Digital Counting of Individual Molecules by Stochastic Attachment of Diverse Labels

• US 20150118680A1
• Filed: 07/08/2014
• Published: 04/30/2015
• Est. Priority Date: 12/15/2009
• Status: Abandoned Application

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Fodor, Stephen P.A., Fu, Glenn K.
• Application Number:
  US14/326,448
• Publication Number:
  US 20150118680A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/10 Gene or protein expression profiling; Expression-ratio estimation or normalisation
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

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.

Claims

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.