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 sequencing, comprising:
 a) attaching a label-tag to both ends of a plurality of target nucleic acid molecules in a genomic sample,   wherein the label-tag comprises natural nucleotide bases and/or chemically or biochemically modified, non-natural, or derivatized nucleotide bases, and   wherein the attaching produces a population of tagged nucleic acid molecules, wherein each of the tagged nucleic acid molecules comprises: (i) a target polynucleotide; (ii) a first label-tag at the 5′ end of the target polynucleotide; and (iii) a second label-tag at the 3′ end of the target polynucleotide,   wherein: in each of the tagged nucleic acid molecules the first label-tag is different from the second label-tag; and   the different tagged nucleic acid molecules in the population have different first label-tags and different second label-tags, relative to one another;   b) amplifying at least some of the tagged nucleic acid molecules, thereby producing a population of amplified tagged nucleic acid molecules; and   c) detecting the plurality of amplified labeled-targets by sequencing, thereby producing a plurality of readouts, wherein the plurality of readouts comprises: (i) at least a portion of the target; (ii) the first label-tag; and (iii) the second label-tag.   
     
     
         2 . The method of  claim 1 , wherein the attaching step is done by ligating a set of adaptors that comprise the label-tag to the plurality of target nucleic acid molecules. 
     
     
         3 . The method of  claim 1 , wherein the attaching is done by extending a set of primers that comprises the label-tags, using an initial nucleic acid sample as a template. 
     
     
         4 . The method of  claim 3 , wherein the initial nucleic acid sample is an amplification product. 
     
     
         5 . The method of  claim 1 , wherein the method comprises, prior to the attaching step (a), enriching for the target nucleic acid molecules from an initial nucleic acid sample. 
     
     
         6 . The method of  claim 5 , wherein the initial nucleic acid sample is an amplification product. 
     
     
         7 . 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. 
     
     
         8 . The method of  claim 7 , 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. 
     
     
         9 . The method of  claim 1 , wherein the tagged nucleic acid molecules further comprise a unique sequence tag that is used to distinguish target DNA molecules from different samples. 
     
     
         10 . The method of  claim 9 , 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. 
     
     
         11 . The method of  claim 9 , wherein each of the different samples is derived from a human subject. 
     
     
         12 . The method of  claim 9 , wherein each of the different samples comprises polynucleotides from tumor cells. 
     
     
         13 . The method of  claim 1 , wherein the genomic sample comprises polynucleotides from a tumor cell. 
     
     
         14 . The method of  claim 1 , wherein the genomic sample comprises polynucleotides from bacteria and/or polynucleotides encoding viral epitopes. 
     
     
         15 . The method of  claim 1 , wherein the genomic sample comprises human genomic DNA. 
     
     
         16 . The method of  claim 1 , wherein detecting the plurality of amplified labeled-targets by sequencing comprises sequencing a plurality of the amplified tagged nucleic acid molecules on a next-generation sequencing platform. 
     
     
         17 . The method of  claim 1 , wherein the amplifying step (b) is done by polymerase chain reaction. 
     
     
         18 . The method of  claim 1 , wherein the label-tag is used to correct estimation errors. 
     
     
         19 . A method of sequencing, comprising:
 a) appending a degenerate base region (DBR) to both ends of a plurality of target nucleic acid molecules in a genomic sample, wherein said DBR comprises a sequence comprising at least one nucleotide base selected from: R, Y, S, W, K, M, B, D, H, V, N and modified versions thereof and wherein said appending produces a population of asymmetrically tagged nucleic acid molecules, wherein each of the asymmetrically tagged nucleic acid molecules comprises:
 (i) a target polynucleotide, 
 (ii) a first DBR sequence at the 5′ end of the target polynucleotide and 
 (iii) a second DBR sequence at the 3′ end of the target polynucleotide, wherein: in each of said asymmetrically tagged nucleic acid molecules the first DBR sequence is different from the second DBR sequence; and the different asymmetrically tagged nucleic acid molecules in said population have different first DBR sequences and different second DBR sequences, relative to one another; 
   b) amplifying at least some of said asymmetrically tagged nucleic acid molecules, thereby producing a population of amplified asymmetrically tagged nucleic acid molecules; and   c) sequencing a plurality of the amplified asymmetrically tagged nucleic acid molecules to produce a plurality of sequences, wherein the sequencing step provides, for each of the asymmetrically tagged nucleic acid molecules that are sequenced:
 (i) the nucleotide sequence of at least a portion of a target polynucleotide; 
 (ii) the nucleotide sequence of the first DBR sequence; and 
 (iii) the nucleotide sequence of the second DBR sequence. 
   
     
     
         20 . The method of  claim 19 , wherein the appending step is done by ligating a set of adaptors that comprise said DBR to said plurality of target nucleic acid molecules. 
     
     
         21 . The method of  claim 19 , wherein said appending is done by extending a set of primers that comprises said DBR, using an initial nucleic acid sample as a template. 
     
     
         22 . The method of  claim 21 , wherein said initial nucleic acid sample is an amplification product. 
     
     
         23 . The method of  claim 19 , wherein the method comprises, prior to the appending step (a), enriching for said target nucleic acid molecules from an initial nucleic acid sample. 
     
     
         24 . The method of  claim 23 , wherein said initial nucleic acid sample is an amplification product. 
     
     
         25 . The method of  claim 19 , wherein said DBR 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. 
     
     
         26 . The method of  claim 25 , wherein said DBR comprises 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. 
     
     
         27 . The method of  claim 19 , wherein said asymmetrically tagged nucleic acid molecules further comprise a unique multiplex identifier (MID) sequence that identifies the source of a nucleic acid molecule to which it is appended. 
     
     
         28 . The method of  claim 27 , wherein said 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. 
     
     
         29 . The method of  claim 27 , wherein each of the sources is derived from a human subject. 
     
     
         30 . The method of  claim 27 , wherein each of the sources is derived from different sections of a tumor. 
     
     
         31 . The method of  claim 27 , wherein each of the sources is derived from different tumors of a subject. 
     
     
         32 . The method of  claim 27 , wherein each of the sources is derived from a subject at different times. 
     
     
         33 . The method of  claim 19 , wherein the genomic sample comprises polynucleotides from a tumor. 
     
     
         35 . The method of  claim 19 , wherein the genomic sample comprises polynucleotides from a microorganism and/or a virus. 
     
     
         35 . The method of  claim 19 , wherein the genomic sample comprises human genomic DNA. 
     
     
         36 . The method of  claim 19 , wherein the sequencing step c) comprises sequencing a plurality of the amplified asymmetrically tagged nucleic acid molecules on a next-generation sequencing platform. 
     
     
         37 . The method of  claim 19 , wherein the amplifying step (b) is done by polymerase chain reaction. 
     
     
         38 . The method of  claim 19 , wherein the DBR comprises an error-correcting code.