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US 10,510,435 B2Granted
Error correction of multiplex imaging analysis by sequential hybridization
OwnerLong Cai, Sheel Shah +2inventors
InventorsLong Cai, Sheel Shah, Eric Lubeck, Wen Zhou
Life of this patent
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Abstract
Disclosed herein are methods and systems for detecting and/or quantifying cellular targets such as nucleic acids in cells, tissues, organs or organisms. Through sequential barcoding, it is possible to perform high-throughput profiling of a large number of targets, such as transcripts and/or DNA loci. In some embodiments, error correction is implemented through use of barcodes that can tolerate mistakes and missing data during sequential hybridization of probes to selected targets.
Claims20
A sequential hybridization method, comprising:
- identifying a plurality of target genes;
- and
- associating, via sequential hybridization of binding probes to the plurality of target genes, a first plurality of unique codes with the plurality of target genes, wherein each target gene in the plurality of target genes is represented by a unique code in the first plurality of unique codes, wherein the sequential hybridization comprises n rounds of hybridization (where n≥2), and wherein each round of hybridization inn rounds of hybridization comprises:
- contacting the plurality of target genes with a plurality of binding probes, wherein each probe in the plurality of binding probes comprises:
- a binding sequence that specifically binds a target sequence in a gene in the plurality of target genes, wherein target genes from the plurality of target genes are spatially transfixed from each other, and wherein each probe is capable of emitting a detectable visual signal upon binding of the probe to a target sequence;
- detecting visual signals that reflect the binding between the plurality of binding probes and the plurality of target genes;
- and
- removing the visual signals, prior to the next round of hybridization;
- wherein probes used in the n rounds of hybridization are capable of emitting at least F types of detectable visual signals (where F≥2 and Fn is greater than the number of target genes in the plurality of target genes),
- wherein a unique code in the first plurality of unique codes for a target gene consists of n components,
- wherein each component is determined by visual signals that reflect the binding between binding probes and the target gene during one of the n rounds of hybridization,
- wherein the n rounds of hybridization include m rounds of error correction n≥m≥1), wherein each m error correction round comprises one round of then rounds of hybridization, and each m round of hybridization is a repeat of one of the remaining one or more (n−1) rounds of the n rounds of hybridization,
- wherein a second plurality of unique codes for the plurality of target genes is generated after each m error correction round is removed from the n rounds of hybridization, and
- wherein each unique code in the second plurality of unique codes consists of (n−m) components and uniquely represents a target gene in the plurality of target genes.
11 more elements
A hybridization method, comprising:
- identifying a plurality of target genes;
- performing sequential hybridization of binding probes to the plurality of target genes, wherein the sequential hybridization comprises n rounds of hybridization (where n≥2), and wherein each round of hybridization inn rounds of hybridization comprises:
- contacting the plurality of target genes with a plurality of binding probes, wherein each probe in the plurality of binding probes comprises:
- a binding sequence that specifically binds a target sequence in a gene in the plurality of target genes, wherein target genes from the plurality of target genes are spatially transfixed from each other, and wherein each probe is capable of emitting a detectable visual signal upon binding of the probe to a target sequence;
- detecting visual signals that reflect the binding between the plurality of binding probes and the plurality of target genes, wherein each target gene in the plurality of target genes is represented by visual signals that are unique for the target gene, and wherein probes used in the n rounds of hybridization are capable of emitting at least F types of detectable visual signals (where F≥2, and Fn is greater than the number of target genes in the plurality of target genes);
- and
- removing the visual signals, prior to the next round of hybridization;
- and
- performing serial hybridizations against one or more target genes, wherein the expression level of each target gene is above a predetermined threshold value, wherein each serial hybridization comprises:
- contacting the one or more target genes with a plurality of binding probes, wherein each probe in the plurality of binding probes comprises:
- a binding sequence that specifically binds a target sequence in a target gene in the one or more target genes, wherein one or more target genes are spatially transfixed from each other,
- wherein each probe is capable of emitting a detectable visual signal upon binding of the probe to the target sequence, and wherein probes binding to target sequences in the same target gene emit the same detectable visual signals;
- and
- detecting visual signals that reflect the binding between the plurality of binding probes and the one or more target gene.
11 more elements
A non-transitory computer-readable medium containing instructions that, when executed by a computer processor, cause the computer processor to:
- associate, via sequential hybridization of binding probes to a plurality of target genes, a first plurality of unique codes with the plurality of target genes, wherein each target gene in the plurality of target genes is represented by a unique code in the first plurality of unique codes, wherein the sequential hybridization comprises n rounds of hybridization (where n≥2), and wherein each round of hybridization inn rounds of hybridization comprises:
- contacting the plurality of target genes with a plurality of binding probes, wherein each probe in the plurality of binding probes comprises:
- a binding sequence that specifically binds a target sequence in a gene in the plurality of target genes, wherein target genes from the plurality of target genes are spatially transfixed from each other, and wherein each probe is capable of emitting a detectable visual signal upon binding of the probe to a target sequence;
- detecting visual signals that reflect the binding between the plurality of binding probes and the plurality of target genes;
- and
- removing the visual signals, when applicable, prior to the next round of hybridization;
- wherein probes used in the n rounds of hybridization are capable of emitting at least F types of detectable visual signals (where F≥2 and Fn is greater than the number of target genes in the plurality of target genes),
- wherein a unique code in the first plurality of unique codes for a target gene consists of n components,
- wherein each component is determined by visual signals that reflect the binding between binding probes and the target gene during one of the n rounds of hybridization,
- wherein the n rounds of hybridization include m error correction round (m≥1),
- wherein a second plurality of unique codes for the plurality of target genes is generated after the m error correction round is removed from the n rounds of hybridization, and
- wherein each unique code in the second plurality of unique codes consists of (n−m) components and uniquely represents a target gene in the plurality of target genes.
9 more elements
A non-transitory computer-readable medium containing instructions that, when executed by a computer processor, cause the computer processor to:
- perform sequential hybridization of binding probes to a plurality of target genes, wherein the sequential hybridization comprises n rounds of hybridization (where n≥2), and wherein each round of hybridization in n rounds of hybridization comprises:
- contacting the plurality of target genes with a plurality of binding probes, wherein each probe in the plurality of binding probes comprises:
- a binding sequence that specifically binds a target sequence in a gene in the plurality of target genes, wherein target genes from the plurality of target genes are spatially transfixed from each other, and wherein each probe is capable of emitting a detectable visual signal upon binding of the probe to a target sequence;
- detecting visual signals that reflect the binding between the plurality of binding probes and the plurality of target genes, wherein each target gene in the plurality of target genes is represented by visual signals that are unique for the target gene, and wherein probes used in the n rounds of hybridization are capable of emitting at least F types of detectable visual signals (where F≥2, and Fn is greater than the number of target genes in the plurality of target genes);
- and
- removing the visual signals, when applicable, prior to the next round of hybridization;
- and
- perform hybridizations against one or more target genes, wherein the expression level of each target gene is above a predetermined threshold value, wherein each hybridization comprises:
- contacting the one or more target genes with a plurality of binding probes, wherein each probe in the plurality of binding probes comprises:
- a binding sequence that specifically binds a target sequence in a target gene in the one or more target genes, wherein one or more target genes are spatially transfixed from each other,
- wherein each probe is capable of emitting a detectable visual signal upon binding of the probe to the target sequence, and wherein probes binding to target sequences in the same target gene emit the same detectable visual signals;
- and
- detecting visual signals that reflect the binding between the plurality of binding probes and the one or more target gene.
10 more elements
Dependencies are inferred from the claim text.