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 estimating a number of target molecules in a sample, comprising:
 a) amplifying a population of different target nucleic acid molecules from a tagged genomic sample thereby producing a population of amplified target DNA molecules, wherein:
 (i) the different target DNA molecules that comprise a target region are tagged with different label-tags; 
 (ii) 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 
 (iii) the amplified target DNA molecules comprise the target region and an associated label-tag of the label-tags; 
   b) detecting the plurality of amplified target DNA molecules by sequencing, thereby producing a plurality of readouts,   wherein the plurality of readouts comprise:
 (i) at least a portion of the target region; and 
 (ii) an associated label-tag of the label-tags; and 
   c) estimating, using a computer, the number of target nucleic acid molecules that comprise the target region in the tagged sample based on:
 (i) a determination of the number of the different label-tags that are associated with the target region; and 
 (ii) a determination of the number of readouts that comprise each of the different label-tags that are associated with the target region. 
   
     
     
         2 . The method of  claim 1 , wherein the estimating of step c) comprises performing a statistical analysis. 
     
     
         3 . The method of  claim 1 , wherein the population of different target nucleic acid molecules is produced by ligating a set of adaptors that comprise the label-tags to an initial nucleic acid sample. 
     
     
         4 . The method of  claim 3 , wherein the initial nucleic acid sample comprises mRNA or cDNA. 
     
     
         5 . The method of  claim 3 , wherein the initial nucleic acid sample comprises genomic DNA. 
     
     
         6 . The method of  claim 3 , wherein the initial nucleic acid sample is an amplification product. 
     
     
         7 . The method of  claim 1 , wherein the population of different target nucleic acid molecules is produced by extending a set of primers that comprises the label-tags, using an initial nucleic acid sample as a template. 
     
     
         8 . The method of  claim 7 , wherein the initial nucleic acid sample comprises mRNA or cDNA. 
     
     
         9 . The method of  claim 7 , wherein the initial nucleic acid sample comprises genomic DNA. 
     
     
         10 . The method of  claim 7 , 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-tag comprises 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 1 , 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. 
     
     
         14 . The method of  claim 1 , wherein the label-tag is used to correct estimation errors. 
     
     
         15 . The method of  claim 1 , wherein the target nucleic acid molecules are further tagged with a unique sequence tag that that is used to distinguish target DNA molecules from different samples. 
     
     
         16 . The method of  claim 15 , 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. 
     
     
         17 . The method of  claim 1 , wherein the tagged genomic sample comprises mammalian genomic DNA and the target region is a region that varies in copy number. 
     
     
         18 . The method of  claim 1 , wherein the detecting the plurality of amplified target DNA molecules by sequencing step b) comprises sequencing the amplified target DNA molecules by next-generation sequencing. 
     
     
         19 . The method of  claim 1 , wherein the amplifying step is done by polymerase chain reaction. 
     
     
         20 . The method of  claim 1 , wherein the target nucleic acid molecules are cDNA molecules, and the estimating step c) provides an estimate of the abundance of cDNA molecules that comprise the target region. 
     
     
         21 . The method of  claim 1 , wherein the target nucleic acid molecules are synthetic DNA molecules. 
     
     
         22 . The method of  claim 1 , wherein the label-tag comprises 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. 
     
     
         23 . A method for estimating the number of initial target nucleic acid molecules in a sample, comprising:
 a) amplifying a population of initial target nucleic acid molecules from a tagged sample thereby producing a population of amplified target DNA molecules, wherein the initial target nucleic acid molecules that comprise a target region are tagged with 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 wherein the amplified target DNA molecules comprise said target region and an associated DBR sequence of said DBR sequences;   b) sequencing a 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 target region and 
 (ii) an associated DBR sequence of said DBR sequences; and 
   c) estimating, using a computer, the number of initial target nucleic acid molecules that comprise said target region in said tagged sample based on:
 (i) a determination of the number of said different DBR sequences that are associated with said target region in the sequence reads; and 
 (ii) a determination of the number of sequence reads that comprise each of the different DBR sequences that are associated with said target region. 
   
     
     
         24 . The method of  claim 23 , wherein said estimating step c) is done using a maximum likelihood method. 
     
     
         25 . The method of  claim 23 , wherein said population of initial target nucleic acid molecules is made by ligating a set of adaptors that comprise said DBR sequences to an initial nucleic acid sample. 
     
     
         26 . The method of  claim 25 , wherein said initial nucleic acid sample comprises mRNA or cDNA. 
     
     
         27 . The method of  claim 25 , wherein said initial nucleic acid sample comprises genomic DNA. 
     
     
         28 . The method of  claim 25 , wherein said initial nucleic acid sample is an amplification product. 
     
     
         29 . The method of  claim 23 , wherein said population of initial target nucleic acid molecules is made by extending a set of primers that comprises said DBR sequences, using an initial nucleic acid sample as a template. 
     
     
         30 . The method of  claim 29 , wherein said initial nucleic acid sample comprises mRNA or cDNA. 
     
     
         31 . The method of  claim 29 , wherein said initial nucleic acid sample comprises genomic DNA. 
     
     
         32 . The method of  claim 29 , wherein said initial nucleic acid sample is an amplification product. 
     
     
         33 . The method of  claim 23 , wherein the method comprises, prior to the amplifying step (a), enriching said population of initial target DNA molecules from an initial nucleic acid sample. 
     
     
         34 . The method of  claim 23 , 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. 
     
     
         35 . The method of  claim 23 , 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, K, M, B, D, H, V, N, and modified versions thereof. 
     
     
         36 . The method of  claim 23 , wherein said DBR sequences comprise an error-correcting code. 
     
     
         37 . The method of  claim 23 , wherein the initial target nucleic acid molecules are further tagged with a unique multiplex identifier (MID) sequence that identifies the source of a nucleic acid molecule to which it is tagged. 
     
     
         38 . The method of  claim 37 , wherein said tagged 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. 
     
     
         39 . The method of  claim 23 , wherein the tagged sample comprises mammalian genomic DNA and said target region is a region that varies in copy number. 
     
     
         40 . The method of  claim 23 , wherein the sequencing step b) comprises sequencing said plurality of amplified target DNA molecules on a next-generation sequencing platform. 
     
     
         41 . The method of  claim 23 , wherein the amplifying step is done by polymerase chain reaction. 
     
     
         42 . The method of  claim 23 , wherein said initial target nucleic acid molecules are cDNA molecules, and the estimating step c) provides an estimate of the abundance of cDNA molecules that comprise said target region. 
     
     
         43 . The method of  claim 23 , wherein said initial target nucleic acid molecules are synthetic DNA molecules. 
     
     
         44 . The method of  claim 23 , 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.