Digital counting of individual molecules by stochastic attachment of diverse labels
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.
1. A method, comprising:
(a) obtaining a sample comprising a plurality of occurrences of a first target molecule and a plurality of occurrences of a second target molecule;
(b) contacting the sample with a set of diverse labels comprising m labels with different label sequences, thereby attaching to each occurrence of the first target molecule and the second target molecule in the sample a label randomly selected from the set of diverse labels to generate, for each occurrence of the first target molecule and each occurrence of the second target molecule, a new first molecule and a new second molecule, respectively,
wherein each of the new first molecules comprises a copy of the first target molecule and a copy of the second target molecule or a complement thereof, and a label,
wherein each of the new second molecules comprises a copy of the second target molecule or a complement thereof, and a label,
wherein n 1 and n 2 are numbers of occurrences of the plurality of occurrences of first target molecule and the second target molecule, respectively, and
wherein the ratio of the greater of n 1 and n 2 to m is smaller than 0.2;
(c) detecting the new first molecules and the new second molecules by detecting the label sequence present on the new first and second molecules or derivatives thereof, wherein detecting the labels present in the new first molecules, or the new second molecules, comprises sequencing the new first molecules, or the new second molecules, respectively, and wherein the number of labels with distinct sequences present in the new first molecules, or the new second molecules, indicates the number of the new first molecules, or the new second molecules, respectively.
2. The method of claim 1 , wherein the labels attached to the occurrences of the first target molecule or the second target molecule comprise labels with k different label sequences,
wherein n is n 1 or n 2 ,
wherein m and n are related by E(k),
wherein
E
(
k
)
=
m
[
1
-
(
1
-
1
m
)
n
]
,
and wherein E(k) denotes the mean of k.
3. The method of claim 1 , wherein the labels attached to the occurrences of the first target molecule or the second target molecule comprise labels with k different label sequences,
wherein n is n 1 or n 2 ,
wherein m and n are related by Var(k),
wherein
Var
[
k
]
=
m
[
1
-
(
1
-
1
m
)
n
]
(
1
-
1
m
)
n
+
m
(
m
-
1
)
[
(
1
-
2
m
)
n
-
(
1
-
1
m
)
2
n
]
,
and wherein Var(k) denotes the variance of k.
4. The method of claim 1 , wherein the ratio of the greater of n 1 and n 2 to m is smaller than 0.02.
5. The method of claim 1 , wherein each of the m labels is 6 or more nucleotides in length.
6. The method of claim 1 , wherein the set of diverse labels comprises at least 4000 labels with different sequences.
7. The method of claim 1 , further comprising mapping sequencing reads that are obtained from sequencing the new first molecules, or the new second molecules, to a reference sequence.
8. The method of claim 1 , further comprising aligning sequencing reads obtained from sequencing the new first molecules, or the new second molecules.
9. The method of claim 1 , further comprising detecting a mismatch in the label sequence, or the copy of the first target molecule or the second target molecule, in sequencing reads obtained from sequencing the new first molecules, or the new second molecules, respectively.
10. The method of claim 1 , wherein sequencing the new first molecules, or the new second molecules, comprises sequencing a portion of one of the label sequences or a portion of one of the new first molecules, or the new second molecules, respectively.
11. The method of claim 1 , comprising scoring a label sequence in sequencing reads obtained from sequencing the new first molecules or new second molecules.
12. The method of claim 1 , comprising adding one or more adaptors to each of the plurality of occurrences of the first target molecule or the second target molecule.
13. The method of claim 1 , wherein the attaching of step (b) comprises reverse transcription and/or polymerase chain reaction (PCR) of the occurrences of the first target molecule or the second target molecule.
14. The method of claim 1 , wherein the attaching of step (b) is performed using a reverse transcriptase, a DNA polymerase, or a combination thereof.
15. The method of claim 1 , wherein the attaching of step (b) comprises attaching to the occurrences of the first target molecule and the second target molecule in the sample (i) a first label with the first label sequence randomly selected from the set of diverse labels, and (ii) a second label with the second label sequence randomly selected from the set of diverse labels, respectively, wherein the first label sequence and the second label sequence are different.
16. The method of 14 , wherein the first label, the second label, or both comprises an oligo-dT sequence.
17. The method of claim 14 , wherein the first label, the second label, or both, comprises a target specific region to the first target molecule, the second target molecule, or both.
18. The method of claim 14 , wherein the first label and/or the second label comprises a randomer sequence.
19. The method of claim 1 , wherein each of m labels from the set of diverse labels comprises an oligo-dT sequence.
20. The method of claim 1 , wherein the first target molecule, the second target molecule, or both, comprises a ribonucleic acid (RNA), deoxyribonucleic acid (DNA), an antibody, or a combination thereof.
21. The method of claim 1 , wherein the first target molecule, the second target molecule, or both, is associated with an antibody.
22. The method of claim 1 , wherein the first target molecule, the second molecule, or both, is an oligonucleotide-labeled antibody.
23. The method of claim 1 , wherein the first target molecule, the second target molecule, or both, cDNA, genomic DNA, mRNA, or fragments thereof.
24. The method of claim 1 , wherein the plurality of occurrences of a first target molecule and the plurality of occurrences of a second target molecule are from a single cell.
25. The method of claim 1 , wherein the first target molecule, the second target molecule, or both comprises DNA or RNA from natural sources, recombinantly produced, artificially synthesized, or mimetics thereof.
26. The method of claim 1 , wherein the sample comprises nucleic acid content from two or more cells.