# US 11,993,814 B2 · Digital counting of individual molecules by stochastic attachment of diverse labels

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- Number: US 11,993,814 B2
- Document ID: US11993814B2
- Status: Granted
- Kind: B2
- Application number: 18/190,884
- Filed: 2023-03-27
- Published: 2023-11-16
- Granted: 2024-05-28
- Earliest claimed: 2009-12-15
- Assignees: Becton, Dickinson and Company
- Applicants: Becton, Dickinson and Company
- Inventors: Stephen P. A. Fodor; Glenn K. Fu
- Web page: https://patentgrep.com/patent/US11993814B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/6851: Quantitative amplification
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- G16B 25/00: ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B 25/20: Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 25/10: Gene or protein expression profiling; Expression-ratio estimation or normalisation
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## 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

### Claim 1

A method of preparing a sample, comprising:
obtaining a plurality of first molecules and a plurality of second molecules,
wherein the obtaining step comprises generating the plurality of first molecules and the plurality of second molecules from occurrences of a first target molecule and occurrences of a second target molecule, respectively, and labels from a set of diverse labels,
wherein the set of diverse labels comprises labels with m different label sequences,
wherein one or more of the plurality of first molecules and the plurality second molecules comprises an occurrence of the first target molecule or the complementary thereof and an occurrence of the second target molecule or the complementary thereof, respectively, and a label sequence or the complementary thereof from the set of diverse labels,
wherein more than 90% of the plurality of first molecules and the plurality of second molecules have a label sequence that is different from the label sequences on the other of the plurality of first molecules and the other of the plurality of second molecules, respectively,
wherein n1 and n2 are a number of occurrences of the first target molecule and a number of occurrences the second target molecule, respectively, and
wherein the ratio of the greater of n1 and n2 to m is smaller than 0.2.

### Claim 2 (depends on 1)

The method of claim 1, wherein the plurality of first molecules or the plurality of second molecules comprises labels with k different label sequences,
wherein n is n1 or n2,
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.

### Claim 3 (depends on 1)

The method of claim 1, wherein the plurality of first molecules or the plurality of second molecules comprises labels with k different label sequences,
wherein n is n1 or n2,
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.

### Claim 4 (depends on 1)

The method of claim 1, wherein the plurality of first molecules or the plurality of second molecules comprises labels with t different label sequences exactly x times,
wherein n is n1 or n2,
wherein m and n are related by E(t),
wherein
E
⁡
(
t
)
=
m
,
n
⁢
t
x
i
(
n
-
x
)
⁢
t
⁢
(
1
m
)
x
⁢
(
1
-
1
m
)
n
-
x
,
and wherein E(t) denotes the mean of t.

### Claim 5 (depends on 1)

The method of claim 1, wherein the plurality of first molecules or the plurality of second molecules comprises the labels exactly x times,
wherein n is n1 or n2,
wherein in and n are related by Var(t),
wherein
Var
⁡
(
t
)
=
A
⁡
(
1
-
A
)
+
(
m
-
1
)
⁢
m
·
(
n
2
⁢
x
)
·
(
2
m
)
2
⁢
x
⁢
(
1
-
2
m
)
n
-
2
⁢
x
⁢
(
2
⁢
x
x
)
⁢
(
1
2
)
2
⁢
x
,
wherein Var(t) denotes the variance of k, and
wherein
A
=
m
·
(
m
x
)
·
(
1
m
)
x
⁢
(
1
-
1
m
)
n
-
x
.

### Claim 6 (depends on 1)

The method of claim 1, wherein the ratio of the greater of n1 and n2 to m is smaller than 0.02.

### Claim 7 (depends on 1)

The method of claim 1, comprising detecting the plurality of first molecules and the plurality of second molecules by detecting the label sequences present on the plurality of first molecules and the plurality of second molecules or products thereof, wherein the detected first molecules and the detected second molecules indicate the number of occurrences of the first target molecule and the number of occurrence of the second target molecule, respectively.

### Claim 8 (depends on 7)

The method of claim 7, wherein detecting the label sequences present on plurality of first molecules comprises obtaining sequences of the plurality of first molecules.

### Claim 9 (depends on 8)

The method of claim 8, wherein obtaining the sequences present on the plurality of first molecules comprises sequencing the plurality of first molecules or products thereof.

### Claim 10 (depends on 9)

The method of claim 9, further comprising mapping sequencing reads that are obtained from sequencing the plurality of first molecules, or products thereof, to a reference sequence.

### Claim 11 (depends on 9)

The method of claim 9, further comprising aligning sequencing reads obtained from sequencing the plurality of first molecules, or products thereof.

### Claim 12 (depends on 9)

The method of claim 9, further comprising detecting a mismatch in the label sequence or the copy of the first target molecule in sequencing reads obtained from sequencing the plurality of first molecules, or products thereof.

### Claim 13 (depends on 9)

The method of claim 9, wherein sequencing the plurality of first molecules comprises sequencing a portion of the label sequence or a portion of the copy of the first target molecule, or a product thereof.

### Claim 14 (depends on 9)

The method of claim 9, wherein the label sequences present on the plurality of first molecules indicates the number of occurrences of the first target molecule.

### Claim 15 (depends on 14)

The method of claim 14, comprising scoring a label sequence in sequencing reads obtained from sequencing the plurality of first molecules or products thereof.

### Claim 16 (depends on 7)

The method of claim 7, wherein the detecting step comprises detecting the label sequences present on amplification products of the plurality of first molecules and the plurality of second molecules.

### Claim 17 (depends on 16)

The method of claim 16, wherein the amplification products comprise polymerase chain reaction (PCR) amplification products.

### Claim 18 (depends on 16)

The method of claim 16, further comprising performing one or more amplification steps to generate the amplification products.

### Claim 19 (depends on 1)

The method of claim 1, wherein the copy of the label sequence of a first molecule of the plurality of first molecules is 3′ to the copy of the first target molecule of the first molecule.

### Claim 20 (depends on 1)

The method of claim 1, wherein the copy of the label sequence of a first molecule of the plurality of first molecules is 5′ to the copy of the first target molecule of the first molecule.

### Claim 21 (depends on 1)

The method of claim 1, wherein the generating step comprises reverse transcription of an occurrence of the first target molecule and/or the second target molecule.

### Claim 22 (depends on 1)

The method of claim 1, wherein the generating step is performed using a reverse transcriptase, a polymerase, or a combination thereof.

### Claim 23 (depends on 1)

The method of claim 1, wherein the copy of the first molecule in a first molecule of the plurality of first molecules comprises a reverse complement of the first molecule, or a portion thereof, and wherein the copy of the label sequence in the first molecule of the plurality of first molecules comprises a reverse complement of a label sequence of a label of the set of diverse labels.

### Claim 24 (depends on 1)

The method of claim 1, wherein the copy of the first molecule in a first molecule of the plurality of first molecules comprises a reverse complement of the first molecule, or a portion thereof, and wherein the copy of the label sequence in the first molecule of the plurality of first molecules comprises a label of the set of diverse labels, or a portion thereof.

### Claim 25 (depends on 1)

The method of claim 1, wherein the first target molecule comprises an oligonucleotide.

### Claim 26 (depends on 25)

The method of claim 25, wherein the oligonucleotide is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

### Claim 27 (depends on 1)

The method of claim 1, wherein the first target molecule comprises RNA, and wherein the second target molecule comprises DNA.

### Claim 28 (depends on 1)

The method of claim 1, wherein one or more of the first target molecule and the second target molecule comprise a RNA, DNA, antibody, or a combination thereof.

### Claim 29 (depends on 1)

The method of claim 1, wherein the first target molecule is associated with an antibody.

### Claim 30

A method of preparing a sample, comprising:
obtaining a plurality of first molecules and a plurality of second molecules,
wherein the plurality of first molecules and the plurality of second molecules are generated from occurrences of a first target molecule and occurrences of a second target molecule, respectively, and labels from a set of diverse labels,
wherein the set of diverse labels comprises labels with m different label sequences,
wherein one or more of the plurality of first molecules and the plurality second molecules comprises an occurrence of the first target molecule or the complementary thereof and an occurrence of the second target molecule or the complementary thereof, respectively, and a label sequence or the complementary thereof from the set of diverse labels,
wherein more than 90% of the plurality of first molecules and the plurality of second molecules have a label sequence that is different from the label sequences on the other of the plurality of first molecules and the other of the plurality of second molecules, respectively,
wherein:
the copy of the label sequence of a first molecule of the plurality of first molecules is 3′ to the copy of the first target molecule of the first molecule; and/or
the copy of the label sequence of a first molecule of the plurality of first molecules is 5′ to the copy of the first target molecule of the first molecule,
wherein n1 and n2 are a number of occurrences of the first target molecule and a number of occurrences the second target molecule, respectively, and
wherein the ratio of the greater of n1 and n2 to m is smaller than 0.2.