US 2020/0140933 A1 · POLYMORPHISM DETECTION WITH INCREASED ACCURACY

Abstract

The invention relates to methods and compositions for the detection and quantification of nucleotide sequence variants, such as genetic polymorphisms, with decreased error and increased sensitivity, including single molecule detection. Detection of genetic polymorphisms, including single nucleotide polymorphisms (SNPs), is highly useful for the study of physiology, disease, phylogeny and forensics. Current methods for the detection and identification of nucleic acid sequence variants, such as genetic polymorphisms, lack the sensitivity to accurately detect low incidence mutations sequence variants or alleles. Detection techniques for highly multiplexed single molecule identification and quantification of analytes using optical systems are disclosed. Analytes include, but are not limited to, nucleic acid, such as DNA and RNA molecules, with and without modifications. Techniques described herein include use of specific and non-specific probes complementary to nucleic acids of interest for detailed characterization of nucleotide sequence variants and highly multiplexed single molecule identification and quantification.

Claims193

Dependent claims

A method of detecting at least one target nucleotide sequence variant suspected of being present in a sample, comprising:

(a) distributing a plurality of oligonucleotides on a substrate such that individual oligonucleotides bind to said substrate at spatially separate regions;
(b) carrying out on said substrate a target nucleotide sequence variant identification assay, wherein the sequence variant identification assay comprises performing at least M detection cycles to generate a signal detection sequence, wherein M is at least two, each cycle comprising:
(i) contacting said plurality of oligonucleotides with a probe comprising a detection label, wherein said probe binds preferentially to one of said at least one target nucleotide sequence variants or a barcode sequence bound to one of said at least one target nucleotide sequence variants;
(ii) washing the surface of the substrate to remove unbound barcode probes;
(iii) detecting the identity and location of the detection label on said substrate, and
(iv) if the cycle number is less than M, removing said barcode probe from said barcode moiety;
and
(c) analyzing the signal detection sequence generated by said M cycles at said spatially separate locations on said substrate to determine the presence or absence of said at least one target nucleotide sequence variant of interest.

5 more elements

A method of identifying at least one target nucleotide sequence variant suspected of being present in a sample, comprising:

(a) distributing a plurality of oligonucleotides comprising N distinct nucleotide sequence variants on a substrate such that each distinct nucleotide sequence variant of the N distinct nucleotide sequence variants is immobilized on a solid substrate in a location that is spatially separate from any other distinct target analyte of the N distinct target analytes
(b) carrying out on said substrate a target nucleotide sequence variant identification assay for identifying at least one of N distinct nucleotide sequence variants, wherein the assay comprises:
(i) obtaining a plurality of ordered probe reagent sets, each of said ordered probe reagent sets comprising one or more probes directed to a defined subset of said N distinct nucleotide sequence variants, wherein each of said probes comprises a sequence complementary to an oligonucleotide comprising one of said nucleotide sequence variants, and wherein each of said probes is detectably labeled such that one probe is configured to detect one distinct nucleotide sequence variants;
(ii) performing at least M cycles of probe binding and signal detection, each cycle comprising one or more passes, wherein a pass comprises use of at least one of said ordered probe reagent sets;
(iii) detecting from said at least M cycles a presence or an absence of a plurality of signals from said spatially separate locations of said substrate;
(iv) determining from said plurality of signals at least K bits of information per cycle for one or more of said N distinct nucleotide sequence variants, wherein said at least K bits of information are used to determine L total bits of information, wherein K×M=L bits of information and L>log2 (n), and wherein said L bits of information are used to determine a presence or an absence of one or more of said N distinct nucleotide sequence variants.

3 more elements

A method of detecting at least one target nucleotide sequence variant suspected of being present in a sample, comprising:

(a) providing a ligation reaction product of a target-dependent oligonucleotide ligation reaction performed on said sample, wherein said ligation reaction product comprises a plurality of oligonucleotides each comprising a substrate binding moiety and a barcode moiety;
(b) distributing said ligation reaction product on a substrate such that individual oligonucleotides bind to the substrate via the substrate binding moiety at spatially separate regions of said substrate;
(c) carrying out on said substrate a target nucleotide sequence variant identification assay, wherein the sequence variant identification assay comprises performing at least M detection cycles to generate a signal detection sequence, wherein M is at least two, each cycle comprising:
(i) contacting said ligation reaction product with a barcode probe comprising a detection label, wherein said barcode probe binds to the barcode moiety when it is present on the substrate;
(ii) washing the surface of the substrate to remove unbound barcode probes;
(iii) detecting the identity and location of said detection label on said substrate;
and
(iv) if the cycle number is less than m, removing said barcode probe from said barcode moiety;
and
(d) analyzing the signal detection sequence generated by said M cycles at said spatially separate locations on said substrate to determine the presence or absence of said at least one target nucleotide sequence variant of interest.

7 more elements

A method of identifying at least one target nucleotide sequence variant suspected of being present in a sample, comprising:

(a) providing a ligation reaction product of a target-dependent oligonucleotide ligation reaction performed on said sample, wherein said ligation reaction product comprises a plurality of oligonucleotides each comprising a substrate binding moiety and a barcode moiety;
(b) distributing said ligation reaction product on a substrate such that individual oligonucleotides bind to the substrate via the substrate binding moiety at spatially separate regions of said substrate;
(c) carrying out on said substrate a target nucleotide sequence variant identification assay for identifying at least one of N nucleotide sequence variants, wherein the assay comprises:
(i) providing at least M sets of barcode probes for performing at least M cycles of said assay, each set comprising N unique barcode binding moieties capable of binding preferentially to a corresponding one of said N barcode moieties, each barcode probe set comprising a detection label for generating K bits of information per cycle;
(ii) performing at least M detection cycles to generate a signal detection sequence at a plurality of locations on said substrate, wherein M is at least two, each cycle comprising
(1) contacting said substrate bound to said ligation reaction products with said barcode probe set corresponding with said cycle number;
(2) washing the surface of the substrate to remove unbound barcode probes;
(3) detecting the presence or absence of a plurality of signals from said spatially separate regions of said substrate;
and
(4) if the cycle number is less than m, performing a denaturation reaction to remove said barcode probe from said barcode moiety;
and
(d) Determining from said at least M detection cycles L total bits of information, wherein K×M=L and L>log2 (n), and wherein said L bits of information are used to identify one or more of said N nucleotide sequence variants.

9 more elements

A method of detecting at least one target nucleotide sequence variant suspected of being present in a sample, comprising:

(a) distributing a sample comprising a plurality of oligonucleotides suspected of comprising at least one target nucleotide sequence variant at a locus on a substrate so that they bind to the substrate at spatially separate regions of said substrate;
(b) carrying out on said oligonucleotides bound to said substrate a target nucleotide sequence variant identification assay comprising performing M number of detection cycles for target nucleotide sequence variant identification, wherein M is at least two, each cycle comprising:
(i) contacting said enriched nucleic acid sample bound to said substrate with a target nucleotide sequence variant binding probe that binds preferentially to said target nucleotide sequence variant at said locus, said variant binding probe comprising a detectable label;
(ii) washing the surface of the substrate to remove unbound variant binding probes;
(iii) detecting the identity and location of said detectable label on said substrate;
and
(iv) if the cycle number is less than m, performing a denaturation reaction to remove bound variant binding probes from said oligonucleotide bound to said substrate;
and
(c) determining from the sequence of detectable labels at said location on said substrate the presence or absence of said target nucleotide sequence variant suspected of being present in said sample.

6 more elements

A method of identifying at least one target oligonucleotide sequence variant suspected of being present in a sample, comprising:

(a) distributing a sample on a substrate such that said plurality of oligonucleotides bind to said substrate at spatially separate regions of said substrate, wherein said oligonucleotides are suspected of comprising at least one target oligonucleotide sequence variant of a plurality of sequence variants at one of a plurality of target loci;
(b) carrying out on said oligonucleotides bound to said substrate a target oligonucleotide sequence variant identification assay for identifying at least one of N nucleotide sequence variants, wherein the assay comprises:
(i) providing at least M sets of sequence variant probes for performing at least M cycles of said assay,
(1) each set comprising sequence variant probes capable of binding preferentially to a single locus comprising one or more of said N nucleotide sequence variants,
(2) wherein each of said sequence variant probes comprise a detection label for generating K bits of information for said corresponding cycle;
(3) wherein for at least 2 of said M cycles, said sequence variant probe set comprises N sequence variant probes each capable of binding preferentially to a corresponding single one of said N nucleotide sequence variants;
and
(ii) performing at least M detection cycles to generate a signal detection sequence at said spatially separate regions of said substrate bound to said oligonucleotides, wherein M is at least 2, each cycle comprising:
(1) contacting said oligonucleotides bound to said substrate with said sequence variant probe set corresponding with said cycle;
(2) washing the surface of the substrate to remove unbound sequence variant probes;
(3) detecting the identity and location of said detection label on said substrate to generate K bits of information at each of said spatially separate regions for said cycle;
and
(4) if the cycle number is less than m, performing a denaturation reaction to remove bound sequence variant probes from said bound oligonucleotides;
and
(c) determining from said at least M detection cycles L total bits of information, wherein the L equals the sum of said K bits of information generated at each of said M detection cycles, wherein L>log2 (n), and wherein said L bits of information are used to identify one or more of said N oligonucleotide sequence variants.

12 more elements

A method of detecting at least one target nucleotide sequence variant suspected of being present in a sample, comprising:

(a) distributing a plurality of oligonucleotides on a substrate so that the plurality of oligonucleotides bind to the substrate at spatially separate regions, wherein said plurality of oligonucleotides are suspected of comprising said at least one target nucleotide sequence variant at least one of a plurality of loci;
(b) carrying out on said substrate a target nucleotide sequence variant identification assay, wherein the sequence variant identification assay comprises performing at least M detection cycles to generate a signal detection sequence, wherein M is at least two, each cycle comprising:
(i) contacting said substrate with a set of primers each capable of binding preferentially to an oligonucleotide sequence immediately 5′ or 3′ to the location of one of said at least one target sequence variants, thereby forming a hybridized primer/oligonucleotide bound to said substrate when said at least one target sequence variant is bound to said substrate;
(ii) contacting said substrate with reagents for performing a single nucleotide extension reaction, said reagents comprising at least one nucleotide comprising a detectable label and a terminator;
(iii) exposing said substrate to conditions that promote a single nucleotide extension reaction at the 3′ terminus of said primer;
(iv) washing the surface of the substrate to remove unbound nucleotides;
(v) detecting the identity and location of said detectable label on said substrate;
and
(vi) if the cycle number is less than m, performing a denaturation reaction to remove said primers bound to said oligonucleotides;
and
(c) determining from the sequence of detectable labels for each cycle at a location on said substrate the presence or absence of said target nucleotide sequence variant suspected of being present in said sample.

8 more elements

A method of identifying at least one target single nucleotide variant suspected of being present in a sample, comprising:

(a) distributing a nucleic acid sample comprising a plurality of oligonucleotides suspected of comprising at least one target single nucleotide variant of a plurality of single nucleotide variants at least one of a plurality of loci on a substrate such that said plurality of oligonucleotides bind to said substrate at spatially separate regions of said substrate;
(b) carrying out on said oligonucleotides bound to said substrate a target single nucleotide variant identification assay for identifying at least one of N single nucleotide variants at least one of a plurality of loci, said assay comprising:
(i) providing a set of primers for each locus comprising at least one of said N single nucleotide variants, each of said set of primers capable of hybridizing to an oligonucleotide sequence immediately 5′ or 3′ to one of the N single nucleotide variants;
(ii) performing at least M detection cycles to generate a signal detection sequence at said spatially separate regions of said substrate bound to said oligonucleotides, wherein M is at least 2, each cycle comprising:
(1) contacting said oligonucleotides bound to said substrate with said set of primers for each locus, thereby hybridizing said each of said sets of primers to the corresponding oligonucleotide sequence immediately 5′ or 3′ to the single nucleotide variant at said locus;
(2) contacting said oligonucleotides hybridized to said primers with a set of nucleotides for generating K bits of information for said corresponding cycle, said nucleotides comprising a terminator and a detectable label, and reagents for performing a single nucleotide extension reaction, each nucleotide comprising detectable label;
(3) exposing said substrate surface to conditions to promote a single nucleotide extension reaction;
(4) washing the surface of the substrate to remove unbound nucleotides;
(5) detecting the identity and location of said detection label on said substrate to generate K bits of information at each of said spatially separate regions for said cycle;
and
(6) if the cycle number is less than m, performing a denaturation reaction to remove said primers bound to said oligonucleotides;
and
(c) determining from said at least M detection cycles L total bits of information, wherein the L equals the sum of said K bits of information generated at each of said M detection cycles, wherein L>log2 (n), and wherein said L bits of information are used to identify one or more of said N oligonucleotide sequence variants.

10 more elements

A method of identifying at least one target nucleotide sequence variant suspected of being present in a sample, comprising:

(a) providing an amplification reaction product of a sequence variant-specific amplification reaction performed on said sample, wherein said amplification reaction product comprises a plurality of oligonucleotides each comprising a substrate binding moiety and a barcode moiety;
(b) distributing said amplification reaction product on a substrate such that individual oligonucleotides bind to the substrate via the substrate binding moiety at spatially separate regions of said substrate;
(c) carrying out on said substrate a target nucleotide sequence variant identification assay, wherein the sequence variant identification assay comprises performing at least M detection cycles to generate a signal detection sequence, wherein M is at least two, each cycle comprising
(i) contacting said ligation reaction product with a barcode probe comprising a detection label, wherein said barcode probe binds to the barcode moiety when it is present on the substrate;
(ii) washing the surface of the substrate to remove unbound barcode probes;
(iii) detecting the identity and location of said detection label on said substrate;
and
(iv) if the cycle number is less than m, removing said barcode probe from said barcode moiety;
and analyzing the signal detection sequence generated by said M cycles at said spatially separate locations on said substrate to determine the presence or absence of said at least one target nucleotide sequence variant of interest.

6 more elements

A method of identifying at least one target nucleotide sequence variant suspected of being present in a sample, comprising:

(a) providing an amplification reaction product of a sequence variant-specific amplification reaction performed on said sample, wherein said amplification reaction product comprises a plurality of oligonucleotides each comprising a substrate binding moiety and a barcode moiety;
(b) distributing said amplification reaction product on a substrate such that individual oligonucleotides bind to the substrate via the substrate binding moiety at spatially separate regions of said substrate;
(c) carrying out on said substrate a target nucleotide variant identification assay for identifying at least one of N nucleotide sequence variants, wherein the assay comprises:
(i) providing at least M sets of barcode probes for performing at least M cycles of said assay, each set comprising N unique barcode binding moieties capable of binding preferentially to a corresponding one of said N barcode moieties for generating K bits of information per cycle;
(ii) performing at least M detection cycles to generate a signal detection sequence at a plurality of said spatially separate regions on said substrate, wherein M is at least one, each cycle comprising:
(1) contacting said substrate bound to said allele specific amplification reaction products with said barcode probe set corresponding with said cycle number;
(2) washing the surface of the substrate to remove unbound barcode probes;
(3) detecting the presence or absence of a plurality of signals from said spatially separate regions of said substrate;
and
(4) if the cycle number is less than m, performing a denaturation reaction to remove said barcode probe from said barcode moiety;
and
(d) determining from said at least M detection cycles L total bits of information, wherein K×M=L and L>log2 (n), and wherein said L bits of information are used to identify one or more of said N nucleotide sequence variants.

9 more elements

Dependencies are inferred from the claim text.

Polymorphism detection with increased accuracy

Life of this patent

Polymorphism detection with increased accuracy

Life of this patent

Abstract

Claims193