample thereby producing a population of amplified target DNA molecules, wherein the distinct initial target DNA molecules that comprise a polymorphic target sequence are tagged with:
 (i) different degenerate base region (DBR) sequences, wherein said DBR sequences comprise at least one nucleotide base selected from: R, Y, S, W, K, M, B, D, H, V, N and modified versions thereof and 
 (ii) a unique multiplex identifier (MID) sequence that identifies a source for each of the initial target DNA molecules to which it is associated, and wherein each of a plurality of the amplified target DNA molecules comprises said polymorphic target sequence, an associated DBR sequence of said different DBR sequences and said unique MID sequence; and 
   b) sequencing the plurality of the amplified target DNA molecules, thereby producing a plurality of sequence reads, wherein the sequencing step provides, for each of the amplified target DNA molecules that are sequenced: the nucleotide sequence of:
 (i) at least a portion of the polymorphic target sequence; 
 (ii) an associated DBR sequence of said DBR sequences; and 
 (iii) said unique MID sequence. 
   
     
     
         28 . The method of  claim 27 , further comprising: c) assessing the presence of an allele of said polymorphic target sequence region in said tagged genomic sample based on: (i) a determination of the number of said different DBR sequences that are associated with said allele; (ii) a determination of the number of said sequence reads that comprise each of the different DBR sequences that are associated with said allele. 
     
     
         29 . The method of  claim 28 , wherein the assessing step is done by a computer that is programmed to perform the assessing step. 
     
     
         30 . The method of  claim 28 , wherein: the assessing step further comprises independently assessing the presence of an additional allele of the polymorphic target sequence in said tagged genomic sample based on: (i) a determination of the number of said different DBR sequences that are associated with the additional allele of said polymorphic target sequence; and (ii) a determination of the number of said sequence reads that comprise each of the different DBR sequences that are associated with the additional allele. 
     
     
         31 . The method of  claim 28 , wherein the assessing step comprises performing a maximum likelihood analysis. 
     
     
         32 . The method of  claim 28 , wherein the method further comprises determining the amount of the allele in the tagged genomic sample. 
     
     
         33 . The method of  claim 27 , wherein said population of distinct initial target DNA molecules is made by ligating a set of adaptors that comprise said DBR sequences to an initial nucleic acid sample. 
     
     
         34 . The method of  claim 33 , wherein said initial nucleic acid sample is an amplification product. 
     
     
         35 . The method of  claim 27 , wherein said population of distinct initial target DNA molecules is made by extension of a set of primers that comprises said DBR sequences, using an initial nucleic acid sample as a template. 
     
     
         36 . The method of  claim 35 , wherein said initial nucleic acid sample is an amplification product. 
     
     
         37 . The method of  claim 27 , wherein the method comprises, prior to the amplifying step (a), enriching said population of distinct initial target DNA molecules from an initial nucleic acid sample. 
     
     
         38 . The method of  claim 27 , wherein said DBR sequences comprise 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. 
     
     
         39 . The method of  claim 38 , wherein the DBR sequences comprise 10 or more nucleotide bases, wherein each of the 10 or more nucleotide bases is selected from: R, Y, S, W, K, M, B, D, H, V, N, and modified versions thereof. 
     
     
         40 . The method of  claim 38 , wherein the DBR sequences comprise from 3 to 10 nucleotide bases, wherein each of the 3 to 10 nucleotide bases is selected from: R, Y, S, W, M, B, D, H, V, N, and modified versions thereof. 
     
     
         41 . The method of  claim 27 , wherein the DBR sequences comprise an error correcting code. 
     
     
         42 . The method of  claim 27 , wherein said tagged genomic sample is a pooled sample comprising nucleic acid molecules from several different sources, where each of said sources is associated with a different MID sequence. 
     
     
         43 . The method of  claim 42 , wherein each of the sources is derived from a human subject. 
     
     
         44 . The method of  claim 42 , wherein each of the sources is derived from different sections of a tumor. 
     
     
         45 . The method of  claim 42 , wherein each of the sources is derived from different tumors of a subject. 
     
     
         46 . The method of  claim 32 , wherein each of the sources is derived from a subject at different times. 
     
     
         47 . The method of  claim 27 , wherein the tagged genomic sample comprises polynucleotides from a tumor. 
     
     
         48 . The method of  claim 27 , wherein the tagged genomic sample comprises polynucleotides from a microorganism and/or a virus. 
     
     
         49 . The method of  claim 27 , wherein the tagged genomic sample comprises human genomic DNA and said polymorphic target sequence comprises a single nucleotide polymorphism of the human genome. 
     
     
         50 . The method of  claim 27 , wherein the sequencing step b) comprises sequencing said plurality of amplified target DNA molecules on a next-generation sequencing platform. 
     
     
         51 . The method of  claim 27 , wherein the amplifying step is done by polymerase chain reaction.