# US 2021/0340618 A1 · Method of detecting target nucleic acid molecules

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- Number: US 2021/0340618 A1
- Document ID: US20210340618A1
- Status: Published application
- Kind: A1
- Application number: 17/335,931
- Filed: 2021-06-01
- Published: 2021-11-04
- Earliest claimed: 2019-05-31
- Assignees: 10x Genomics, Inc.
- Applicants: 10x Genomics, Inc.
- Inventors: Malte KÜHNEMUND; Toon VERHEYEN
- Web page: https://patentgrep.com/patent/US20210340618A1

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6813: Hybridisation assays
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 2537/1373: Displacement by a nucleic acid
- 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

## Abstract

The present application provides methods for detecting a nucleic acid molecule involving the use of a signal code sequence which corresponds to said nucleic acid molecule and a plurality of labelled detection probes which yield signals which make up the signal code sequence. In particular, the invention provides a sequential barcoding and decoding scheme which utilises a sequencing-by-hybridisation (SBH) strategy to sequence and decode a nucleotide barcode sequence, and to differentiate the nucleotide barcode sequence from other nucleotide barcode sequences. In an extension of the method, the application also provides a new coding scheme for providing a target nucleic acid with a detectable “colour” (or similar signal)-based code.

## Claims

### Claim 1

A method of detecting a nucleic acid molecule, wherein the method comprises:
(a) assigning a unique signal code sequence to the nucleic acid molecule, wherein the signal code sequence is specific to that molecule, wherein the signal code sequence may be derived by interrogating a target nucleotide sequence within the nucleic acid molecule with a sequence of detection probes, each detection probe yielding a signal and the signals together make up the signal code sequence, and wherein the target nucleotide sequence comprises first and second domains, each domain comprising a first subunit and a second subunit, wherein the second subunit of the first domain at least partially overlaps with the first subunit of the second domain in the target nucleotide sequence;
(b) contacting the nucleic acid molecule with a first detection probe for generating a signal code for the first position of the signal code sequence, and allowing the detection probe to hybridise to the nucleic acid molecule, wherein the detection probe comprises a recognition sequence that is complementary to the first and second subunits of the first domain and a reporter;
(c) detecting a signal from the reporter of the first detection probe which has hybridized to the nucleic acid molecule at the first domain;
(d) identifying the signal code for the first position of the signal code sequence from the signal detected in (c);
(e) contacting the nucleic acid molecule with a subsequent detection probe for generating a signal code for the next position of the signal code sequence, and allowing the detection probe to hybridise to the nucleic acid molecule, wherein the subsequent detection probe comprises a recognition sequence that is complementary to the first and second subunits of the second domain, and a reporter, and wherein the subsequent detection probe hybridises to a sequence in the second subunit of the preceding domain, and thereby initiates a strand displacement reaction which displaces the hybridised preceding detection probe;
(f) detecting a signal from the reporter of the subsequent detection probe which has hybridized to the nucleic acid molecule at the adjacent domain;
(g) identifying the signal code for the next position of the signal code sequence from the signal detected in (f);
(h) repeating steps (e)-(g) to identify signal codes for subsequent signal code sequence positions until sufficient signal codes have been identified to detect the nucleic acid molecule, wherein in repeated steps the detection probes for identifying the subsequent signal codes in the signal code sequence respectively have recognition sequences that are complementary to the subunits that are present at the first and second domains of the target nucleotide sequence such that detection probes successively hybridise to and displace preceding detection probes from the first and second domains;
wherein the first domain can be the 5′ or the 3′ domain in the target nucleotide sequence.

### Claim 2 (depends on 1)

The method of claim 1, wherein the target nucleotide sequence is a specific target sequence which occurs within a DNA or RNA molecule to be detected.

### Claim 3 (depends on 2)

The method of claim 2, wherein the target nucleotide sequence is a specific target sequence which is present in a native genomic DNA or in a naturally occurring RNA molecule, or in a cDNA, or in an amplification product generated from any of the foregoing nucleic acid molecules.

### Claim 4 (depends on 1)

The method of claim 1, wherein the second subunit from each domain fully overlaps with the first subunit of the adjacent domain.

### Claim 5 (depends on 1)

The method of claim 1, for detecting different nucleic acid molecules present in a sample, wherein each nucleic acid molecule comprises a different target nucleotide sequence, and wherein a different series of first and subsequent detection probes is provided for each different target nucleotide sequence, each different series of detection probes yielding a different signal code sequence.

### Claim 6 (depends on 5)

The method of claim 5, wherein each target nucleotide sequence comprises: (i) a first common region adjacent to the first subunit of the first domain, wherein the recognition sequence of the first detection probe comprises a sequence which is complementary to the first common region; and (ii) a second common region adjacent to the second subunit of the second domain, wherein the recognition sequence of the subsequent detection probe comprises a sequence which is complementary to the second common region.

### Claim 7 (depends on 1)

The method of claim 1, wherein the first detection probes and the subsequent detection probes each comprise a displacer toehold overhang region.

### Claim 8 (depends on 7)

The method of claim 7, wherein all of the detection probes which comprise a recognition sequence which is complementary to the first domain comprise a first displacer toehold overhang sequence, and all of the detection probes which comprise a recognition sequence which is complementary to the second domain comprise a second displacer toehold overhang sequence.

### Claim 9 (depends on 8)

The method of claim 8, wherein step (e) further comprises contacting the nucleic acid molecule with either a first displacer probe which comprises a displacer toehold binding region complementary to the first displacer toehold overhang and a sequence complementary to the first common region; or a second displacer probe which comprises a displacer toehold binding region complementary to the second displacer toehold overhang and a sequence complementary to the second common region.

### Claim 10 (depends on 1)

The method of claim 1, wherein the target nucleotide sequence is a nucleotide barcode sequence provided within the nucleic acid molecule.

### Claim 11 (depends on 1)

The method of claim 1, wherein the nucleic acid molecule is linked to an antibody.

### Claim 12

A method of decoding a nucleotide barcode sequence in a nucleic acid molecule, wherein the nucleotide barcode sequence corresponds to a specific signal code sequence that may be derived by interrogating the nucleotide barcode sequence with sequential detection probes each yielding a signal and the signals together make up the signal code sequence, and wherein the nucleotide barcode sequence comprises at least two adjacent barcode positions, each barcode position comprising a barcode subunit pair comprising a first barcode subunit and a second barcode subunit, wherein the second barcode subunit from each barcode position at least partially overlaps with the first barcode subunit of the adjacent barcode position in the sequence, said method comprising:
(i) contacting the nucleic acid molecule with a first detection probe for generating a signal code for the first position of the signal code sequence, and allowing the detection probe to hybridise to the nucleic acid molecule, wherein the detection probe comprises a recognition sequence that is complementary to the barcode subunit pair that is present at the first barcode position and a reporter;
(ii) detecting a signal from the reporter of the first detection probe which has hybridized to the nucleotide barcode sequence at the first barcode position;
(iii) identifying the signal code for the first position of the signal code sequence from the signal detected in (ii);
(iv) contacting the nucleotide barcode sequence with a subsequent detection probe for generating a signal code for the next position of the signal code sequence, and allowing the detection probe to hybridise to the nucleic acid molecule, wherein the subsequent detection probe comprises a recognition sequence that is complementary to the barcode subunit pair that is present at the adjacent barcode position, and a reporter, and wherein the subsequent detection probe which hybridises to the barcode subunit at the adjacent position hybridises to a sequence within the overlapping portion of the preceding barcode position, and thereby initiates a strand displacement reaction which displaces the hybridised preceding detection probe;
(v) detecting a signal from the reporter of the subsequent detection probe which has hybridized to the nucleotide barcode sequence at the adjacent barcode position;
(vi) identifying the signal code for the next position of the signal code sequence from the signal detected in (v);
(vii) repeating steps (iv)-(vi) to identify signal codes for subsequent signal code sequence positions until sufficient signal codes have been identified to decode the nucleotide barcode sequence, wherein detection probes for identifying the subsequent signal codes in the signal code sequence have (a) recognition sequences that are complementary to barcode subunit pairs that are present at subsequent sequential barcode positions in a nucleotide barcode sequence comprising multiple sequential adjacent barcode positions, or (b) recognition sequences that are complementary to the barcode subunit pairs that are present at the first and second barcode positions of a nucleotide barcode sequence comprising two adjacent barcode positions, and a reporter;
wherein the first barcode position can be any barcode position in the nucleotide barcode sequence.

### Claim at position 13

13-42. (canceled)

### Claim 43

A method of detecting a target nucleic acid molecule, wherein the target nucleic acid molecule is detected using a padlock probe specific for said target molecule which is circularised upon hybridisation to a target sequence in the target nucleic acid molecule and which is provided with a nucleotide barcode sequence to identify the padlock probe, and wherein the circularised padlock probe is amplified by rolling circle amplification (RCA) to produce a rolling circle product (RCP), the RCP containing multiple complementary copies of the nucleotide barcode sequence, characterised in that the method comprises:
(a) assigning a unique signal code sequence to the target nucleic acid molecule,
wherein the signal code sequence is specific to that molecule;
(b) providing the padlock probe with a nucleotide barcode sequence such that a RCP is formed comprising multiple complementary copies of the nucleotide barcode sequence that each corresponds to the signal code sequence, wherein the signal code sequence may be derived by interrogating the complementary copies of the nucleotide barcode sequence with sequential detection probes each yielding a signal and the signals together make up the signal code sequence;
(c) contacting the RCP with a first detection probe for generating a signal code for the first position of the signal code sequence and allowing the detection probe to hybridise to the RCP at the complementary copies of the nucleotide barcode sequence, wherein the detection probe comprises a recognition sequence complementary to the nucleotide barcode sequence and a reporter;
(d) detecting a signal from the reporter of the first detection probe which has hybridized to the RCP;
(e) identifying the first signal code for the first position in the signal code sequence for the RCP from the signal detected in (d);
(f) removing the first detection probe from the RCP;
(g) contacting the RCP with a subsequent detection probe for generating a signal code for the next position of the signal code sequence, and allowing the detection probe to hybridise to the nucleic acid molecule at the complementary copies of the nucleotide barcode sequence, wherein the detection probe comprises a recognition sequence complementary to the nucleotide barcode sequence and a reporter;
(h) detecting a signal from the reporter of the subsequent detection probe which has hybridized to the RCP;
(i) identifying the signal code for the next position in the signal code sequence for the RCP from the signal detected in (g);
(j) removing the subsequent detection probe from the RCP;
(k) repeating steps (g)-(j) to identify signal codes for subsequent signal code positions until sufficient signal codes have been identified to identify the nucleotide barcode sequence and thereby to detect the nucleic acid molecule.

### Claim 44 (depends on 43)

The method of claim 43, wherein the reporter of each detection probe is a binding site for a reporter probe comprising a detectable label, said binding site being contained in an overhang region of the detection probe which does not hybridise to the RCP, and wherein the signal is detected from said label.

### Claim 45 (depends on 44)

The method of claim 44, wherein said detectable label is a fluorophore and the signal code sequence is a unique fluorophore sequence.

### Claim 46 (depends on 44)

The method of claim 44, wherein the detection probes for generating the signal code sequence for a target nucleic acid molecule are provided as a set of detection probes each comprising the same recognition sequence and a reporter binding site, which may be the same or different, and which is specific for a reporter probe comprising a detectable label which gives rise to a given signal code, and wherein the detection probes are used sequentially in a pre-determined sequence which corresponds to the signal code sequence assigned to the target nucleic acid molecule.

### Claim 47 (depends on 43)

The method of claim 43, for detecting multiple different nucleic acid molecules present in a sample, wherein each different nucleic acid molecule is assigned a different signal code sequence and is detected using a specific padlock probe with a different nucleotide barcode sequence.

### Claim 48 (depends on 47)

The method of claim 47, wherein a different set of subsequent detection probes is provided for each different nucleic acid molecule, each different set of detection probes yielding a different signal code sequence.

### Claim 49 (depends on 43)

The method of claim 43, wherein said target nucleic acid molecule is detected in situ in a cell sample.

### Claim 50 (depends on 49)

The method of claim 49, wherein the target nucleic acid molecule is detected in a cell culture, or in a tissue sample.