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US 12,421,540 B2Granted
Sequential probing of molecular targets based on pseudo-color barcodes with embedded error correction mechanism
AssigneeCalifornia Institute of Technology
InventorsLong Cai, Chee Huat Eng, Sheel Shah
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Abstract
The present invention, among other things, provides technologies for detecting and/or quantifying nucleic acids in cells, tissues, organs, or organisms. Pre-designed barcodes are associated with specific molecular targets through sequential hybridization experiments. A pseudo-color based barcoding scheme is described that overcomes the limitations in the previous generation of the technology. The current method can be applied to both in vitro and in situ analysis.
Claims27
A method of barcoding molecular targets in a cell, comprising:
- (a) identifying N molecular targets in a cell, wherein N is greater than 500;
- (b) performing n sequential barcoding rounds, wherein n≥2, and wherein each barcoding round comprises m serial hybridizations of probes collectively hybridized to the N molecular targets, and wherein m≥2, and wherein m serial hybridizations of probes in each barcoding round are generated by:
- (i) contacting one or more groups of probes to a subset of the N molecular targets, the total number of groups of probes corresponding to the number of molecular targets in the subset, wherein probes in each group comprise one or more binding sequences capable of hybridizing to the molecular targets in the subset, wherein each probe is capable of generating at least one detectable signal representing hybridizations of the probes to the molecular targets in the subset, and wherein probes in the one or more groups generate one or more different detectable signals corresponding to the number of molecular targets in the subset;
- (ii) detecting detectable visual signals corresponding to the hybridizations between the one or more groups of probes and the subset of the N molecular targets in an image;
- (iii) super-localizing the detectable visual signals in the image;
- (iv) removing the detectable visual signals, prior to the next serial hybridization;
- (v) repeating steps (i)-(iv), each time with a new group of probes capable of hybridizing to a new subset of the N molecular targets;
- (c) for each barcoding round in n sequential barcoding rounds, generating a composite image by superimposing m images corresponding to m serial hybridizations, wherein m≥2, and wherein the m images are aligned based on one or more alignment references whose positions remain constant relative to the cell, and wherein the detectable signals in the composite image corresponding to the molecular targets are assigned S pseudo-color symbols;
- (d) combining the S pseudo-color symbols from each barcoding round in n sequential barcoding rounds to generate barcodes for the molecular targets, wherein the barcode to each molecular target consists of u unique components, and wherein u≥2, and wherein each unique component in the u unique components is assigned from S unique pseudo-color symbols, and wherein S is an integer that is equal to or greater than
- N
- n
- ,
- and wherein the molecular targets can be differentiated by one or more differences in their barcodes.
10 more elements
A method of barcoding molecular targets in a cell, comprising:
- (a) identifying N molecular targets in a cell, wherein Nis greater than 500;
- (b) performing n sequential barcoding rounds, wherein n≥2, and wherein each barcoding round comprises m serial bindings of probes collectively bound to the N molecular targets, and wherein m≥2, and wherein m serial bindings of probes in each barcoding round are generated by:
- i. contacting one or more groups of probes to a subset of the N molecular targets, the total number of groups of probes corresponding to the number of molecular targets in the subset, wherein probes in each group comprise one or more binding sequences capable of binding to the molecular targets in the subset, wherein each probe is capable of generating at least one detectable signal representing bindings of the probes to the molecular targets in the subset, and wherein probes in the one or more groups generate one or more different detectable signals corresponding to the number of molecular targets in the subset;
- ii. detecting detectable visual signals corresponding to the bindings between the one or more groups of probes and the subset of the N molecular targets in an image;
- iii. super-localizing the detectable visual signals in the image;
- iv. removing the detectable visual signals, prior to the next serial bindings;
- v. repeating steps (i)-(iv), each time with a new group of probes capable of binding to a new subset of the N molecular targets;
- (c) for each barcoding round in n sequential barcoding rounds, generating a composite image by superimposing m images corresponding to m serial bindings, wherein m≥2, and wherein the m images are aligned based on one or more alignment references whose positions remain constant relative to the cell, and wherein the detectable signals in the composite image corresponding to the molecular targets are assigned S pseudo-color symbols;
- (e) combining the S pseudo-color symbols from each barcoding round in n sequential barcoding rounds to generate barcodes for the molecular targets, wherein the barcode to each molecular target consists of u unique components, and wherein u≥2, and wherein each unique component in the u unique components is assigned from S unique pseudo-color symbols, and wherein S is an integer that is equal to or greater than
- N
- n
- ,
- and wherein the molecular targets can be differentiated by one or more differences in their barcodes.
10 more elements
A method of barcoding molecular targets in a cell, comprising:
- (a) identifying N molecular targets in a cell;
- (b) performing n sequential barcoding rounds, wherein n≥2, and wherein each barcoding round comprises m serial bindings of probes collectively bound to the N molecular targets, and wherein m≥2, and wherein m serial bindings of probes in each barcoding round are generated by:
- i. contacting one or more groups of probes to a subset of the N molecular targets, the total number of groups of probes corresponding to the number of molecular targets in the subset, wherein probes in each group comprise one or more binding sequences capable of binding to the molecular targets in the subset, wherein each probe is capable of generating at least one detectable signal representing bindings of the probes to the molecular targets in the subset, and wherein probes in the one or more groups generate one or more different detectable signals corresponding to the number of molecular targets in the subset;
- ii. detecting detectable visual signals corresponding to the bindings between the one or more groups of probes and the subset of the N molecular targets in an image;
- iii. removing the detectable visual signals, prior to the next serial bindings;
- iv. repeating steps (i)-(iii), each time with a new group of probes capable of binding to a new subset of the N molecular targets;
- (c) for each barcoding round in n sequential barcoding rounds, generating a composite image by superimposing m images corresponding to m serial bindings, wherein m≥2, and wherein the m images are aligned based on one or more alignment references whose positions remain constant relative to the cell, and wherein the detectable signals in the composite image corresponding to the molecular targets are assigned S pseudo-color symbols;
- (f) combining the S pseudo-color symbols from each barcoding round in n sequential barcoding rounds to generate barcodes for the molecular targets, wherein the barcode to each molecular target consists of u unique components, and wherein u≥2, and wherein each unique component in the u unique components is assigned from S unique pseudo-color symbols, and wherein S is an integer that is equal to or greater than
- N
- u
- ,
- and wherein the molecular targets can be differentiated by one or more differences in their barcodes.
9 more elements
Dependencies are inferred from the claim text.