Multiplex drop-off digital polymerase chain reaction method

Abstract

The present application provides multiplex digital polymerase chain reaction (dPCR) assays, e.g., multiplex drop-off dPCR assays, methods, systems, and kits. The methods described herein can be used for a variety of applications, e.g., detection of microsatellite instability and quantification of site-specific genome editing products.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to European Patent Application No. 19306765.9, filed December 23, 2019, and U.S. Patent Application No. 17 / 013,222, filed September 4, 2020, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence list of submitted ASCII text files

[0004] The contents of the following submitted ASCII text file are incorporated herein by reference in their entirety: Sequence List in Computer-Readable Form (CRF) (filename: 186142000641SEQLIST.TXT, record date: December 14, 2020, size: 3KB). Technical Field

[0005] This application relates to multiplex digital polymerase chain reaction (PCR) assays (e.g., multiplex drop-off dPCR assays), methods, and systems, including methods for assessing microsatellite instability (MSI) and genome editing products. Background Technology

[0006] Digital polymerase chain reaction (dPCR) is a powerful and sensitive method for detecting rare mutations in nucleic acid samples. It utilizes allele-specific fluorescent TAQMAN assays. TM Digital PCR assays using probes have been developed for detecting somatic mutations in biomarker genes. In conventional dPCR assays, a wild-type probe that recognizes wild-type alleles and a mutant probe that recognizes specific mutant alleles are used. After the wild-type or mutant probe hybridizes to an amplicon in a dPCR region, the probe releases its fluorophore via the exonuclease activity of DNA polymerase. The fluorophore released from the wild-type probe is detected through a different fluorescence detection channel than that released from the mutant probe. This assay requires a dPCR instrument with an R detection channel to detect R mutations at one or more gene loci. In contrast, drop-off assays allow for the quantification of any number of mutations occurring at mutation hotspots by using two probes in the dPCR reaction: a drop-off probe that recognizes wild-type sequences at mutation hotspots and a reference probe that recognizes sequences at low-mutation regions on the same amplicon. See, Decraene C. et al., Clinical Chemistry 64(2):317-328 (2017). In drop-off assays, two detection channels are required to detect any number of mutations at a single gene locus.

[0007] Because dPCR instruments have a limited number of fluorescence detection channels, there is a need to increase the number of multiplex dPCR assays targeting different mutations and different gene loci. Such assays are particularly useful in clinical and other applications involving limited sample sizes. Robust methods for quantifying different genetic subtypes based on data from multiplex dPCR assays are also needed. Summary of the Invention

[0008] This application provides methods, apparatus, systems, and compositions for detecting and / or quantifying wild-type and mutant sequences at multiple target regions in a nucleic acid sample using multiplex dPCR assays (e.g., multiplex drop-off dPCR assays). The assays described herein can be used to assess microsatellite instability (MSI) and detect genome editing products (e.g., CRISPR-Cas system editing products).

[0009] One aspect of this application provides a method for quantifying wild-type and / or mutant sequences at multiple target regions in a sample containing nucleic acid molecules, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, and wherein substantially all partitions each comprise:

[0010] Multiple probe groups corresponding to multiple target regions, wherein each of the multiple probe groups includes:

[0011] A drop-off probe comprising a drop-off tag and an oligonucleotide drop-off sequence, wherein the oligonucleotide drop-off sequence is complementary to a wild-type sequence at a target region corresponding to a respective probe set;

[0012] A reference probe comprising a reference marker and an oligonucleotide reference sequence, the oligonucleotide reference sequence being complementary to a wild-type sequence located upstream or downstream of a target region corresponding to a particular probe set;

[0013] In the plurality of probe groups, the reference marker and drop-off marker of each probe group can be detected through different detection channels; in the plurality of probe groups, the reference markers can be detected through different detection channels; in the plurality of probe groups, the drop-off markers can be detected through different detection channels; and in the plurality of probe groups, at least one reference marker and at least one drop-off marker can be detected through the same detection channel.

[0014] The method includes detecting hybridization of reference probes of the plurality of probe sets with a nucleic acid molecule or its amplicon containing a wild-type sequence at a reference region in a plurality of partitions; and detecting hybridization of drop-off probes of the plurality of probe sets with a nucleic acid molecule or its amplicon containing a wild-type sequence at a target region in the plurality of partitions; thereby providing quantification of wild-type and / or mutant sequences at a plurality of target regions in a sample. In some embodiments, detection of signals from a reference label and signals from a drop-off label in a probe set indicates the presence of a wild-type sequence at the target region corresponding to the probe set, and detection of signals from a reference label but no signals from a drop-off label in a probe set indicates the presence of a mutant sequence at the target region corresponding to the probe set. In some embodiments, each of the plurality of probe sets is a probe pair, and the total number of detection channels is less than twice the total number of probe sets. In some embodiments, the total number of detection channels is equal to the total number of probe sets.

[0015] In some embodiments according to any of the above methods, the plurality of probe sets are R probe pairs,

[0016] where the first probe pair among the R probe pairs includes:

[0017] a first reference probe that includes a first reference sequence (r1) and a first reference label detectable through a first detection channel (X1), and

[0018] a first drop-off probe that includes a first drop-off sequence (w1) and a first drop-off label detectable through a second detection channel (X2);

[0019] where the second probe pair among the R probe pairs includes:

[0020] a second reference probe that includes a second reference sequence (r2) and a second reference label detectable through the second detection channel (X2), and

[0021] a second drop-off probe that includes a second drop-off sequence (w2) and a second drop-off label detectable through a third detection channel (X3);

[0022] where, if (e.g., when) R is strictly greater than 3, the ith probe pair (2 < i < R) among the R probe pairs includes:

[0023] an ith reference probe that includes an ith reference sequence (r i ) and an ith reference label detectable through an ith detection channel (X i ), and

[0024] The i-th drop-off probe contains the i-th drop-off sequence (w i ) and can be detected through the (i+1)th detection channel (X) i+1 The i-th drop-off marker detected;

[0025] Wherein, if (for example, when) R is strictly greater than 2, then the Rth probe pair of the R probe pairs includes:

[0026] The R-th reference probe contains the R-th reference sequence (r R ) and with the Rth detection channel (X R The related R-th reference marker, and

[0027] The R-th drop-off probe contains the R-th drop-off sequence (w R ) and the Rth drop-off tag that can be detected by the first detection channel (X1);

[0028] The method includes:

[0029] Through detection channel X1-X R Each of the R probe pairs in the assay detects hybridization between a reference probe and a nucleic acid molecule or its amplicon containing a wild-type sequence at a reference region in multiple partitions; and

[0030] Through detection channel X1-X R Each of the R probe pairs is detected by hybridization of the drop-off probe with a nucleic acid molecule or its amplicon containing a wild-type sequence at a target region in multiple partitions.

[0031] In some implementations, the method further includes: obtaining a first count of one or more partitions, each partition generating a positive signal through the i-th detection channel and through detection channels X1-X. R Any other detection channel in the array generates a negative signal; a second count is obtained for one or more partitions, each partition passing through all detection channels X1-X. R Generate a negative signal; and calculate the mutation probability that a given partition contains a mutated sequence at the target region corresponding to the i-th probe pair. The mutation probability is based on the ratio between the first count and the sum of the first and second counts. In some embodiments, the method further includes determining an estimated concentration of the mutated sequence in the sample corresponding to the target region of the i-th probe pair based on the mutation probability. In some embodiments, the estimated concentration of the mutated sequence in the sample corresponding to the target region of the i-th probe pair is determined according to the following:

[0032]

[0033] in This represents the estimated concentration of the mutant sequence in the target region corresponding to the i-th probe pair in the sample.

[0034] Where v represents the volume of the partition, and

[0035] in This represents the mutation probability that a given partition in the sample contains a mutated sequence at the target region corresponding to the i-th probe pair. In some embodiments, the method further includes determining a confidence interval and / or uncertainty measure related to the estimated concentration of the mutated sequence at the target region corresponding to the i-th probe pair in the sample.

[0036] In some embodiments of any of the methods described above, wherein the plurality of probe groups are R probe pairs, the method further includes calculating the wild-type probability that a given partition contains a wild-type sequence at a target region corresponding to the i-th probe pair, wherein the wild-type probability is based on the mutation probability corresponding to the i-th probe pair and the mutation probability corresponding to the (i+1)-th probe pair, wherein if (e.g., when) i = R, then the (i+1)-th probe pair refers to the first probe pair. In some embodiments, the wild-type probability is calculated according to the following:

[0037]

[0038] in This represents the wild-type probability that a given partition contains a wild-type sequence at the target region corresponding to the i-th probe pair.

[0039] Where n i,(i+1) This represents the count of one or more partitions, each represented by X. i The detection channel generates a positive signal, which is transmitted through X. i+1 The detection channel generates a positive signal and passes through detection channel X1-X. R Any other detection channel in the process generates a negative signal;

[0040] Where if (for example, when) i = R, n i,(i+1) It refers to n R,1 ;

[0041] Where n0 represents the count of one or more partitions, each of which passes through all detection channels X1-X. R Generates a negative signal;

[0042] Where n i This represents the count of one or more partitions, each of which generates a positive signal through the Xi detection channel and through the detection channels X1-X. R Any other detection channel in the process generates a negative signal;

[0043] Where n i+1 This represents the count of one or more partitions, each represented by X. i+1 The detection channel generates a positive signal and passes through detection channel X1-X. R Any other detection channel in the process generates a negative signal;

[0044] Where if (for example, when) i = R, n i+1 This refers to n1.

[0045] in This represents the mutation probability that a given partition contains a mutated sequence at the target region corresponding to the i-th probe pair.

[0046] in This represents the mutation probability that a given partition contains a mutated sequence at the target region corresponding to the (i+1)th probe pair, and

[0047] Where if (for example, when) i = R, It refers to In some embodiments, the method further includes determining an estimated concentration of the wild-type sequence in the sample corresponding to the target region of the i-th probe pair based on the wild-type probability. In some embodiments, the estimated concentration of the wild-type sequence in the sample corresponding to the target region of the i-th probe pair is determined according to the following:

[0048]

[0049] in This represents the estimated concentration of the wild-type sequence in the target region corresponding to the i-th probe pair in the sample.

[0050] Where v represents the volume of the partition, and

[0051] in This represents the wild-type probability that a given partition in the sample contains a wild-type sequence at the target region corresponding to the i-th probe pair. In some embodiments, the method further includes determining a confidence interval and / or uncertainty measure related to the estimated concentration of the wild-type sequence at the target region corresponding to the i-th probe pair in the sample.

[0052] In some embodiments of any of the methods described above, wherein the plurality of probe sets are R probe pairs, the method further includes adjusting the concentration of nucleic acid molecules in the sample based on partition counting, each partition being detected through three or more detection channels X1-X. RA positive signal is generated, wherein: (i) if (e.g., when) the count is greater than a predetermined value, the adjustment is made by reducing the concentration of nucleic acid molecules in the sample through dilution; or (ii) if (e.g., when) the count is less than a predetermined value, the adjustment is made by increasing the concentration of nucleic acid molecules in the sample through concentration. In some embodiments, the method further includes passing each of the detection channels X1-X R The quality control metric is determined by comparing the count of each partition that generates a positive signal with an estimated count, wherein the estimated count is based on the respective counts generated by the detection channels X1-X. R The partition count is the count of each partition that generates a positive signal, in addition to the partition count of the partitions in the table.

[0053] In some embodiments of any of the above methods, the plurality of probe groups are R probe pairs, where R is between 2 and 6, for example, 3.

[0054] In some embodiments of any of the methods described above, wherein the plurality of probe groups are three probe pairs, the method includes obtaining a first count (n) of one or more partitions. 100 Each partition generates a positive signal through detection channel X1, a negative signal through detection channel X2, and a negative signal through detection channel X3; a second count (n) of one or more partitions is obtained. 000 Each of the partitions generates a negative signal on all detection channels X1-X3, and the mutation probability of a given partition containing a mutated sequence at the target region corresponding to the first probe pair is calculated. The mutation probability is based on the first count (n) 100 ) and the first count (n) 100 ) and second count (n 000 The ratio between the sums of the mutation probabilities. In some embodiments, the method further includes a ratio based on the mutation probability. Determine the estimated concentration of the mutant sequence in the sample corresponding to the target region of the first probe pair. In some embodiments, the method further includes determining the estimated concentration in the sample. The relevant confidence intervals and / or uncertainty measures.

[0055] In some embodiments of any of the methods described above, wherein the plurality of probe groups are three probe pairs, the method further includes calculating the wild-type probability that a given partition in the sample contains a wild-type sequence at a target region corresponding to the first probe pair. The wild-type probability is based on Calculation. In some implementations, the wild-type probability is based on... and Calculation. In some implementations, the wild-type probability... based on Sure,

[0056] Where n 110 This represents the count of one or more partitions, each of which generates a positive signal through a first detection channel, a positive signal through a second detection channel, and a negative signal through a third detection channel.

[0057] Where n 010 This represents the count of one or more partitions, each of which generates a negative signal through a first detection channel, a positive signal through a second detection channel, and a negative signal through a third detection channel.

[0058] in This represents the probability that a given partition contains a mutated sequence at the target region corresponding to the second probe pair.

[0059] In some implementations of any of the methods described above, substantially all partitions further include:

[0060] Allele-specific (AS) probes comprising an AS marker and an oligonucleotide AS sequence complementary to the allele sequence at the target region.

[0061] The AS marker can be detected via a detection channel different from that of the reference probe and drop-off probe corresponding to multiple probe groups; and

[0062] The method further includes detecting hybridization of the AS probe with a nucleic acid molecule or its amplicon containing an allele sequence at a target region in the sample, thereby providing quantification of the allele sequence at the target region in the sample.

[0063] In some embodiments of any of the methods described above, each of the reference probe and drop-off probe has a single detectable label. In some embodiments, the reference label and drop-off label are fluorophores. In some embodiments, one or more different detection channels have different excitation wavelength ranges and / or different emission wavelength ranges. In some embodiments, one or more different detection channels share the same excitation and / or emission wavelength ranges but are associated with different fluorescence intensities. In some embodiments, a probe set corresponding to different target regions within the target gene comprises drop-off probes having drop-off labels associated with different detection channels sharing the same excitation and / or emission wavelength ranges, wherein the drop-off probes are detected at different fluorescence intensities. In some embodiments, the reference label and drop-off label are selected from luciferin, FAM, and YAKIMA. Cy3, HEX, VIC, ROX, CY5, CY5.5, ALEXA 647, ALEXA The group consists of 448 and Quasar705. In some embodiments, the plurality of probe groups are three probe pairs, wherein the first reference marker, the second reference marker, and the third reference marker are selected from the group consisting of Cy3, FAM, and Cy5, or wherein the first reference marker, the second reference marker, and the third reference marker are selected from the group consisting of FAM, HEX, and Cy5.

[0064] In some embodiments of any of the above methods, the target region is a mutation hotspot region selected from one or more genes comprising the group consisting of EGFR, NRAS, KRAS, ESR1, and BRAF.

[0065] In some embodiments according to any of the above methods, each partition further includes:

[0066] (a) Multiple primer sets corresponding to multiple target regions, and

[0067] (b) DNA-dependent DNA polymerase;

[0068] Each of the plurality of primer sets comprises a forward oligonucleotide primer and a reverse oligonucleotide primer, suitable for amplifying a target fragment containing a target region corresponding to the primer set and a reference region corresponding to the target region; the method comprises amplifying the target fragment from nucleic acid molecules in the plurality of partitions; and the detection comprises detecting hybridization of a reference probe and a drop-off probe with an amplicon of the target fragment. In some embodiments, the DNA-dependent DNA polymerase comprises 5' to 3' exonuclease activity and the detection comprises detecting an increase in fluorescence in the plurality of partitions caused by digestion of a reference label from a hybridized reference probe and / or a drop-off label from a hybridized drop-off probe by a 5' to 3' exonuclease.

[0069] In some embodiments according to any of the methods described above, the length of the amplicon is about 100 to about 200 nucleotides. In some embodiments, the reference region is not related to the single nucleotide polymorphism.

[0070] In some embodiments of any of the above methods, the method further includes forming a plurality of partitions having a predetermined volume.

[0071] In some embodiments of any of the methods described above, the nucleic acid molecule is a genomic DNA molecule, a tumor DNA molecule, or a cDNA molecule. In some embodiments, the method further includes extracting the nucleic acid molecule from a biological sample. In some embodiments, the nucleic acid molecule is obtained from a formalin-fixed paraffin-embedded (FFPE) sample or a liquid biopsy sample. In some embodiments, the method includes fragmenting the nucleic acid molecule in the biological sample to provide a sample containing the nucleic acid molecule.

[0072] In some embodiments of any of the above methods, the plurality of target regions are microsatellite sequence sites.

[0073] In some embodiments of any of the methods described above, the nucleic acid molecule is genomic DNA in a cell sample, wherein the cells have been contacted with a site-specific genome editing agent configured to cleave target sites in multiple target regions, and wherein the mutated sequence is a non-homologous end joining (NHEJ) edited sequence at the multiple target regions. In some embodiments, the site-specific genome editing agent includes a Cas nuclease, TALEN, or a zinc finger nuclease. In some embodiments, the method further includes contacting the cells with the site-specific genome editing agent.

[0074] In some embodiments according to any of the above methods, each of the plurality of probe groups further includes:

[0075] Allele-specific (AS) probes comprising an AS marker and an oligonucleotide AS sequence, wherein the oligonucleotide AS sequence is complementary to the allele sequence at the target region corresponding to the respective probe set.

[0076] The AS marker can be detected through a detection channel different from the detection channel of the corresponding reference probe or the detection channel of the corresponding drop-off probe, and the AS markers of the multiple probe groups can be detected through different detection channels from each other.

[0077] The method further includes detecting hybridization of the AS probes of the plurality of probe sets with nucleic acid molecules or their amplicones containing allele sequences at target regions in the plurality of partitions; thereby providing quantification of allele sequences at multiple target regions in the sample. In some embodiments, each of the plurality of probe sets is a probe triplet, and the total number of detection channels is less than three times the total number of probe sets. In some embodiments, the total number of detection channels is one more than the total number of probe sets. In some embodiments, the nucleic acid molecule is genomic DNA in a cell sample, wherein the cells have been contacted with a site-specific genome editing reagent and a homology-directed repair (HDR) template nucleic acid, the HDR template containing an HDR substitution sequence, wherein the site-specific genome editing reagent is configured to cleave target sites in the plurality of target regions, wherein the mutation sequence is a non-homologous end joining (NHEJ) editing sequence at the plurality of target regions, and wherein the allele sequence is an HDR substitution sequence inserted at the plurality of target regions. In some embodiments, the site-specific genome editing reagent includes a Cas nuclease, TALEN, or a zinc finger nuclease. In some embodiments, the method further includes contacting the cells with the site-specific genome editing reagent.

[0078] One aspect of this application provides a method for quantifying mutations at multiple microsatellite sequence sites in a sample containing nucleic acid molecules, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, and wherein substantially all partitions each comprise:

[0079] Multiple primer sets corresponding to multiple microsatellite sequence sites, wherein each of the multiple primer sets includes:

[0080] Suitable for both forward and reverse oligonucleotide primers for amplifying target fragments from nucleic acid molecules.

[0081] Each target fragment contains microsatellite sequence sites corresponding to the primer set and adjacent reference regions upstream or downstream of the microsatellite sequence sites;

[0082] Multiple probe pairs corresponding to multiple microsatellite sequence sites, wherein each of the multiple probe pairs includes:

[0083] Drop-off probes comprise drop-off tags and oligonucleotide drop-off sequences, the oligonucleotide drop-off sequences being complementary to the wild-type sequences at microsatellite sequence sites corresponding to the respective probe pairs.

[0084] A reference probe comprises a reference marker and an oligonucleotide reference sequence, wherein the oligonucleotide reference sequence is complementary to the wild-type sequence corresponding to the reference region of the respective probe pair.

[0085] The reference marker and drop-off marker of each of the plurality of probe pairs can be detected through different detection channels;

[0086] The reference markers of the plurality of probe pairs can be detected through different detection channels.

[0087] The drop-off markers of the multiple probe pairs can be detected through different detection channels;

[0088] At least one reference marker of the plurality of probe pairs and at least one drop-off marker of the plurality of probe pairs can be detected through the same detection channel;

[0089] The method includes amplifying target fragments in multiple partitions; and detecting hybridization of reference probes and drop-off probes of the multiple probe pairs with amplicones at target fragments in the multiple partitions, thereby providing quantification of mutations at multiple microsatellite sequence sites in the sample.

[0090] One aspect of this application provides a method for quantifying unmodified, homology-directed repair (HDR) edited and / or non-homologous end joining (NHEJ) edited sequences at multiple target regions in nucleic acid molecules from a cell sample, wherein the cells have been contacted with a site-specific genome editing reagent and an HDR template nucleic acid containing an HDR substitution sequence, wherein the site-specific genome editing reagent is configured to cleave target sites in multiple target regions, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, and wherein substantially all partitions each comprise:

[0091] Multiple probe groups corresponding to multiple target regions, wherein each of the multiple probe groups includes:

[0092] An HDR probe comprising an HDR marker and an oligonucleotide HDR sequence, the oligonucleotide HDR sequence being complementary to an HDR substitution sequence inserted at a target region corresponding to a respective probe set.

[0093] An NHEJ drop-off probe comprises an NHEJ drop-off tag and an oligonucleotide drop-off sequence, wherein the oligonucleotide drop-off sequence is complementary to a wild-type sequence corresponding to a target region of a corresponding probe set, and wherein the drop-off sequence does not hybridize with an NHEJ-edited mutant sequence at the target region corresponding to the corresponding probe set.

[0094] A reference probe comprises a reference marker and an oligonucleotide reference sequence, the oligonucleotide reference sequence being complementary to a wild-type sequence located upstream or downstream of a target region corresponding to a specific probe set.

[0095] In the plurality of probe groups, the HDR marker, NHEJ drop-off marker, and reference marker of each probe group can be detected through different detection channels; in the plurality of probe groups, the HDR markers can be detected through different detection channels; in the plurality of probe groups, the NHEJ drop-off markers can be detected through different detection channels; in the plurality of probe groups, the reference markers can be detected through different detection channels; in the plurality of probe groups, at least one reference marker and at least one NHEJ drop-off marker can be detected through the same detection channel, and / or at least one reference marker and / or at least one HDR marker of the plurality of probe groups can be detected through the same detection channel.

[0096] The method includes:

[0097] Hybridization of a reference probe with multiple probe sets with a nucleic acid molecule or its amplicon containing a wild-type sequence in a reference region in multiple partitions;

[0098] Detecting hybridization of HDR probes from multiple probe sets with nucleic acid molecules or their amplicons containing HDR substitution sequences at target regions in multiple partitions; and

[0099] The hybridization of NHEJ drop-off probes with multiple probe sets to nucleic acid molecules or their amplicon containing wild-type sequences at target regions in multiple partitions was detected.

[0100] This provides quantification of unmodified, HDR-edited, and / or NHEJ-edited sequences at multiple target regions in a sample. In some embodiments, the plurality of probe sets are (R-1) probe triplets, wherein the first probe triplet of the (R-1) probe triplets comprises:

[0101] The first reference probe includes a first reference sequence (m1) and a first reference marker detectable by a first detection channel (X1);

[0102] The first NHEJ drop-off probe comprises a first NHEJ drop-off sequence (r1) and a first NHEJ drop-off marker detectable by a second detection channel (X2); and

[0103] The first HDR probe contains a first HDR sequence (w1) and a first HDR marker detectable by the third channel (X3);

[0104] The second probe triplet of the (R-1) probe triplets includes:

[0105] A second reference probe, which includes a second reference sequence (m2) and a second reference label detectable through a second detection channel (X2);

[0106] A second NHEJ drop-off probe, which includes a second drop-off sequence (r2) and a second NHEJ drop-off label detectable through a third detection channel (X3); and

[0107] A second HDR probe, which includes a second HDR sequence (w2) and a second HDR label detectable through a fourth detection channel (X4);

[0108] wherein, if (e.g., when) R is strictly greater than 3, the i-th probe triplet (2 < i < R - 1) of (R - 1) probe triplets includes:

[0109] The i-th reference probe, which includes the i-th reference sequence (m i ) and the i-th reference label detectable through the i-th detection channel (X i );

[0110] The i-th NHEJ drop-off probe, which includes the i-th drop-off sequence (r i ) and the i-th NHEJ drop-off label detectable through the (i + 1)-th detection channel (X i+1 ); and

[0111] The i-th HDR probe, which includes the i-th HDR sequence (w i ) and the i-th HDR label detectable through the (i + 2)-th detection channel (X i+2 );

[0112] wherein, if (e.g., when) R is strictly greater than 3, the (R - 1)-th probe triplet of (R - 1) probe triplets includes:

[0113] The (R - 1)-th reference probe, which includes the (R - 1)-th reference sequence (m R-1 ) and the R-th reference label detectable through the R-th detection channel (X R );

[0114] The (R - 1)-th NHEJ drop-off probe, which includes the (R - 1)-th drop-off sequence (r R-1 ) and the (R - 1)-th NHEJ drop-off label detectable through the (R - 1)-th detection channel (X R-1 ); and

[0115] The (R-1)th HDR probe contains the (R-1)th HDR sequence (w R-1 ) and the (R-1)th HDR marker that can be detected by the first detection channel (X1);

[0116] The method includes detecting channels X1-X R-2 and X R Each detection (R-1) probe triplet hybridizes with a reference probe and a nucleic acid molecule or its amplicons containing wild-type sequences at a reference region in multiple partitions; via detection channels X2-X R-1 Each detection (R-1) probe triplet in the NHEJ drop-off probe hybridizes with a nucleic acid molecule or its amplicon containing a wild-type sequence at a target region in multiple partitions; and through detection channels X1 and X3-X R Each detection (R-1) probe triplet hybridizes with an HDR probe and a nucleic acid molecule or its amplicons containing an HDR substitution sequence at a target region in multiple partitions. In some embodiments, the method further includes:

[0117] If (for example, when) 1≤i≤R-2:

[0118] Obtain the first count of one or more partitions, each partition via X. i The detection channel generates a positive signal and passes through detection channel X1-X. R Any other detection channel in the array generates a negative signal; a second count is obtained for one or more partitions, each of which passes through all detection channels X1-X. R Generates a negative signal;

[0119] Or if (for example, when) i is R-1:

[0120] Obtain the first count of one or more partitions, each partition via X. R The detection channel generates a positive signal and passes through detection channel X1-X. R Any other channel in the signal generates a negative signal; a second count is obtained for one or more partitions, each of which passes through all detection channels X1-X. R Generate negative signals; and

[0121] Calculate the NHEJ editing probability of a given partition containing an NHEJ-edited sequence at the target region corresponding to the i-th probe triplet. The NHEJ edit probability is based on the ratio between a first count and the sum of the first and second counts. In some embodiments, the method further includes:

[0122] If (for example, when) 1≤i≤R-2:

[0123] Obtain the first count of one or more partitions, each partition via X. i The detection channel generates a positive signal, which is transmitted through X. i+1 The detection channel generates a positive signal and passes through detection channel X1-X. R Any other detection channel in the process generates a negative signal; a second count is obtained for one or more partitions, each of which is detected through detection channel X1-X. i-1 Each of them generates a negative signal and passes through the detection channel X. i+2 -X R Each of the components generates a negative signal; and the probability that a given partition contains an unmodified wild-type sequence at the target region corresponding to the i-th probe triplet is calculated. The unmodified probability is based on The ratio between the first count and the sum of the first and second counts;

[0124] Or if (for example, when) i is R-1:

[0125] Obtain the first count of one or more partitions, each partition via X. R The detection channel generates a positive signal in X. R-1 A positive signal is generated at the detection channel and passes through detection channel X1-X R Any other detection channel in the process generates a negative signal; a second count is obtained for one or more partitions, each of which is detected through detection channel X1-X. R-2 Each of the components generates a negative signal; and the probability that a given partition contains an unmodified wild-type sequence at the target region corresponding to the (R-1)th probe triplet is calculated. The unmodified probability is based on the ratio between the first count and the sum of the first and second counts. In some embodiments, the method further includes:

[0126] If (for example, when) 1≤i≤R-2:

[0127] Obtain the first count of one or more partitions, each partition via X. i The detection channel generates a positive signal, which is transmitted through X. i+2 The detection channel generates a positive signal and passes through detection channel X1-X. R Any other detection channel in the process generates a negative signal; a second count is obtained for one or more partitions, each of which is detected through detection channel X1-X. i-1 Each of them generates a negative signal in X. i+1 A negative signal is generated and passes through the detection channel X. i+3 -X REach of the components generates a negative signal; and the probability of HDR editing is calculated for a given partition containing an HDR permutation sequence at the target region corresponding to the i-th probe triplet. The HDR editing probability is based on And the ratio between the first count and the sum of the first and second counts;

[0128] Or if (for example, when) i is R-1:

[0129] Obtain the first count of one or more partitions, each partition via X. R The detection channel generates a positive signal, producing a positive signal at the X1 detection channel and passing through the detection channel X1-X. R Any other detection channel in the process generates a negative signal; a second count is obtained for one or more partitions, each of which is detected through detection channel X2-X. R-1 Each of the components generates a negative signal; and the probability of HDR editing of a given partition containing a wild-type sequence at the target region corresponding to the (R-1)th probe triplet is calculated. The HDR editing probability is based on And the ratio between the first count and the sum of the first and second counts.

[0130] One aspect of this application provides a method for quantifying wild-type and / or allele sequences at R target regions in a sample containing nucleic acid molecules, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, wherein substantially all partitions each contain R probe triplets corresponding to the R target regions.

[0131] The first probe triplet of the R probe triplets includes:

[0132] A first reference probe corresponding to the first reference sequence (w1) and a first reference marker detectable by the first detection channel (X1),

[0133] The first AS probe (“first AS probe 1”) of the first probe triplet corresponding to the first allele sequence (r1) and the first AS marker (“first AS marker 1”) of the first probe triplet detectable by the first detection channel (X1), and

[0134] The second AS probe (“second AS probe 1”) of the first probe triplet corresponding to the first allele sequence (r1) and the second AS marker (“second AS marker 1”) of the first probe triplet detectable by the second detection channel (X2);

[0135] The second probe triplet of the R probe triplets includes:

[0136] A second reference probe corresponding to a second reference sequence (w2) and a second reference label detectable through a second detection channel (X2),

[0137] A first AS probe (“first AS probe 2”) of a second probe triplet corresponding to a second allele sequence (r2) and a first AS label (“AS label 2”) of the second probe triplet detectable through a second detection channel (X2), and

[0138] A second AS probe (“second AS probe 2”) of a second probe triplet corresponding to a second allele sequence (r2) and a second AS label (“second AS label 2”) of the second probe triplet detectable through a third detection channel (X3);

[0139] Wherein, if (for example, when) R is strictly greater than 3, the i-th probe triplet (2 < i < R) of the R probe triplets includes:

[0140] The i-th reference probe corresponding to the i-th reference sequence (w i ) and the i-th reference label detectable through the i-th detection channel (X i ),

[0141] A first AS probe (“first AS probe i”) of the i-th probe triplet corresponding to the i-th allele sequence (r i ) and a first AS label (“first AS label i”) of the i-th probe triplet detectable through the i-th detection channel (X i ), and

[0142] A second AS probe (“second AS probe i”) of the i-th probe triplet corresponding to the i-th allele sequence (r i ) and a second AS label (“second AS label i”) of the i-th probe triplet detectable through the (i + 1)-th detection channel (X i+1 );

[0143] Wherein, if (for example, when) R is strictly greater than 2, the R-th probe triplet of the R probe triplets includes:

[0144] The R-th reference probe corresponding to the R-th reference sequence (w R ) and the R-th reference label detectable through the R-th detection channel (X R ),

[0145] A first AS probe (“first AS probe R”) of the R-th probe triplet corresponding to the R-th allele sequence (r R ) and a first AS label (“first AS label R”) of the R-th probe triplet detectable through the R-th detection channel (X RThe first AS marker (“first AS marker R”) of the Rth probe triplet detected, and

[0146] Corresponding to the Rth allele sequence (r R The second AS probe (“second AS probe R”) of the Rth probe triplet and the second AS marker (“second AS marker R”) of the Rth probe triplet that can be detected by the first detection channel (X1);

[0147] In each probe triplet, the first AS probe and the second AS probe hybridize at the target region corresponding to the corresponding probe triplet with the same allele sequence, different parts of the same allele sequence, or their complementary sequence.

[0148] The reference sequence for each probe triplet is located in the reference region corresponding to the corresponding probe triplet.

[0149] Among them, detection channels X1-X R They are different;

[0150] The method includes detecting channels X1-X R Each of the R probe triplet detection methods involves hybridization between a reference probe and a nucleic acid molecule or its amplicons containing the reference sequence or its complementary sequence at a reference region in multiple partitions; and hybridization via detection channels X1-X. R In each of the R probe triplet detection methods, the first and second AS probes hybridize with a nucleic acid molecule or its amplicons containing an allele sequence or its complementary sequence at a target region in multiple partitions; thereby providing quantification of wild-type and / or allele sequences at R target regions in the sample. In some embodiments, the reference region of the probe triplet is adjacent (e.g., upstream or downstream) to the target region corresponding to the respective probe triplet. In some embodiments, R is 3. In some embodiments, the allele sequence is associated with copy number variation (CNV). In some embodiments, the allele sequence is associated with rare alleles. In some embodiments, the allele sequence is associated with one or more mutant allele fractions (MAF). In some embodiments, the allele sequence is associated with one or more variant allele fractions (VAF). In some embodiments, the method is used to simultaneously detect CNV, determine multiple allele frequencies (MAF), or variant allele fractions (VAF).

[0151] Compositions, systems, kits, and articles for use in any of the above methods are also provided. Attached Figure Description

[0152] Figure 1A-1DA schematic diagram of an exemplary triplet drop-off PCR assay is shown, which can detect one wild-type sequence and all mutant sequences at each of six genetic populations, namely three gene loci (e.g., KRAS, NRAS, and EGFR mutation hotspots), using three fluorescence detection channels. Figure 1A A three-dimensional plot of fluorescence signals from dPCR reaction droplets from samples that have wild-type sequences only at the EGFR, NRAS, and KRAS gene loci is shown. Figure 1B A three-dimensional plot of fluorescence signals from dPCR reaction droplets from samples containing wild-type sequences at NRAS and KRAS sites, and samples containing both wild-type and mutant sequences at EGFR sites, is shown. Figure 1C A three-dimensional plot showing the fluorescence signal of dPCR reaction droplets from samples with wild-type sequences at EGFR and KRAS sites and with both wild-type and mutant sequences at NRAS sites. Figure 1D A three-dimensional plot of fluorescence signals from dPCR reaction droplets from samples containing wild-type sequences at EGFR and NRAS sites, and samples containing both wild-type and mutant sequences at KRAS sites, is shown.

[0153] Figure 2 An exemplary data analysis method is illustrated. Using spatial segmentation methods, such as 3D rectangles or 3D polygons, droplet populations corresponding to different feature signal readings are identified in a three-dimensional plot. The number of each droplet population is counted in each spatial segment (e.g., specified as n for 3D rectangular or polygonal segments). 000 n 100 n 110 n 101 n 011 n 010 n 001 and n 111 The counts are used to determine the concentration of wild-type or mutant populations at each of the three tested gene loci (e.g., designated as C). Mut1 C Mut2 C Mut3 C WT1 C WT2 and C WT3 ).

[0154] Figure 3A three-dimensional plot of fluorescence signals from dPCR reaction droplets from a sample is shown in a triple drop-off dPCR assay used to detect KRAS, NRAS, and EGFR mutations. The dPCR assay was performed on a sample containing three mutant DNA species mixed with wild-type DNA molecules at different concentrations. The first mutant DNA species had a G13D mutation in KRAS, the second mutant DNA species had a Q61K mutation in NRAS, and the third DNA species had an E19 deletion mutation in EGFR. Table 12 shows an exemplary set of forward and reverse primers, reference probes, and drop-off probes used in the triple drop-off dPCR assay for KRAS, NRAS, and EGFR. Table 13 shows the expected concentration, measured concentration, and standard deviation of the measured concentration for each genetic population in the sample.

[0155] Figures 4A-4B Two exemplary designs are shown for multiplex drop-off dPCR assays that can detect wild-type and mutant sequences at three KRAS mutation hotspots, two NRAS mutation hotspots, and one BRAF mutation hotspot using only three fluorescence detection channels. Figure 4A This study demonstrates designs for two distinct multiplex drop-off dPCR assays. One assay uses three probe pairs to detect wild-type and mutant sequences at the A146, G12 / 13, and Q61 sites of KRAS. The second assay detects wild-type and mutant sequences at the G12 / 13 and Q61 sites of NRAS, as well as the V600E and V600K mutations of BRAF. In the second assay, the BRAF V600E and V600K mutations can be detected using either drop-off probes or allele-specific probes. Figure 4B This demonstrates a single multiplex assay design that can detect all KRAS, NRAS, and BRAF mutations using three fluorescence detection channels. The same fluorescence detection channel is used to detect wild-type and mutant sequences at different sites within the same gene. However, to distinguish different sites within the same gene and detect probe pairs associated with different sites using varying fluorescence intensities, the fluorescence intensity can be adjusted by using primers and probe pairs of different concentrations.

[0156] Figure 5AA schematic diagram of an exemplary probe pair for detecting mutant sequences at a target region in a nucleic acid molecule is shown. The probe pair includes: (a) a drop-off probe that hybridizes to a wild-type sequence at the target region, and (b) a reference probe that hybridizes to a wild-type sequence at a reference region adjacent to the target region. Both the target and reference regions are present in the same template nucleic acid molecule or its amplicons. The reference region can be upstream or downstream of the target region. The reference probe and drop-off probe can be designed to hybridize to the same strand of the nucleic acid molecule or its amplicons, or to a different strand of the nucleic acid molecule or its amplicons. The reference region is associated with a low mutation frequency. Therefore, the reference probe hybridizes to the nucleic acid molecule or its amplicons regardless of whether the nucleic acid molecule contains a wild-type or mutant sequence at the target region. A forward primer (F primer) and a reverse primer (R primer) can be used to amplify a target fragment containing both the target and reference regions in the nucleic acid molecule.

[0157] Figure 5B A schematic diagram of an exemplary probe pair for detecting NHEJ-edited sequences at a target region in a nucleic acid molecule is shown. The probe pair comprises: (a) an NHEJ drop-off probe that hybridizes to an unmodified sequence (i.e., a wild-type sequence) at the target region, and (b) a reference probe that hybridizes to a wild-type sequence at a reference region adjacent to the target region. Both the target and reference regions are present in the same template nucleic acid molecule or its amplicons. The reference region may be upstream or downstream of the target region. The reference probe and the NHEJ drop-off probe may be designed to hybridize to the same strand of the nucleic acid molecule or its amplicons, or to a different strand of the nucleic acid molecule or its amplicons. The target region contains the target site of a site-specific genome editing agent (e.g., CRISPR-Cas), which can be cleaved and repaired by NHEJ. The reference region is associated with a low mutation frequency. Therefore, the reference probe hybridizes to the nucleic acid molecule or its amplicons regardless of whether the nucleic acid molecule contains an unmodified sequence or a mutated NHEJ-edited sequence at the target region. Forward primers (F primers) and reverse primers (R primers) can be used to amplify target fragments containing target and reference regions in nucleic acid molecules.

[0158] Figure 5CA schematic diagram of an exemplary probe triplet for detecting NHEJ-edited and HDR-edited sequences at target regions in nucleic acid molecules is shown. The probe triplet comprises: (a) an NHEJ drop-off probe that hybridizes to an unmodified sequence (i.e., a wild-type sequence) at the target region; (b) an HDR probe that hybridizes to an HDR substitution sequence at the target region; and (c) a reference probe that hybridizes to a wild-type sequence at a reference region adjacent to the target region. Both the target and reference regions are present in the same template nucleic acid molecule or its amplicons. The reference region may be upstream or downstream of the target region. The reference probe, NHEJ drop-off probe, and HDR probe may be designed to hybridize to the same strand of the nucleic acid molecule or its amplicons, or to different strands of the nucleic acid molecule or its amplicons. The target region contains a target site for a site-specific genome editing agent (e.g., CRISPR-Cas), which can be cleaved and repaired by NHEJ or HDR. The reference region is associated with a low mutation frequency. Therefore, the reference probe hybridizes with the nucleic acid molecule or its amplicon, regardless of whether the nucleic acid molecule contains an unmodified sequence, a mutated NHEJ-edited sequence, or an HDR substitution sequence in the target region. Forward primers (F primers) and reverse primers (R primers) can be used to amplify target fragments containing both the target and reference regions of the nucleic acid molecule.

[0159] Figure 6A This diagram illustrates a multiplex dPCR assay using four detection channels to simultaneously detect unmodified, NHEJ-edited, and HDR-edited sequences at three target genomic sites in cells undergoing CRISPR / Cas genome editing. Each CRISPR-Cas genome editing product is detected using three sets of fluorescently labeled probes. Different numbers represent different fluorophores.

[0160] Figure 6BThis diagram illustrates arrangements of four, six, or ten markers (e.g., fluorophores) from three, five, or nine probe triplets for detecting unmodified, NHEJ-edited, and HDR-edited sequences at three, five, or nine target genomic sites, respectively. Each closed graph with three vertices (represented by gray circles and labeled with numbers) corresponds to a probe triplet with three different markers. Arrows indicate the order of the first marker to the second marker in each probe triplet. For example, in a set of three probe triplets, the probes in the first triplet are labeled with marker 1, marker 2, and marker 3; the probes in the second triplet are labeled with marker 2, marker 3, and marker 4; and the probes in the third triplet are labeled with marker 4, marker 3, and marker 1. In a set of five probe triplets, the probes of the first probe triplet are marked with Mark 1, Mark 2 and Mark 3 respectively; the probes of the second probe triplet are marked with Mark 2, Mark 3 and Mark 4 respectively; the probes of the third probe triplet are marked with Mark 3, Mark 4 and Mark 5 respectively; the probes of the fourth probe triplet are marked with Mark 4, Mark 5 and Mark 6 respectively; and the probes of the fifth probe triplet are marked with Mark 6, Mark 5 and Mark 1 respectively. In a set of nine probe triplets, the probes of the first probe triplet are marked with Mark 1, Mark 2, and Mark 3; the probes of the second probe triplet are marked with Mark 2, Mark 3, and Mark 4; the probes of the third probe triplet are marked with Mark 3, Mark 4, and Mark 5; the probes of the fourth probe triplet are marked with Mark 4, Mark 5, and Mark 6; the probes of the fifth probe triplet are marked with Mark 5, Mark 6, and Mark 7; the probes of the sixth probe triplet are marked with Mark 6, Mark 7, and Mark 8; the probes of the seventh probe triplet are marked with Mark 7, Mark 8, and Mark 9; the probes of the eighth probe triplet are marked with Mark 8, Mark 9, and Mark 10; and the probes of the ninth probe triplet are marked with Mark 10, Mark 9, and Mark 1.

[0161] Figure 7 A flowchart is shown for an exemplary method of data analysis for detecting wild-type and mutant sequences at three target regions using three probes.

[0162] Figure 8 A flowchart is shown for an exemplary method of data analysis for detecting wild-type and mutant sequences at R target regions using R probes.

[0163] Figure 9 A flowchart is shown for an exemplary method of data analysis for detecting unmodified, NHEJ-edited, and HDR-edited sequences at (R-1) target regions using a (R-1) probe triplet.

[0164] Figure 10An exemplary electronic device according to some implementation schemes is depicted.

[0165] Figure 11A A schematic diagram of an exemplary probe set for detecting allele sequences at target regions in nucleic acid molecules is shown. The probe set includes: (a) a first allele-specific (AS) probe with a first label that hybridizes to an allele sequence at the target region (e.g., a sequence containing a mutation, such as one labeled with a cross); (b) a second AS probe with a second label that hybridizes to the same allele sequence at the target region as the first AS probe; and (c) a reference probe that hybridizes to a reference sequence (e.g., a wild-type sequence) at a reference region. The first and second AS probes may hybridize to the same or adjacent sequences containing the allele sequence or its complement. A forward primer (F primer 1) and a reverse primer (R primer 1) can be used to amplify a target fragment containing the target region in the nucleic acid molecule. A forward primer (F primer 2) and a reverse primer (R primer 2) can be used to amplify a reference fragment containing the reference region in the nucleic acid molecule.

[0166] Figure 11B A schematic diagram of an exemplary probe set for detecting allele sequences at target regions in nucleic acid molecules is shown. The probe set includes: (a) an allele-specific (AS) probe with a first and a second label that hybridizes to the allele sequence at the target region (e.g., a sequence containing a mutation), and (c) a reference probe that hybridizes to a reference sequence at a reference region (e.g., a wild-type sequence). The AS probe can hybridize to the same or adjacent sequences containing the allele sequence or its complement. A forward primer (F primer 1) and a reverse primer (R primer 1) can be used to amplify the target fragment containing the target region in the nucleic acid molecule. A forward primer (F primer 2) and a reverse primer (R primer 2) can be used to amplify the reference fragment containing the reference region in the nucleic acid molecule.

[0167] Figure 11CA schematic diagram of an exemplary probe set for detecting allele sequences at a target region in a nucleic acid molecule is shown. The probe set includes: (a) a first allele-specific (AS) probe with a first label that hybridizes to the allele sequence at the target region (e.g., a sequence containing a mutation); (b) a second AS probe with a second label that hybridizes to the same allele sequence at the target region as the first AS probe; and (c) a reference probe that hybridizes to a reference sequence (e.g., a wild-type sequence) at a reference region. Both the target and reference regions are present in the same template nucleic acid molecule or its amplicons. The reference region may be upstream or downstream of the target region. The reference probe and AS probe may be designed to hybridize to the same strand of the nucleic acid molecule or its amplicons, or to a different strand of the nucleic acid molecule or its amplicons. A forward primer (F primer) and a reverse primer (R primer) can be used to amplify a target fragment containing both the target and reference regions in the nucleic acid molecule.

[0168] Figure 11D A schematic diagram of an exemplary probe set for detecting allele sequences at a target region in a nucleic acid molecule is shown. The probe set includes: (a) an allele-specific (AS) probe with a first and a second label that hybridizes to the allele sequence at the target region (e.g., a sequence containing a mutation), and (c) a reference probe that hybridizes to a reference sequence (e.g., a wild-type sequence) at a reference region. Both the target and reference regions are present in the same template nucleic acid molecule or its amplicons. The reference region may be upstream or downstream of the target region. The reference probe and the AS probe may be designed to hybridize to the same strand of the nucleic acid molecule or its amplicons, or to a different strand of the nucleic acid molecule or its amplicons. A forward primer (F primer) and a reverse primer (R primer) are used to amplify a target fragment containing both the target and reference regions in the nucleic acid molecule.

[0169] Figure 11E A schematic diagram of an exemplary probe set for detecting mutant sequences at target regions of nucleic acid molecules is shown. The probe set includes: (a) a first mutation-specific (MS) probe with a first label that hybridizes to the mutant sequence at the target region; (b) a second MS probe with a second label that hybridizes to the same mutant sequence at the target region; and (c) a wild-type-specific (WS) probe that hybridizes to the wild-type sequence at the target region. The WS and MS probes are designed to hybridize to the same or different strands of the nucleic acid molecule or its amplicons. Forward and reverse primers can be used to amplify target fragments containing the target region in the nucleic acid molecule.

[0170] Figure 11FA schematic diagram of an exemplary probe set for detecting mutant sequences at target regions of nucleic acid molecules is shown. The probe set includes: (a) a mutation-specific (MS) probe with a first and a second label that hybridizes to the mutant sequence at the target region; and (b) a wild-type-specific (WS) probe that hybridizes to the wild-type sequence at the target region. The WS and MS probes are designed to hybridize to the same or different strands of the nucleic acid molecule or its amplicons. Forward and reverse primers can be used to amplify target fragments containing the target region in the nucleic acid molecule. Detailed Implementation

[0171] This application provides multiplex digital polymerase chain reaction (dPCR) assays, such as multiplex drop-off dPCR assays, which can use fewer than twice as many R detection channels to detect wild-type and mutant sequences at R different gene loci, for example, by using reference probes and drop-off probes with a shared set of overlapping markers. The assays described herein can be used to assess microsatellite instability (MSI) and genome editing products.

[0172] In some implementations, multiplex drop-off dPCR assays use R probe pairs, each probe pair comprising a reference probe containing a reference marker and a drop-off probe containing a drop-off marker, wherein the reference marker and drop-off marker in each probe pair can be detected through different detection channels. In some implementations, the drop-off marker set and the reference marker set used in the probe pair are cyclically arranged relative to each other, which allows detection of 2R genetic species (i.e., wild-type and mutant at each gene locus) through only R different detection channels. Furthermore, each drop-off probe is capable of detecting all mutant sequences associated with its respective target gene locus, thereby improving the multiplex level of dPCR assays, i.e., the number of detectable mutations per assay.

[0173] In some embodiments, multiplex drop-off dPCR assays use R⁻¹ probe triplets, each triplet comprising a reference probe containing a reference marker, a drop-off probe containing a drop-off marker (e.g., the NHEJ drop-off probe), and an allele-specific probe containing an allele-specific marker (e.g., the HDR probe), wherein the reference marker, drop-off marker, and allele-specific marker in each probe triplet can be detected via different detection channels. In some embodiments, the reference marker set, drop-off marker set, and allele-specific marker set used in the probe triplet are arranged relative to each other (e.g., as shown in the diagram). Figure 6B As shown in the diagram, this allows for the detection of 3(R-1) species using only R detection channels.

[0174] In some embodiments, multiplex dPCR assays use R probe sets, each probe set including a reference probe containing a reference marker, a first allele-specific probe containing a first allele-specific marker, and a second allele-specific probe containing a second allele-specific marker. The second allele-specific probe hybridizes to the same allele sequence or its complement, as with the first allele-specific probe, or the first and second allele-specific probes hybridize to two different portions of the same allele sequence or its complement. The reference marker and the first allele-specific marker in each probe set can be detected through the same detection channel, and the reference marker and the second allele-specific marker in each probe set can be detected through different detection channels. In some embodiments, the second allele-specific marker set and the reference marker set used in the probe sets are cyclically arranged relative to each other, which allows detection of 2R genetic species (i.e., wild-type and allele sequences at each gene locus) through only R different detection channels. Multiplex dPCR assays can be used to detect multiple copy number variations (CNVs), assess multiple allele frequencies (MAFs), and determine multiple variant allele fractions (VAFs) in a sample.

[0175] The multiplex dPCR assays and methods described herein can be used in a variety of applications, such as the detection of microsatellite mutations and the quantification of site-specific genome editing products. Compositions, systems, diagnostic methods, therapeutic methods, screening methods, kits, and products are also provided.

[0176] I. Definition

[0177] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein to refer to polymers of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerases. Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogs. Nucleic acids can be hybrid forms of single-stranded, double-stranded, or higher polymers and may include chemical modifications. “Polynucleotide” or “nucleic acid” may also be used herein to refer to sequences encoded by nucleic acids, including the sense strand (i.e., coding strand) and antisense strand (i.e., non-coding strand) sequences in double-stranded nucleic acid molecules.

[0178] As used herein, "oligonucleotide" generally refers to a short, usually single-stranded, usually synthetic polynucleotide that is typically, but not necessarily no longer than about 200 nucleotides. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides also applies perfectly to oligonucleotides.

[0179] As used herein, the term "sample" means a sample that can withstand the methods described herein, with or without pretreatment, such as nucleic acid extraction, fragmentation, dilution / concentration, or other pretreatment. The sample may be a biological sample or obtained by processing or manipulating a biological sample. In some embodiments, the sample is ready to be loaded onto a digital PCR instrument for analysis. In some embodiments, the sample has been diluted from a biological sample.

[0180] A "probe" is a molecule (e.g., a protein, nucleic acid, aptamer, etc.) that specifically interacts with or binds to a target polynucleotide and thus detects the target polynucleotide. Non-limiting examples of molecules that specifically interact with or bind to a target polynucleotide include nucleic acids (e.g., oligonucleotides), proteins (e.g., antibodies, transcription factors, zinc finger proteins, non-antibody protein scaffolds, etc.), and aptamers. Typically, probes are labeled with a detectable tag. The presence or level of the target polynucleotide can be indicated by an increase or decrease in the signal from the detectable tag. In some embodiments, the probe detects the target polynucleotide in the amplification reaction by digestion with the 5' to 3' exonuclease activity of a DNA-dependent DNA polymerase.

[0181] As used in this article, "set," "pair," and "triplet" refer to an ordered list of members. For example, "set," "pair," and "triplet" correspond to "list," "couple," and "triple" in mathematics, respectively. For instance, each probe pair can have the order {reference probe, drop-off probe}; each probe triplet can have the order {reference probe, drop-off probe, and AS probe}; and the reference probe's tag set can have the order {reference tag of probe set 1, reference tag of probe set 2, ..., reference tag of probe set R}.

[0182] The terms “label” and “detectable label” are used interchangeably herein and refer to reagents that can be detected by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical methods. Useful labels include fluorescent dyes (fluorophores), luminescent agents, electron-dense reagents, enzymes (e.g., commonly used in ELISA), biotin, digoxigenin, etc. 32 P and other isotopes, haptens. The term includes combinations of single-labeled reagents, such as combinations of fluorophores that provide a unique detectable characteristic, for example, at a specific wavelength or combination of wavelengths. Any method known in the art for conjugating labels to desired reagents can be used, for example, the method described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0183] The term "cyclic permutation" refers to the act of rearranging the members (e.g., markers) of a group (e.g., a reference probe group and a drop-off probe group) in a cyclic manner to generate another group with the same members without changing the relative positions of the members, for example, by moving the final element of a linear permutation of the members in the group to the front. Two cyclic permutations are equivalent if one can be rotated into the other (i.e., cyclically without changing the relative positions of the elements). Each group of n members has (n-1)! cyclic permutations. For example, the cyclic permutation of the group {fluorophore 1, fluorophore 2, fluorophore 3} can be {fluorophore 2, fluorophore 3, fluorophore 1} or {fluorophore 3, fluorophore 1, fluorophore 2}.

[0184] The term "arrangement" refers to rearranging members (e.g., markers) of a set (e.g., a reference probe set, a drop-off probe set, and an allele-specific probe set) into a sequence or order. For example, Figure 6B The arrangement of four, six, or ten labeled groups in three, five, or nine probe triplets is shown respectively.

[0185] Primers are typically short, single-stranded polynucleotides, usually with a free 3'-OH group, which binds to the target nucleic acid by hybridizing with the target sequence and then promoting the polymerization of a polynucleotide complementary to the target nucleic acid. Primers can have various lengths and are typically less than 50 nucleotides, for example, 12-30 nucleotides. Primers can be DNA, RNA, or a chimera of DNA and RNA portions. In some cases, primers may include one or more modified or non-natural nucleotide bases.

[0186] When at least two consecutive bases of, for example, a first nucleic acid or primer can combine in an antiparallel binding manner or hybridize with at least one daughter sequence of a second nucleic acid to form a double helix, the nucleic acid sequence is "complementary" to the other nucleic acid. In some embodiments, complementarity refers to the preference for hydrogen-bonded base pairs between the nucleotide bases G, A, T, C, and U, such that when two given polynucleotide or nucleotide sequences anneal to each other, A pairs with T and G pairs with C in DNA, and G pairs with C and A pairs with U in RNA.

[0187] The first nucleic acid sequence “corresponding to” the second nucleic acid sequence is a sequence that is identical to or complementary to the second nucleic acid sequence or a portion thereof, or contains the second nucleic acid sequence or its complementary sequence. When the second nucleic acid sequence contains unique features, such as mutations, the nucleic acid sequence “corresponding to” the second nucleic acid sequence contains a sequence with unique features or its complementary sequence.

[0188] The terms “hybridization” and “annealing” used interchangeably herein refer to reactions in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonds between the bases of the nucleotide residues. Hydrogen bonds can occur through Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific pathway. A nucleic acid, or a portion thereof, “hybridizes” with another nucleic acid in a physiological buffer (e.g., pH 6–9, 25–150 mM chloride salt) under conditions of minimal nonspecific hybridization at a defined temperature. In some embodiments, the defined temperature for specific hybridization is room temperature. In some embodiments, the defined temperature for specific hybridization is above room temperature. In some embodiments, the defined temperature for specific hybridization is at least about 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, or 80 °C. In some embodiments, the defined temperature for specific hybridization is or is about 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, or 80 °C.

[0189] As used herein, "target region," "target site," or "target gene site" refers to a unique genomic location that defines the location of a single target nucleic acid sequence, comprising one or more consecutive nucleotides. In some embodiments, the region or site is a single target nucleotide location. In some embodiments, the region or site is any one of at least about 2, 3, 5, 10, 15, 20, or 25 consecutive nucleotides. A gene may contain multiple target regions. The sequence at the target region may refer to the sense or antisense sequence of the target region, and may be a wild-type sequence or a mutant sequence. In some embodiments, the target region is associated with one or more variant sequences. In some embodiments, the target region is susceptible to mutation and is associated with one or more mutant sequences.

[0190] As used herein, a “reference region” refers to a unique genomic location that defines a nucleic acid sequence (i.e., a “reference sequence”) associated with a wild-type sequence. In some embodiments, the reference region is not associated with a mutation or variant. Those skilled in the art can select and validate reference regions and reference sequences. Different target regions may require different reference regions. In some embodiments, the target region and reference region corresponding to a probe set overlap or are identical.

[0191] As used herein, "target fragment" refers to a nucleic acid molecular fragment amplified by a primer set corresponding to a corresponding target region. "Reference fragment" refers to a nucleic acid molecular fragment amplified by a primer set corresponding to a corresponding reference region. In some embodiments, the target fragment includes a target region (e.g., a mutation hotspot, microsatellite sequence site, or target gene site in genomic DNA that is subject to site-specific genome editing) and an adjacent reference region upstream or downstream of the target region, which provides a sequence that a reference probe can hybridize to. In this case, the target fragment and the reference fragment are identical. In some embodiments, the target region and the reference region are located in different fragments amplified using separate primer pairs (i.e., the target fragment and the reference fragment, respectively). As used herein, a target fragment may also refer to an amplicon of a target fragment, and a reference fragment may refer to an amplicon of a reference fragment.

[0192] As used in this article, "adjacent" target region refers to a region in the target fragment or its amplicon that may partially overlap with or be outside the target region.

[0193] As used herein, an "allele" refers to one of several alternative forms of a gene or DNA sequence at a specific genomic location (locus). In humans, at each autosomal locus, an individual possesses two alleles, one inherited from the father and one from the mother. As used herein, an "allele sequence" refers to the sequence of a specific allele. Allele sequences can be longer than or shorter than the sequence of an allele-specific (AS) probe. The AS probe hybridizes with its corresponding allele sequence or a portion thereof.

[0194] As used interchangeably herein, “mutated sequence” and “variant sequence” refer to any sequence change in the target sequence compared to a reference sequence. “Wild-type sequence” and “reference sequence” are used interchangeably herein to refer to the sequence to which the target sequence is intended for comparison, such as a sequence corresponding to the dominant allele of a gene, or an unmodified gene locus sequence. Mutated sequences include, but are not limited to, insertions, deletions, and substitutions, including single nucleotide changes and changes of more than one nucleotide in the sequence.

[0195] "Mutation hotspots" are gene loci known to have naturally occurring mutations, for example, in diseased tissues or disease states. As used herein, the term "single nucleotide variant," or simply "SNV," refers to a change in a single nucleotide at a specific location in a genome sequence. When an alternative allele appears at a considerable frequency in a population (e.g., at least 1% of the population), an SNV is also called a "single nucleotide polymorphism" or "SNP."

[0196] As used in this article, "specificity" in the context of primers or probes that are specific to a target nucleic acid is the level of complementarity between the primer and the target such that there exists an annealing temperature at which the primer or probe will anneal to the target nucleic acid and mediate its amplification, without annealing to or mediating the amplification of non-target sequences present in the sample.

[0197] As used herein, "amplification" generally refers to the process of producing two or more copies of a desired sequence. Components of an amplification reaction may include, but are not limited to, primers, polynucleotide templates, polymerases, nucleotides, dNTPs, etc.

[0198] "Polymerase chain reaction" or "PCR" refers to a method by which amplifies a specific fragment or subsequence of a target double-stranded DNA in geometric progression. PCR is well known to those skilled in the art; see, for example, U.S. Patent Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, edited by Innis et al., 1990. Exemplary PCR reaction conditions typically consist of two or three cycles. A two-step cycle includes a denaturation step followed by a hybridization / extension step. A three-step cycle includes a denaturation step followed by a hybridization step, followed by a separate extension step. Polymerase chain reactions performed without thermal cycling are also considered herein, including but not limited to isothermal PCR and loop-mediated isothermal amplification (LAMP).

[0199] An "amplifier" is a nucleic acid fragment formed as a product of a PCR amplification reaction. It is a copy of a specific target nucleic acid, for example, containing... Figure 5A The target fragment of the target region is shown. As described herein, the amplicon is typically double-stranded DNA, although its single strand can be referenced.

[0200] As used herein, “digital PCR” refers to a PCR assay in which a sample is divided into numerous partitions and a PCR reaction is performed in each partition. Signals from each partition are detected to allow for the quantification of nucleic acids through statistical analysis. See, for example, Sykes et al., 1992 Quantitation of targets for PCR by use of limiting dilution. BioTechniques 13, 444-449; Vogelstein and Kinzler 1999 Digital PCR. ProcNatl Acad Sci USA, 96:9236-9241; and Pohl and Shihle 2004 Principle and applications of digital PCR. Expert Rev Mol Diagn, 4:41-47; and Monya Baker 2012 Nature Methods 9, 541-544.

[0201] As used herein, the term "partition" or "partitioned" refers to dividing a sample into multiple portions or "partitions." Partitions are typically physical, such that the sample in one partition does not, or substantially does not, mix with the sample in adjacent partitions. Partitions can be solid or fluid. In some embodiments, partitions are solid partitions, such as micropores. In some embodiments, partitions are fluid partitions, such as droplets. In some embodiments, fluid partitions (e.g., droplets) are the result of a mixture of immiscible fluids (e.g., water and oil). In some embodiments, fluid partitions (e.g., droplets) are aqueous droplets surrounded by an immiscible carrier liquid (e.g., oil). As used herein, "substantially all partitions" means at least about any one of 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more of the total number of partitions.

[0202] A “microsatellite sequence site” is a region of genomic DNA containing short repetitive sequence elements of 1 to 7 (e.g., 1 to 5, or 1 to 4) base pairs in length. Each sequence repeated at least once within a microsatellite site is referred to herein as a “repetition unit.” Each microsatellite site typically contains at least seven repetition units, for example at least ten repetition units, or at least twenty repetition units.

[0203] "Site-specific genome editing reagents" refer to components or groups of components that can be used for site-specific genome editing. Typically, such reagents contain a targeting module and a nuclease module. Exemplary targeting modules include nucleic acids, such as guide RNA, as those used in CRISPR / Cas systems. Alternatively, the targeting module can be or be derived from transcription factor domains or TAL effector DNA-binding domains. For example, zinc finger domains can be used as the targeting portion. Exemplary nuclease modules include, but are not limited to, IIS-type restriction endonucleases (e.g., FokI), Cas nucleases (e.g., Cas9), or derivatives thereof. In some cases, site-specific genome editing reagents use a combination of guide RNA, a "dead" Cas nuclease, and an IIS-type restriction endonuclease. Other variations are known in the art. Typically, site-specific genome editing reagents target genomic regions and induce double-strand cleavage ("split") into the DNA within the target region. Repair of the cleavage can be achieved through two alternative pathways. In non-homologous end joining (NHEJ), the cleaved ends of the DNA strand are directly joined without the need for homologous template nucleic acids. NHEJ can result in the addition, deletion, and / or substitution of one or more nucleotides at the repair site, and the resulting sequence is referred to herein as an "NHEJ-edited sequence." In homology-directed repair (HDR), the ends of DNA strand cleavages are repaired by the polymerization of homologous template nucleic acids, and the resulting sequence is referred to herein as an "HDR-edited sequence."

[0204] The terms “individual” or “subject” are used interchangeably in this text to refer to an animal; for example, a mammal. In some instances, “individual” or “subject” refers to an individual or subject who requires treatment for a disease or condition.

[0205] It should be understood that the embodiments of the present invention described herein include embodiments that are “composed of” and / or “substantially composed of”.

[0206] This document mentions "approximately" a value or parameter, including (and describing) the variation with respect to that value or parameter itself. For example, a description mentioning "approximately X" includes a description of "X". For example, the value of approximately X can be in the range of (i.e., ±) 10%, 5%, 2%, 1%, or less of X.

[0207] As used in this article, mentioning “not” a value or parameter usually means and describes “except” a value or parameter.

[0208] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise.

[0209] Where a numerical range is provided, it should be understood that every intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within the specified range, is included within the scope of this disclosure. Where the specified range includes an upper or lower limit, the range excluding any of those included limits is also included in this disclosure.

[0210] It should be understood that, for clarity, certain features of the invention described in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention described in the context of individual embodiments may also be provided individually or in any suitable sub-combination. All combinations of embodiments relating to multiplex dPCR assays (e.g., multiplex drop-off assays) and methods are specifically included in the invention and disclosed herein as if each combination were disclosed individually and explicitly. Furthermore, all sub-combinations of multiplex dPCR assays (e.g., multiplex drop-off dPCR assays) and methods listed in embodiments describing these variables are also specifically included in the invention and disclosed herein as if each such sub-combination of multiplex dPCR assays (e.g., multiplex drop-off dPCR assays) and methods were disclosed individually and explicitly herein.

[0211] II. Multiplex dPCR method

[0212] This application provides a method for quantifying reference (e.g., wild-type) sequences and / or variant sequences (e.g., mutant sequences) at two or more target regions in a nucleic acid sample using any of the probe set designs described in the “Probe Sets” section, said probe set designs comprising reference probes and drop-off probes with overlapping marker sets, and reference probes and allele-specific probes with overlapping marker sets. The methods described herein can be used as multiplex drop-off digital PCR assays.

[0213] Multiplex drop-off dPCR method

[0214] In some embodiments, a method is provided for quantifying wild-type and / or mutant sequences at multiple target regions in a sample containing nucleic acid molecules, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, wherein substantially all partitions (e.g., all partitions) each contain multiple probe sets corresponding to multiple target regions, wherein each of the multiple probe sets comprises:

[0215] A drop-off probe comprising a drop-off tag and an oligonucleotide drop-off sequence, wherein the oligonucleotide drop-off sequence is complementary to a wild-type sequence at a target region corresponding to a respective probe set;

[0216] A reference probe comprising a reference marker and an oligonucleotide reference sequence, the oligonucleotide reference sequence being complementary to a wild-type sequence located upstream or downstream of a target region corresponding to a particular probe set;

[0217] The reference marker and drop-off marker of each of the plurality of probe groups can be detected by different detection channels; the reference markers of the plurality of probe groups can be detected by different detection channels from each other; the drop-off markers of the plurality of probe groups can be detected by different detection channels from each other.

[0218] At least one reference marker of the plurality of probe groups and at least one drop-off marker of the plurality of probe groups can be detected through the same detection channel;

[0219] The method includes: detecting hybridization of reference probes from the plurality of probe sets with nucleic acid molecules or their amplicones containing wild-type sequences at reference regions in the plurality of partitions; and detecting hybridization of drop-off probes from the plurality of probe sets with nucleic acid molecules or their amplicones containing wild-type sequences at target regions in the plurality of partitions; thereby providing quantification of wild-type and / or mutant sequences at multiple target regions in the sample. In some embodiments, detection of signals from reference markers and drop-off markers in the probe sets indicates the presence of wild-type sequences in target regions corresponding to the probe sets, and detection of signals from reference markers but no signals from drop-off markers in the probe sets indicates the presence of mutant sequences in target regions corresponding to the probe sets. In some embodiments, the reference marker sets and drop-off marker sets of the plurality of probe sets have overlapping markers. In some embodiments, the reference marker sets and drop-off marker sets of the plurality of probe sets are cyclically arranged relative to each other.

[0220] In some embodiments, a method is provided for quantifying wild-type and / or mutant sequences at R target regions in a sample containing nucleic acid molecules, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, wherein substantially all partitions (e.g., all partitions) each include R probe pairs corresponding to the R target regions.

[0221] The first probe pair of the R probe pairs includes:

[0222] The first reference probe includes a first reference sequence (r1) and a first reference marker detectable by a first detection channel (X1).

[0223] The first drop-off probe, which comprises a first drop-off sequence (w1) and a first drop-off label detectable through a second detection channel (X2);

[0224] Wherein the second probe pair of the R probe pairs comprises:

[0225] A second reference probe, which comprises a second reference sequence (r2) and a second reference label detectable through a second detection channel (X2), and

[0226] A second drop-off probe, which comprises a second drop-off sequence (w2) and a second drop-off label detectable through a third detection channel (X3);

[0227] Wherein, if (for example, when) R is strictly greater than 3, the i-th probe pair (2 < i < R) of the R probe pairs comprises:

[0228] The i-th reference probe, which comprises the i-th reference sequence (r i ) and the i-th reference label detectable through the i-th detection channel (X i ), and

[0229] The i-th drop-off probe, which comprises the i-th drop-off sequence (w i ) and the i-th drop-off label detectable through the (i + 1)-th detection channel (X i+1 );

[0230] Wherein, if (for example, when) R is strictly greater than 2, the R-th probe pair of the R probe pairs comprises:

[0231] The R-th reference probe, which comprises the R-th reference sequence (r R ) and the R-th reference label associated with the R-th detection channel (X R ), and

[0232] The R-th drop-off probe, which comprises the R-th drop-off sequence (w R ) and the R-th drop-off label detectable through a first detection channel (X1);

[0233] Wherein the drop-off sequence of each probe pair is complementary to the wild-type sequence at the target region corresponding to the respective probe pair;

[0234] Wherein the reference sequence of each probe pair is complementary to the wild-type sequence at an adjacent reference region upstream or downstream of the target region corresponding to the respective probe pair;

[0235] Wherein the detection channels X1 - XR They are different;

[0236] The method includes detecting channels X1-X R Each detection of R probe pairs involves the hybridization of a reference probe with a nucleic acid molecule or its amplicon containing a wild-type sequence at a reference region in multiple partitions; and the detection via channels X1-X R Each detection of R probe pairs involves the drop-off probe hybridizing with a nucleic acid molecule or its amplicons containing wild-type sequences at target regions in multiple partitions; thereby providing quantification of wild-type and / or mutant sequences at R target regions in the sample. R can be any suitable integer of 2 or greater. In some embodiments, R is between 2 and 6. In some embodiments, the detection of signals from a reference marker and a drop-off marker in a probe pair indicates the presence of a wild-type sequence in the target region corresponding to the probe pair, and the detection of a signal from a reference marker but no signal from a drop-off marker in a probe pair indicates the presence of a mutant sequence in the target region corresponding to the probe pair.

[0237] In some embodiments, a method is provided for quantifying wild-type and / or mutant sequences at three target regions in a sample containing nucleic acid molecules, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, wherein substantially all partitions (e.g., all partitions) each contain three probe pairs corresponding to the three target regions, wherein the three probe pairs comprise:

[0238] The first probe pair comprises:

[0239] A first reference probe, comprising a first reference sequence and a first reference marker detectable by a first detection channel, and

[0240] The first drop-off probe includes a first drop-off sequence and a first drop-off marker detectable by a second detection channel;

[0241] The second probe pair includes:

[0242] The second reference probe includes a second reference sequence and a second reference marker detectable by a second detection channel.

[0243] The second drop-off probe contains a second drop-off sequence and a second drop-off marker that can be detected by a third detection channel;

[0244] The third probe pair includes:

[0245] The third reference probe comprises a third reference sequence and a third reference marker detectable by a third detection channel.

[0246] The third drop-off probe contains a third drop-off sequence and a third drop-off marker that can be detected by the first detection channel;

[0247] The drop-off sequence of each probe pair is complementary to the wild-type sequence at the target region corresponding to the corresponding probe pair; the reference sequence of each probe pair is complementary to the wild-type sequence at the adjacent reference region upstream or downstream of the target region corresponding to the corresponding probe pair; and the first detection channel, the second detection channel, and the third detection channel are different from each other.

[0248] The method includes hybridization of a reference probe detecting three probe pairs in each of the first, second, and third detection channels with a nucleic acid molecule or its amplicons containing wild-type sequences at a reference region in a plurality of partitions; and hybridization of a drop-off probe detecting three probe pairs in each of the first, second, and third detection channels with a nucleic acid molecule or its amplicons containing wild-type sequences at a target region in a plurality of partitions; thereby providing quantification of wild-type and / or mutant sequences at three target regions in a sample. In some embodiments, the detection of signals from a reference marker and from a drop-off marker in a probe pair indicates the presence of a wild-type sequence in the target region corresponding to the probe pair, and the detection of a signal from a reference marker but no signal from a drop-off marker in a probe pair indicates the presence of a mutant sequence in the target region corresponding to the probe pair. In some embodiments, the target region is a mutation hotspot region selected from one or more genes comprising the group consisting of EGFR, NRAS, KRAS, ESR1, and BRAF. In some embodiments, the method is used to quantify wild-type and mutant sequences in mutation hotspot regions of EGFR, NRAS, and KRAS. In some embodiments, the method is used to quantify wild-type and mutant sequences in mutation hotspot regions of NRAS, KRAS, and BRAF. In some embodiments, the method is used to quantify wild-type and mutant sequences at G12 / 13, Q61, and / or A146 in KRAS, G12 / 13 and / or Q61 in NRAS, E19 in EGFR, and / or V600 in BRAF.

[0249] In some embodiments, a method is provided for quantifying wild-type, mutant, and / or allele sequences at multiple target regions in a sample containing nucleic acid molecules, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, wherein substantially all partitions (e.g., all partitions) each contain multiple probe sets corresponding to multiple target regions, wherein each of the multiple probe sets comprises:

[0250] A drop-off probe comprising a drop-off tag and an oligonucleotide drop-off sequence, wherein the oligonucleotide drop-off sequence is complementary to a wild-type sequence at a target region corresponding to a respective probe set;

[0251] A reference probe comprising a reference marker and an oligonucleotide reference sequence, the oligonucleotide reference sequence being complementary to a wild-type sequence located upstream or downstream of a target region corresponding to a particular probe set;

[0252] Allele-specific (AS) probes comprising an AS marker and an oligonucleotide AS sequence, wherein the oligonucleotide AS sequence is complementary to the allele sequence at the target region corresponding to the respective probe set.

[0253] The reference marker, drop-off marker, and AS marker of each of the plurality of probe groups can be detected through different detection channels; the reference markers of the plurality of probe groups can be detected through different detection channels; the drop-off markers of the plurality of probe groups can be detected through different detection channels; and the AS markers of the plurality of probe groups can be detected through different detection channels.

[0254] In the case that at least one reference mark of the plurality of probe groups and at least one drop-off mark of the plurality of probe groups can be detected by the same detection channel, and / or at least one reference mark of the plurality of probe groups and / or at least one AS mark of the plurality of probe groups can be detected by the same detection channel;

[0255] The method includes: detecting hybridization of reference probes from the plurality of probe sets with nucleic acid molecules or their amplicons containing wild-type sequences at reference regions in the plurality of partitions; detecting hybridization of drop-off probes from the plurality of probe sets with nucleic acid molecules or their amplicons containing wild-type sequences at target regions in the plurality of partitions; detecting hybridization of AS probes from the plurality of probe sets with nucleic acid molecules or their amplicons containing allele sequences at target regions in the plurality of partitions; thereby providing quantification of wild-type sequences, mutant sequences, and / or allele sequences at multiple target regions in the sample. In some embodiments, detecting signals from reference markers and drop-off markers in the probe sets indicates the presence of wild-type sequences in target regions corresponding to the probe sets; detecting signals from reference markers but not from drop-off markers in the probe sets indicates the presence of mutant sequences in target regions corresponding to the probe sets; and detecting signals from AS markers indicates the presence of AS sequences in target regions corresponding to the probe sets. In some embodiments, the reference marker sets, drop-off marker sets, and AS marker sets of the plurality of probe sets have overlapping markers. In some implementations, the reference marker group, the drop-off marker group, and the AS marker group of the plurality of probe groups are arranged relative to each other.

[0256] In some embodiments, a method is provided for quantifying wild-type, mutant, and / or allele sequences at (R-1) target regions in a sample containing nucleic acid molecules, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, wherein substantially all partitions (e.g., all partitions) each contain (R-1) probe triplets corresponding to (R-1) target regions.

[0257] The first probe triplet of the (R-1) probe triplets includes:

[0258] The first reference probe includes a first reference sequence (m1) and a first reference marker detectable by a first detection channel (X1);

[0259] The first drop-off probe comprises a first drop-off sequence (r1) and a first drop-off marker detectable by a second detection channel (X2); and

[0260] The first allele-specific (AS) probe contains a first AS sequence (w1) and a first AS marker detectable via a third channel (X3);

[0261] The second probe triplet of the (R-1) probe triplets includes:

[0262] A second reference probe, which includes a second reference sequence (m2) and a second reference label detectable through a second detection channel (X2);

[0263] A second drop-off probe, which includes a second drop-off sequence (r2) and a second drop-off label detectable through a third detection channel (X3); and

[0264] A second AS probe, which includes a second AS sequence (w2) and a second AS label detectable through a fourth detection channel (X4);

[0265] wherein, if (e.g., when) R is strictly greater than 3, the i-th probe triplet (2 < i < R - 1) in the (R - 1) probe triplets includes:

[0266] The i-th reference probe, which includes the i-th reference sequence (m i ) and the i-th reference label detectable through the i-th detection channel (X i );

[0267] The i-th drop-off probe, which includes the i-th drop-off sequence (r i ) and the i-th drop-off label detectable through the (i + 1)-th detection channel (X i+1 ); and

[0268] The i-th AS probe, which includes the i-th AS sequence (w i ) and the i-th AS label detectable through the (i + 2)-th detection channel (X i+2 );

[0269] wherein, if (e.g., when) R is strictly greater than 3, the (R - 1)-th probe triplet in the (R - 1) probe triplets includes:

[0270] The (R - 1)-th reference probe, which includes the (R - 1)-th reference sequence (m R-1 ) and the R-th reference label detectable through the R-th detection channel (X R );

[0271] The (R - 1)-th drop-off probe, which includes the (R - 1)-th drop-off sequence (r R-1 ) and the (R - 1)-th drop-off label detectable through the (R - 1)-th detection channel (X R-1 ); and

[0272] The (R - 1)-th AS probe, which includes the (R - 1)-th AS sequence (w R-1) and the (R-1)th AS mark that can be detected by the first detection channel (X1);

[0273] The drop-off sequence of each probe triplet is complementary to the wild-type sequence at the target region corresponding to the corresponding probe triplet; the reference sequence of each probe triplet is complementary to the wild-type sequence at the adjacent reference region upstream or downstream of the target region corresponding to the corresponding probe triplet; the AS sequence of each probe triplet is complementary to the allele sequence at the target region corresponding to the corresponding probe triplet; and the detection channels X1-X R They are different;

[0274] The method includes detecting channels X1-X R Each detection (R-1) probe triplet hybridizes with a reference probe and a nucleic acid molecule or its amplicon containing a wild-type sequence at a reference region in multiple partitions; through detection channels X1-X R Each detection (R-1) probe triplet hybridizes the AS probe with a nucleic acid molecule or its amplicons containing allele sequences at the target region in multiple partitions; and through detection channels X1-X R Each detection (R-1) of the drop-off probes in the probe triad hybridizes with a nucleic acid molecule or its amplicons containing a wild-type sequence at a target region in multiple partitions; thereby providing quantification of wild-type, mutant, and / or allele sequences at multiple target regions in the sample. R can be any integer of 3 or greater. In some embodiments, R is 4. In some embodiments, the detection of signals from a reference marker and a drop-off marker in the probe set indicates the presence of a wild-type sequence in a target region corresponding to the probe set; the detection of a signal from a reference marker but no signal from a drop-off marker in the probe set indicates the presence of a mutant sequence in a target region corresponding to the probe set; and the detection of a signal from an AS marker indicates the presence of an AS sequence in a target region corresponding to the probe set.

[0275] The methods described herein may further include one or more steps such as partitioning, amplification, and / or sample preparation, as described in the “Digital PCR” section below. In some embodiments, the method further includes forming multiple partitions. In some embodiments, the method further includes dispensing a composition comprising nucleic acid molecules and multiple probe sets into multiple partitions. In some embodiments, the method further includes amplifying nucleic acid molecules in multiple partitions using multiple primer sets corresponding to multiple target regions. In some embodiments, substantially all partitions (e.g., all partitions) each contain multiple primer pairs corresponding to multiple target regions, wherein each primer pair contains a forward oligonucleotide primer and a reverse oligonucleotide primer suitable for amplifying a target fragment comprising a target region corresponding to the primer set and a reference region corresponding to the target region. In some embodiments, the method further includes dispensing a composition comprising nucleic acid molecules, multiple probe sets, and optionally multiple primer sets into multiple partitions.

[0276] The method may further include data analysis steps, as described in the subsections “Implementation with three (3) probe pairs,” “Implementation with R probe pairs,” and “Implementation with (R-1) probe triplet.” In some embodiments, the quantification includes providing estimated concentrations of wild-type and / or mutant sequences at multiple target regions in the sample. In some embodiments, the quantification includes providing confidence intervals for the estimated concentrations of wild-type and / or mutant sequences at multiple target regions in the sample. In some embodiments, the quantification includes providing a measure of uncertainty for wild-type and / or mutant sequences at multiple target regions. The confidence intervals and / or uncertainty measures may be at any given confidence level, such as any one of approximately 80%, 85%, 90%, 95%, 98%, 99%, or higher. The quantification of wild-type and / or mutant sequences at multiple target regions in the sample may be further converted to the quantification of wild-type and / or mutant sequences at multiple target regions in a biological sample, if the sample was prepared by diluting a biological sample, for example, by multiplying by a dilution factor from the biological sample.

[0277] Figure 1A-1DThis describes an exemplary triple drop-off dPCR assay for detecting wild-type and mutant sequences at target gene loci in EGFR, NRAS, and KRAS, respectively. For each target gene locus, a pair of forward and reverse primers, and a pair of reference and drop-off probes were designed. The reference and drop-off probes hybridize with the same amplicon amplified from the template nucleic acid in the sample via the forward and reverse primers. The drop-off probe hybridizes with the wild-type sequence at the corresponding target gene locus but not with the mutant sequence at the target gene locus. The reference probe hybridizes with the wild-type sequence adjacent to but not overlapping with the target gene locus. The reference and drop-off probes are TAQMAN probes labeled with different fluorophores. TM The probes, specifically the fluorophores, can be detected via different fluorescence detection channels. For nucleic acids containing wild-type sequences at the target gene site, both the reference probe and the drop-off probe hybridize with the amplicon, resulting in positive signals in the fluorescence channels corresponding to the reference probe and the drop-off probe, respectively. For nucleic acids containing mutant sequences at the target gene site, only the reference probe hybridizes with the amplicon, resulting in a positive signal in the fluorescence channel corresponding only to the reference probe, while no signal is detected in the fluorescence channel corresponding to the drop-off probe. Figure 2 As shown, signals from dPCR droplets can be plotted in three dimensions. Spatial segments corresponding to different signal clusters were identified, and the number of droplets in each segment was counted. These counts were used to estimate the concentration for each wild-type and mutant population. Exemplary primers and probes corresponding to KRAS, NRAS, and EGFR gene loci are shown in Table 12.

[0278] The method described herein can be further multiplexed with conventional allele-specific dPCR assays by including allele-specific probes with markers detectable via different detection channels used with multiple probe sets, including probe pairs and probe triplets. Allele-specific (“AS”) probes hybridize to specific allele sequences, including wild-type sequences, mutant sequences, or SNPs at the target region. AS probes are designed to confer the ability to bind correctly to specific allele sequences at the target region while preventing hybridization of AS probes in the presence of any other sequences at the target region. In some embodiments, each AS probe is also used with a dark probe to increase the stringency of the assay by binding to the wild-type sequence of the allele rather than the allele sequence associated with the AS probe.

[0279] For example, Figure 4AAn exemplary assay combining two probe pairs is described, the probe pairs including a reference probe and a drop-off probe (one set for detecting wild-type and mutant sequences at Q61 of NRAS, and a second set for detecting wild-type and mutant sequences at G12 / 13 of NRAS), and having allele-specific probes for detecting V600E and V600K mutations in BRAF.

[0280] In some embodiments, the probes (e.g., reference probes, drop-off probes, AS probes) each have a single detectable marker. In some embodiments, the markers (e.g., reference markers, drop-off markers, AS markers) are fluorophores. In some embodiments, different detection channels have different excitation wavelength ranges and / or different emission wavelength ranges.

[0281] The method described herein for distinguishing probes based on excitation wavelength and / or emission wavelength or wavelength ranges associated with different fluorophores can be further combined with multiplexing methods that distinguish probes having the same fluorophore but depending on different fluorescence intensities. In some embodiments, one or more detection channels are associated with different excitation wavelengths or wavelength ranges and / or emission wavelengths or wavelength ranges, and one or more detection channels are associated with different fluorescence intensities. In some embodiments, probe sets corresponding to different target regions within the same target gene are labeled with the same set of fluorophores, which are detected by different fluorescence intensities. In some embodiments, a probe set corresponding to different target regions within the target gene includes: a reference probe having a reference marker detectable by a detection channel sharing the same excitation and / or emission wavelength or wavelength range, wherein the reference probe is detected at different fluorescence intensities relative to each other; a drop-off probe having a drop-off marker detectable by different detection channels sharing the same excitation and / or emission wavelength or wavelength range, wherein the drop-off probe is detected at different fluorescence intensities relative to each other; and / or an AS probe having an AS marker detectable by different detection channels sharing the same excitation and / or emission wavelength or wavelength range, wherein the AS probe is detected at different fluorescence intensities relative to each other.

[0282] For example, Figure 4BThis illustrates an exemplary assay that can simultaneously quantify twelve different genetic species (i.e., wild-type and mutant sequences at G12 / G13, Q61, and A146 of KRAS; wild-type and mutant sequences at G12 / G13 and Q61 of NRAS; and V600E and V600K at BRAF) by combining cyclic arrangement of probe labels with different fluorescence intensities. For example, genetic species associated with the same gene (e.g., KRAS) can use probe pairs with the same reference marker and drop-off label, but each probe pair can use different concentrations of different probes and corresponding primers, so that signals from different probe pairs can be distinguished based on their fluorescence intensities.

[0283] probe group

[0284] The methods described herein use multiple probe sets to detect wild-type and mutant sequences at multiple target regions. Each probe set may include 2, 3, 4, 5, 6, or more probes. In some embodiments, the multiple probe sets are multiple probe pairs, each probe pair containing a reference probe that always hybridizes with the target fragment and a drop-off probe that hybridizes with the target fragment containing a wild-type sequence at the target region. In some embodiments, the multiple probe sets are multiple probe triplets, each probe triplet containing a reference probe, a drop-off probe, and an allele-specific (“AS”) probe that hybridizes with a specific allele sequence at the target region. Any probe set (including probe pairs and probe triplets) described herein may be used with one or more “independent” AS probes that are not part of said multiple probe sets; for example, one or more AS probes may hybridize to a target region different from the target region corresponding to the multiple probe sets. In some embodiments, at least 1, 2, 3, 4, 5, 6, or more independent AS probes are used. Each probe contains a detectable marker.

[0285] In some embodiments, the multiple probe sets are multiple probe triplets comprising a reference probe, a first AS probe, and a second AS probe, wherein the first AS probe and the second AS probe hybridize to the same specific allele sequence or its complementary sequence, or different portions of the same specific allele sequence. In some embodiments, the allele sequence is at the junction of two repeats of a CNV-associated mutant gene at the target region. In some embodiments, the reference probe and one of the first AS probe and the second AS probe in each probe triplet have the same detectable label, and the other of the first AS probe and the second AS probe has a different detectable label that can be detected through a detection channel different from the detectable label of the reference probe. In some embodiments, the multiple probe sets are multiple probe pairs comprising a reference probe with a single detectable label and an AS probe with two detectable labels, wherein one of the two detectable labels of the AS probe is the same as the detectable label of the reference probe, and the other detectable label of the AS probe can be detected through a detection channel different from the detectable label of the reference probe. The first AS probe and the second AS probe in the multiple probe triplets, or the AS probe with two detectable labels in the multiple probe pairs, are referred to herein as "dual-labeled AS probes".

[0286] In some implementations, the AS probe hybridizes with the mutant sequence at the target region, and the reference probe hybridizes with the wild-type sequence at the same target region or at a reference region overlapping with the target region.

[0287] In some implementations, the probe (e.g., a reference probe, drop-off probe, or AS probe) is an oligonucleotide probe. Exemplary probes include oligonucleotide primers with a hairpin structure, wherein the fluorescent molecule remains near the fluorescence quencher until forcibly separated by primer extension, e.g., Whitecombe et al., Nature Biotechnology, 17:804-807 (1999) (AMPLIFLUOR) TM (hairpin primers). Exemplary probes may alternatively comprise oligonucleotides linked to a fluorophore and a fluorescence quencher, wherein the fluorophore and quencher are close together until the oligonucleotide specifically binds to the amplification product, e.g., Gelfand et al., U.S. Patent No. 5,210,015 (TAQMAN). TMPCR probes; Nazarenko et al., Nucleic Acids Research, 25:2516-2521 (1997) (“scorpion probes”); and Tyagi et al., Nature Biotechnology, 16:49-53 (1998) (“molecular beacons”). These probes can be used to measure the total amount of reaction products at the end of the reaction or to measure the production of amplification products during the amplification reaction. In some embodiments, the probe (e.g., a reference probe, drop-off probe, or AS probe) is a TAQMAN probe. TM Probe.

[0288] Probes from the same probe set described herein (e.g., reference probes, drop-off probes, or AS probes) hybridize within the same target fragment or its amplicons. However, independent AS probes that are not part of the multiple probe sets may or may not hybridize with any probe from any of the multiple probe sets within the same target fragment or its amplicons. Probes are designed according to established practices in the art to minimize PCR artifacts and hybridize specifically with the target sequence. The specificity of oligonucleotide probe hybridization with the target fragment or its amplicons can be achieved by varying the probe length, GC content, or amplification and / or detection conditions (e.g., temperature, salt content, etc.).

[0289] In some embodiments, the probe has a nucleotide sequence length of about 10 to about 50 nucleotides. In some embodiments, the probe has a nucleotide sequence length of any one of about 15-40, 25-50, 15-35, 20-40, 30-50, or 30-40 nucleotides. The probe may further include modifications to increase the specificity of the probe to its target sequence, for example by increasing the probe's melting temperature (Tm) and stabilizing the probe-target hybrid. In some embodiments, one or more probes include a minor groove binding (MGB) portion at their 3' end. In some embodiments, the probe comprises chemically modified nucleotides, such as locked nucleic acids (LNAs).

[0290] The drop-off probe hybridizes to the wild-type sequence at a target region in a nucleic acid molecule or its amplicon. The target region can be a mutation hotspot in the target gene, including microsatellite sequence sites. Alternatively, the target region can be a genomic site edited by a site-specific genome editing agent (e.g., CRISPR-Cas). Preferably, the drop-off probe covers the entire wild-type sequence of the target region and extends further at each end by several nucleotides (typically 1 to 10 nucleotides, e.g., 2 to 8, 2 to 6, 2 to 5, or 2 to 4) to confer correct binding ability and instability in the presence of mutated sequences. In other words, the probe size is designed to confer correct binding to the wild-type sequence in the target region while preventing hybridization of the drop-off probe in the presence of mutations in the target region.

[0291] The reference probe hybridizes with the wild-type sequence in a reference region. The reference region is a region associated with low mutation or single nucleotide polymorphism frequencies. In some embodiments, the reference probe hybridizes at a reference region located on a fragment or amplicon different from the AS probe. In some embodiments, the reference probe hybridizes at a reference region adjacent to a target region located on the same target fragment as the nucleic acid molecule or its amplicon.

[0292] For drop-off assays, the reference probe hybridizes with a wild-type sequence at an adjacent reference region within the target fragment of a nucleic acid molecule or its amplicons. The reference region is located upstream or downstream of the target region and does not overlap with it. The reference probe is designed to confer the ability to bind to substantially all target fragments or their amplicons associated with its corresponding target region, regardless of the mutational state of the target region.

[0293] Figure 5A An exemplary reference probe and drop-off probe pair is illustrated.

[0294] Allele-specific (AS) probes in a probe set hybridize to a specific allele sequence at the target region where they hybridize with the corresponding drop-off probe and the corresponding reference probe. Each independent AS probe, not part of the plurality of probe sets, hybridizes to a specific allele sequence at the target region, which may or may not overlap with the target region corresponding to any of the plurality of probe sets. AS probes can be used to detect specific sequences in a target gene (e.g., wild-type sequences, mutant sequences, SNPs, or amplifications), or HDR-edited sequences at target genomic sites edited by site-specific genome editing agents (e.g., CRISPR-Cas). AS probes are designed to confer the ability to correctly bind to a specific allele sequence at the target region while preventing hybridization of the AS probe in the presence of any other sequences at the target region. In some embodiments, the AS probe has a single detectable marker. In some embodiments, the AS probe has two different detectable markers. In some embodiments, two AS probes with different detectable markers are used to detect a specific allele sequence at the target region.

[0295] In some embodiments, the probe set containing the AS probe further includes a dark probe that binds to the wild-type sequence of the allele but not to the allele sequence associated with the AS probe. In some embodiments, the AS probe, which is not part of the plurality of probe sets, is used in combination with the dark probe that binds to the wild-type sequence of the allele but not to the allele sequence associated with the AS probe. The dark probe can increase the stringency of the assay by reducing the false signal provided by the binding of the AS probe to the wild-type target gene site. Typically, the dark probe is designed to include a non-extending 3' end. Exemplary non-extending 3' ends include, but are not limited to, 3' terminal phosphate. Alternative non-extending 3' ends include, for example, those disclosed in International Patent Application Publication No. WO 2013 / 026027.

[0296] Furthermore, since dPCR is performed as an endpoint reaction (PCR runs to completion before fluorescence measurement), target molecules isolated with single or near-single (e.g., 2, 3, 4, 5, 6 copies, etc., e.g., a Poisson distribution of the target molecule into each compartment containing 0, 1, 2, 3, 4, 5, or more copies of the target molecule) allow for multiplexing based on probe intensity (Zhong, Bhattacharya et al., 2011 Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR. Lab Chip, 11:21 67-2 174). For example, by adding target-specific fluorescence at a limiting concentration, the compartment with the first target will be PCR positive, but with limited brightness at the PCR endpoint. To count the second target type, different target-specific probes with the same "color" (i.e., the same fluorophore) are added at different concentrations. The compartment with the second target will have a brighter signal at the PCR endpoint than the compartment with the first target, thus providing a separate cloud and enabling separate counting for each target. Therefore, combinations of probes of different colors and concentrations can be used for higher levels of multiplexing. Alternatively, different primer concentrations can be used for different target fragments to generate different signal intensities for different probe sets, thus allowing probe intensity-based multiplexing.

[0297] In some implementations, the probe (e.g., a reference probe, drop-off probe, or AS probe) is detectably labeled with a fluorophore, which may be selected from, for example, FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, FITC, IRD-700 / 800, Cy3, Cy5, Cy3.5, Cy5.5, HEX, TET (5-tetrachlorofluorescein), TAMRA, JOE, ROX, BODIPYTMR, Oregon Green, Rhodamine Green, Rhodamine Red, ALEXA FLUOR. BIOSEARCH BLUE TM MARINA BOTHELL ALEXA 350FAM TM , Green 1, EvaGreen TM ALEXA 488 JOE TM 25 VIC TM HEX TM TET TM CAL Gold 540, YAKIMA ROX TM CAL Red 610, Cy3.5 TM TEXAS ALEXA 568CRY5 TM QUASAR TM 670, Light Cycler ALEXA 633 QUASAR TM 705, Light Cycler ALEXA 680, SYT09, LC LC Plus+ and EVAGREEN TM The group consists of [various components]. In some embodiments, the reference marker, drop-off marker, and / or AS marker are selected from fluorescein, FAM, YAKIMA, etc. Cy3, HEX, VIC, ROX, Cy5, Cy5.5, ALEXA 647, ALEXA The group consisting of 448 and Quasar705. In some embodiments, the reference marker, drop-off marker, and / or AS marker are selected from the group consisting of Cy3, FAM, and Cy5. In some embodiments, the reference marker, drop-off marker, and / or AS marker are selected from the group consisting of FAM, HEX, and Cy5.

[0298] In some embodiments, each fluorophore is detected via a detection channel having a characteristic excitation range and a characteristic emission range. In some embodiments, the different fluorophores used in the probe set have non-overlapping excitation wavelength ranges and / or non-overlapping emission wavelength ranges. Table 1 below shows exemplary detection channels and compatible fluorophores useful in methods with three probe pairs.

[0299] Table 1

[0300]

[0301] The methods described herein use a total number of detection channels less than the total number of probes, including reference probes, drop-off probes, and AS probes. In some embodiments, the plurality of probe groups are multiple probe pairs (e.g., a reference probe and a drop-off probe in each probe pair, or a reference probe and an AS probe with two different labels in each probe pair), and the total number of detection channels is less than twice the total number of probe pairs. In some embodiments, R probe pairs are used, and R is 2 or more, and the total number of detection channels is R, R+1, R+2, ..., or 2R-1. In some embodiments, the total number of detection channels is equal to the total number of probe pairs. In some embodiments, the plurality of probe groups are multiple probe triplets (e.g., a reference probe, a drop-off probe, and an AS probe in each probe triplet), and the total number of detection channels is less than three times the total number of probe triplets. In some embodiments, R probe triplets are used, and R is 2 or more, and the total number of detection channels is R+1, R+2, R+3, ..., or 3R-1. In some embodiments, the total number of detection channels is equal to the total number of probe triplets plus 1. In some embodiments, the plurality of probe sets are plurality of probe triplets (e.g., a reference probe, a first AS probe, and a second AS probe that hybridize to the same allele sequence in each probe triplet), and the total number of detection channels is less than twice the total number of probe triplets. In some embodiments, R probe triplets are used, and R is 2 or more, and the total number of detection channels is R, R+1, R+2, ..., or 2R-1. In some embodiments, the total number of detection channels is equal to the total number of probe triplets.

[0302] The quencher can be an internal quencher or a quencher located at the 3' end of the probe. Typical quenchers include, but are not limited to, tetramethylrhodamine, TAMRA, and BLACK HOLE. (BHQ; e.g., BHQ-1, BHQ-2, BHQ-3) and non-fluorescent quenchers (NFQ). Hydrolysis probes available according to the invention are well known in the art. In some embodiments, the hydrolysis probe has a fluorophore and a quencher covalently linked to the 5' end of the oligonucleotide probe. The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by a light source typically via FRET (Forster resonance energy transfer). Quenching suppresses any fluorescence signal as long as the fluorophore and quencher are in close proximity. In some embodiments, the probes (e.g., reference probes, drop-off probes, or AS probes) are designed such that they anneal within the target fragment amplified by a specific primer set. When a DNA polymerase (e.g., Taq polymerase) extends the primers and synthesizes the nascent strand, the inherent 5'-3' exonuclease activity in the DNA polymerase separates the 5' reporter gene from the 3' quencher, thereby providing a fluorescence signal proportional to the amplicon yield.

[0303] Furthermore, as mentioned above, probe multiplexing can be based on probe strength, for example, by varying probe and / or primer concentrations. See, Zhong, Bhattacharya et al., 2011 Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR. Lab Chip, 11:21 67-2174. Therefore, for reference probes, drop-off probes, and AS probes in multiple probe sets, combinations of overlapping label groups (e.g., arrangements of labels between different types of probes) and different concentrations of probes and / or primers can be used for multiplexing at a higher level.

[0304] Digital PCR

[0305] The method described in this article can be performed in the form of digital PCR, where virtually all partitions contain 0, 1, or close to 1 target molecule.

[0306] In some embodiments, each partition contains 0 or 1 target molecules. In some embodiments, multiple partitions have a Poisson distribution of target molecules, wherein each partition has 0, 1, 2, 3, 4, 5 or more target molecules, and wherein the average number of target molecules in each partition is close to 1. In some embodiments, the average number of target molecules in each partition is any one of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0. For example, the optimal conditions for the determination described herein may have a Poisson distribution of target molecules in multiple partitions, wherein the average number of target molecules is about 1.6. In some embodiments, approximately 20% of the partitions each contain 0 target molecules; approximately 32.3% of the partitions each contain 1 target molecule; approximately 25.8% of the partitions each contain 2 target molecules; approximately 13.8% of the partitions each contain 3 target molecules; approximately 5.5% of the partitions each contain 4 target molecules; approximately 1.8% of the partitions each contain 5 target molecules; approximately 0.47% of the partitions each contain 6 target molecules; and approximately 0.12% of the partitions each contain 7 or more target molecules. In some embodiments, the number of target molecules mentioned in this paragraph refers to target molecules having the target sequence in the target region. In some embodiments, the number of target molecules mentioned in this paragraph refers to target molecules having a wild-type sequence in the target region. In some embodiments, the number of target molecules mentioned in this paragraph refers to target molecules having one or more mutant sequences in the target region. In some embodiments, the number of target molecules mentioned in this paragraph refers to target molecules having either wild-type or mutant sequences in the target region.

[0307] In some embodiments, no more than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, or 60% of the multiple partitions are occupied by one or more target molecules. In some embodiments, about 60%-95%, 60%-70%, 70%-80%, 80%-90%, 70%-90%, 75%-85%, 76%-84%, 77%-83%, 78%-82%, or 79%-81% of the multiple partitions are occupied by one or more target molecules. In some embodiments, about 80% of the multiple partitions are occupied by one or more target molecules. In some embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the multiple partitions each have 0 target molecules. In some embodiments, approximately 5%-40%, 10%-20%, 20%-30%, 30%-40%, 10%-30%, 15%-25%, 16%-24%, 17%-23%, 18%-22%, or 19%-21% of each of the multiple partitions each have 0 target molecules. In some embodiments, the number of target molecules mentioned in this paragraph refers to target molecules having the target sequence in the target region. In some embodiments, the number of target molecules mentioned in this paragraph refers to target molecules having a wild-type sequence in the target region. In some embodiments, the number of target molecules mentioned in this paragraph refers to target molecules having one or more mutant sequences in the target region. In some embodiments, the number of target molecules mentioned in this paragraph refers to target molecules having either a wild-type or mutant sequence in the target region.

[0308] Because different target sequences (e.g., different alleles) in the target region may exist at different frequencies, in some embodiments, a first dPCR is performed to detect a first target sequence, wherein the target molecule has a first distribution in multiple partitions, and a second dPCR is performed to detect a second target sequence, wherein the target molecule has a second distribution in multiple partitions, for example, by diluting the sample and redistributing the diluted sample in multiple partitions.

[0309] Techniques applicable to digital PCR include PCR amplification on microfluidic chips (Warren et al., 2006, Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. Proc Natl Acad Sci USA 103, 17807-1781 2; Ottesen et al., 2006, Microfluidic digital PCR enables multigene analysis of individual environmental bacteria. Science 314, 1464-1467; Fan and Quake 2007, Detection of anepoloidy with digital polymerase chain reaction. Anal Chem 79, 7576-7579). Other systems involve separation to microarrays (Morrison et al., 2006 Nanoliter high-throughput quantitative PCR. Nucleic Acids Res 34, e123) or spinning microfluidic disks (Sundberg et al., 2010 Spinning disk platform for microfluidic digital polymerase chain reaction. Analytical Chem 82, 1546-1550) and droplet technology based on oil-water emulsions (Hindson, Benjamin et al., 2011 High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number. Analytical Chemistry 83(22):8604-8610; J. Madic et al., 2016, Three-Color crystal digital PCR, Biomolecular Detection and Quantification, 10:34-36). Typically, digital PCR is selected from DROPLET DIGITAL. TM PCR (ddPCR), CRYSTAL DIGITAL TMPCR, chamber-based (e.g., microwell-based) digital PCR, beaming-based (bead, emulsion, amplification, and magnetism) digital PCR, and microfluidic chip-based digital PCR. In some implementations, dPCR is droplet digital. TM PCR. In some implementations, dPCR is CRYSTAL DIGITAL. TM PCR.

[0310] Examples of suitable digital PCR systems include Stilla Technologies' NAICA. TM CRYSTALDIGITAL TM A PCR system that separates samples into droplets of 25,000-30,000 nanoliters; Bio-Rad's QX100 TM DROPLET DIGITAL TM A PCR system that separates samples containing nucleic acid templates into droplets of 20,000 nanoliters; and RainDance's RAINDROP. TM Digital PCR systems that aliquot samples containing nucleic acid templates into droplets ranging in size from 1,000,000 to 10,000,000 picoliters. Droplet PCR systems have been described, for example, in US10501789B2, the contents of which are incorporated herein by reference in their entirety.

[0311] In a typical digital PCR experiment, the PCR solution is prepared similarly to that of a classic TaqMan probe assay. The PCR solution typically contains a DNA sample, a fluorescence quencher probe (i.e., a hydrolysis probe), primers, and a PCR master mixture, which usually contains optimal concentrations of DNA polymerase, dNTPs, MgCl2, and reaction buffer. The PCR solution is then randomly assigned to discrete (i.e., individual) partitions, some containing no target DNA and others containing one or more copies of the target DNA; for example, each partition contains an average of approximately 1.6 copies of the target DNA. Each partition is amplified individually to the terminal stationary phase (or endpoint) of the PCR, and fluorescence is then read to determine the fraction of positive partitions.

[0312] If the partition has a uniform volume, the number of target DNA molecules present can be calculated from the fraction of a positive endpoint reaction using Poisson statistics according to the following equation:

[0313] λ = -ln(1-p)

[0314] Where λ is the average number of target DNA molecules in each partition (i.e., replication reaction), and p is the fraction of positive endpoint reactions. From λ, along with the volume of each partition and the total number of partitions analyzed, an estimate of the absolute target DNA concentration is calculated.

[0315] The method described herein uses multiple probe groups, where different types of probes share overlapping groups of markers (e.g., circularly arranged groups, or such as...). Figure 6B (As shown in the arrangement). Therefore, positive signals generated by two or more detection channels can be ambiguous because partitions with two or more distinct target regions can produce positive signals generated by each detection channel, which may add up to provide a composite signal that appears to correspond to a specific genetic species within a single target region. The data analysis methods described herein discard or otherwise interpret errors caused by such ambiguous signals at some steps in order to estimate the concentration of mutant sequences at the target region.

[0316] Primer set and amplification

[0317] Nucleic acid molecules within each partition can be amplified. Each partition may include multiple primer sets corresponding to multiple target regions. In some embodiments, one or more primer sets each further include forward and reverse primers for amplifying a reference region corresponding to the target region. Each primer pair in a primer set includes a forward primer and a reverse primer. In some embodiments, the forward and reverse primers are oligonucleotide primers annealed to the opposite strand of the nucleic acid molecule and located flanking the target region and reference region (i.e., the target fragment). The primer sets allow the generation of target fragment-specific amplicons during the PCR reaction. The corresponding probe sets can then hybridize with the amplicons.

[0318] In some embodiments, substantially all partitions (e.g., all partitions) each contain (a) multiple primer sets corresponding to multiple target regions, and (b) a DNA-dependent DNA polymerase; wherein each of the multiple primer sets contains a forward oligonucleotide primer and a reverse oligonucleotide primer, suitable for amplifying a target fragment containing a target region corresponding to the primer set and a reference region corresponding to the target region; wherein the method includes amplifying the target fragment from nucleic acid molecules in the multiple partitions; and wherein the method includes detecting hybridization of a reference probe, a drop-off probe, and / or an AS probe with the amplicons of the target fragment. In some embodiments, the method includes detecting hybridization of the reference probe and the drop-off probe with the amplicons of the target fragment.

[0319] In some embodiments, substantially all partitions (e.g., all partitions) each contain (a) multiple primer sets corresponding to multiple target regions and reference regions, and (b) a DNA-dependent DNA polymerase; wherein each of the multiple primer sets contains a forward oligonucleotide primer and a reverse oligonucleotide primer adapted to amplify a target fragment containing a target region corresponding to the primer set; wherein each of the multiple primer sets contains a forward oligonucleotide primer and a reverse oligonucleotide primer adapted to amplify a reference fragment containing a reference region corresponding to the target region; wherein the method includes amplifying target fragments from nucleic acid molecules and reference fragments from nucleic acid molecules in the multiple partitions; and wherein the method includes detecting hybridization of a reference probe with an amplicon of the reference fragment, and detecting hybridization of an AS probe (e.g., a first AS probe and a second AS probe) with an amplicon of the target fragment.

[0320] Primers can have any suitable length and GC content. In some implementations, multiple primer sets can be designed using available computer programs to predict that the resulting amplicons will have the same melting temperature during amplification.

[0321] Primers are designed to provide amplicons of suitable length, such that the amplicons are long enough to allow hybridization of their respective reference probes, drop-off probes (and, in some experiments, AS probes), but short enough to avoid excessive nonspecific binding of any probe in the reaction mixture. In some embodiments, the amplicons are at least any one of about 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 base pairs. In some embodiments, the amplicons are no longer than any one of about 500, 450, 400, 350, 300, 250, 200, 150, or 100 base pairs. In some embodiments, the amplicons are of any one of about 100-500, 100-400, 100-300, 100-250, 100-200, 150-250, or 150-300 base pairs.

[0322] Each partition may contain a polymerase, which is an enzyme that performs template-guided synthesis of polynucleotides such as DNA and / or RNA. The term "polymerase" includes a full-length polypeptide and a domain having polymerase activity. DNA polymerases are well known to those skilled in the art, including but not limited to DNA polymerases isolated or derived from *Pyrococcus furiosus*, *Thermococcus litoralis*, and *Thermotoga maritime*, or modified forms thereof. Other examples of commercially available polymerases include, but are not limited to, Klenow fragments (New England Biolabs Inc.), Taq DNA polymerase (QIAGEN), 9°WM DNA polymerase (New England Biolabs Inc.), and Deep Vent. TM DNA polymerases (New England Biolabs Inc.), Manta DNA polymerase (Enzymatics), Bst DNA polymerase (New England Biolabs Inc.), and phi29 DNA polymerase (New England Biolabs Inc.). In some embodiments, the polymerase is a DNA-dependent polymerase. In some embodiments, the polymerase is an RNA-dependent polymerase, such as a reverse transcriptase.

[0323] The droplets support PCR amplification of template molecules, using similar homogenization assay chemistry and workflows to those widely used in real-time PCR applications (Hinson et al., 2011, Anal. Chem. 83:8604-8610; Pinheiro et al., 2012, Anal. Chem. 84:1003-1011). Once the droplets are generated, they can be transferred to a PCR plate, and emulsified PCR reactions can be run on a thermal cycler using classic PCR programs. Alternatively, in NAICA... TM The droplets generated on the Sapphire chip of the system can be thermally cycled using a classic PCR procedure. The thermal cycle continues until the endpoint is reached.

[0324] To circumvent the technical challenges associated with amplifying low-complexity sequences such as microsatellite sequences, the annealing temperature and / or extension time of the amplification step can be increased. For example, a typical annealing temperature is 55°C, while for microsatellite site detection, the annealing temperature can be increased by 3 to 15°C.

[0325] PCR data collection steps typically use optical detectors (e.g., Stilla's NAICA). TMThe PRISM3 system, or the Bio-Rad QX-100 droplet reader, can be used. A detection system with an appropriate number of detection channels, such as a three-color detection system, should be employed.

[0326] partition

[0327] The partitioning described in this article can be in any suitable format. Microplates, capillaries, oil emulsions, and microcompartment arrays with nucleic acid-binding surfaces can be used to divide samples into different partitions or droplets. Therefore, the digital PCR used in this article includes various forms, including compartment digital PCR, droplet digital PCR, and more. TM PCR (ddPCR), CRYSTAL DIGITAL TM PCR, BEAMing-based (bead, emulsion, amplification, and magnetism) digital PCR, and microfluidic chip-based digital PCR.

[0328] Samples can be partitioned into multiple mixture partitions. The use of partitions can facilitate reduced background amplification, reduced amplification bias, increased throughput, provision of absolute or relative quantitative detection, or a combination thereof. Partitions can include any of a variety of types, including solid partitions (e.g., pores or tubes) or fluid partitions (e.g., aqueous droplets within an oil phase). In some embodiments, the partitions are droplets. In some embodiments, the partitions are micropores. In some embodiments, the partitions are two-dimensional monolayer droplets within microchambers. For example, methods and compositions for dispensing samples are described in published patent applications WO2010 / 036352, US2010 / 0173394, US2011 / 0092373, US2011 / 0092376, and US10501789B2, the entire contents of which are incorporated herein by reference.

[0329] In some cases, the sample is dispensed and the detection reagent (e.g., probe, enzyme, etc.) is incorporated into the dispensed sample. In other cases, the sample is contacted with the detection reagent (e.g., probe, enzyme, etc.) before dispensing. In some embodiments, reagents such as probes, primers, buffers, enzymes, substrates, nucleotides, salts, etc., are mixed together before dispensing, and then the sample is dispensed. In some cases, the sample is dispensed shortly after the reagents are mixed so that substantially all or most of the reaction (e.g., DNA amplification, DNA cleavage, etc.) occurs after dispensing. In other cases, the reagents are mixed at a temperature where the reaction proceeds slowly or not at all, then the sample is dispensed, and the reaction temperature is adjusted to allow the reaction to proceed. For example, the reagents may be combined on ice, below 5°C, or at approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, or 30-35°C. Generally, those skilled in the art will know how to select temperatures at which one or more reactions are inhibited. In some cases, a combination of temperature and time is used to prevent a substantial reaction from occurring before dispensing. In some implementations, one or more hot-start enzymes, such as hot-start DNA-dependent DNA polymerases, can be used to mix reagents and samples. Thus, the sample and one or more buffers, salts, nucleotides, probes, labels, enzymes, etc., can be mixed and then dispensed. Subsequently, the reaction catalyzed by the hot-start enzyme can be initiated by heating the mixture partition to activate one or more hot-start enzymes.

[0330] In some embodiments, samples and reagents (e.g., one or more of buffers, salts, nucleotides, probes, labels, enzymes, etc.) can be mixed together without requiring one or more reagents necessary to initiate the intended reaction (e.g., DNA amplification). The mixture can then be dispensed into a first partitioned mixture group, and one or more basic reagents can be provided by fusing the first partitioned mixture group with a second partitioned mixture group that provides the basic reagents. In some embodiments, the basic reagents can be added to the first partitioned mixture without forming the second partitioned mixture. For example, the basic reagents can diffuse into water-in-oil droplets of the first partitioned mixture group. As another example, lost reagents can be directed to a microchannel group containing the first partitioned mixture group.

[0331] In some embodiments, the sample is aliquoted into multiple droplets. In some embodiments, the droplets comprise an emulsion composition, i.e., a mixture of immiscible fluids (e.g., water and oil). In some embodiments, the droplets are aqueous droplets surrounded by an immiscible carrier liquid (e.g., oil). In some embodiments, the droplets are oil droplets surrounded by an immiscible carrier liquid (e.g., an aqueous solution). In some embodiments, the droplets described herein are relatively stable and exhibit minimal coalescence between two or more droplets. In some embodiments, droplets of less than 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% from the sample coalesce with other droplets. Emulsions can also have limited flocculation, a process in which the dispersed phase precipitates from a suspension in the form of flakes.

[0332] In some embodiments, droplets are formed by flowing an oil phase through an aqueous sample containing the nucleic acid molecules to be detected. In some embodiments, droplets are formed by flowing an aqueous sample through a microchannel comprising a channel that diverges under the surface tension of the solution to separate droplets of the aqueous sample into wall portions within a storage region, wherein the oil phase carrier is located in a microfluidic device. The oil phase may contain a fluorinated base oil, which may be further stabilized by combination with a fluorinated surfactant such as a perfluorinated polyether. In some embodiments, the oil phase further contains additives for adjusting oil properties such as vapor pressure, viscosity, or surface tension.

[0333] In some embodiments, the emulsion is formulated to produce highly monodisperse droplets with a liquid-like interfacial membrane, which can be converted into microcapsules with a solid-like interfacial membrane by heating; these microcapsules can act as bioreactors, capable of retaining their contents during incubation. Conversion to microcapsule form can occur upon heating. For example, this conversion can occur at temperatures greater than about 40, 50, 60, 70, 80, 90, or 95°C. During heating, a fluid or mineral oil coating can be used to prevent evaporation. Excess continuous phase oil may or may not be removed prior to heating. The microcapsules are resistant to coalescence and / or flocculation during a wide range of thermal and mechanical processing. In some embodiments, these capsules can be used to store or transport partitioned mixtures. For example, samples can be collected at one location, aliquoted into droplets containing enzymes, buffers, probes, and / or primers, optionally subjected to one or more amplification reactions, and then the partitions can be heated for microencapsulation, and the microcapsules can be stored or transported for further analysis.

[0334] In some implementations, the sample is divided into at least 500 partitions, at least 1000 partitions, at least 2000 partitions, at least 3000 partitions, at least 4000 partitions, at least 5000 partitions, at least 6000 partitions, at least 7000 partitions, at least 8000 partitions, at least 10,000 partitions, at least 15,000 partitions, at least 20,000 partitions, at least 30,000 partitions, and so on. At least 40,000 partitions, at least 50,000 partitions, at least 60,000 partitions, at least 70,000 partitions, at least 80,000 partitions, at least 90,000 partitions, at least 100,000 partitions, at least 200,000 partitions, at least 300,000 partitions, at least 400,000 partitions, at least 500,000 partitions, at least 600,000 partitions, at least 70 0,000 partitions, at least 800,000 partitions, at least 900,000 partitions, at least 1,000,000 partitions, at least 2,000,000 partitions, at least 3,000,000 partitions, at least 4,000,000 partitions, at least 5,000,000 partitions, at least 10,000,000 partitions, at least 20,000,000 partitions, at least 30,000,000 partitions 00 partitions, at least 40,000,000 partitions, at least 50,000,000 partitions, at least 60,000,000 partitions, at least 70,000,000 partitions, at least 80,000,000 partitions, at least 90,000,000 partitions, at least 100,000,000 partitions, at least 150,000,000 partitions, or at least 200,000,000 partitions.

[0335] In some implementations, NAICA TM The dPCR platform is used to implement the methods described herein. In some implementations, NAICA TM The Sapphire chip in the dPCR platform is used for sample dispensing. Typically, a Sapphire chip contains four microchambers, each containing a two-dimensional monolayer droplet. In some embodiments, data from dPCR reactions in droplets from different microchambers within the Sapphire chip are combined to provide quantification of the genetic species (e.g., wild-type and mutant sequences at multiple target regions) for which the method is designed to detect.

[0336] In some embodiments, the sample is divided into a sufficient number of partitions such that at least a majority of partitions have no more than 1-5 target regions or their amplicones (e.g., no more than about 1, 2, 3, 4, or 5 target regions or their amplicones). In some embodiments, each partition contains an average of about 0.5, 1, 2, 3, 4, or 5 target regions or their amplicones. In some embodiments, no more than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, or less of all partitions each contain at least one target region or its amplicon. In some embodiments, at least one partition does not contain a target region or its amplicon (the partition is "empty"). In some implementations, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 25%, 30%, or 40% of the partitions do not contain the target region or its amplicons. Typically, the partitions may contain excess enzymes, probes, and primers, such that each mixture partition is likely to successfully amplify any target region present in the partition.

[0337] In some embodiments, the generated droplets are substantially uniform in shape, size, and / or volume. For example, in some embodiments, the average diameter of the droplets is substantially uniform. In some embodiments, the generated droplets have an average diameter of about 0.001 micrometers, about 0.005 micrometers, about 0.01 micrometers, about 0.05 micrometers, about 0.1 micrometers, about 0.5 micrometers, about 1 micrometer, about 5 micrometers, about 10 micrometers, about 20 micrometers, about 30 micrometers, about 40 micrometers, about 50 micrometers, about 60 micrometers, about 70 micrometers, about 80 micrometers, about 90 micrometers, about 100 micrometers, about 150 micrometers, about 200 micrometers, about 300 micrometers, about 400 micrometers, about 500 micrometers, about 600 micrometers, about 700 micrometers, about 800 micrometers, about 900 micrometers, or about 1000 micrometers. In some embodiments, the generated droplets have an average diameter of less than about 1000 micrometers, less than about 900 micrometers, less than about 800 micrometers, less than about 700 micrometers, less than about 600 micrometers, less than about 500 micrometers, less than about 400 micrometers, less than about 300 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 50 micrometers, or less than about 25 micrometers. In some embodiments, the generated droplets are inconsistent in shape and / or size.

[0338] In some implementations, the resulting droplets have substantially uniform volumes. For example, the standard deviation of the droplet volume may be less than about 1 picoliter, 5 picoliters, 10 picoliters, 100 picoliters, 1 nL, or less than about 10 nL. In some cases, the standard deviation of the droplet volume may be less than about 10-25% of the average droplet volume. In some implementations, the resulting droplets have volumes of about 0.001 nL, about 0.005 nL, about 0.01 nL, about 0.02 nL, about 0.03 nL, about 0.04 nL, about 0.05 nL, about 0.06 nL, about 0.07 nL, about 0.08 nL, about 0.09 nL, about 0.1 nL, about 0.2 nL, about 0.3 nL, about 0.4 nL, about 0.5 nL, about 0.6 nL, about 0.7 nL, about 0.8 nL, and about 0. 9 nL, about 1 nL, about 1.5 nL, about 2 nL, about 2.5 nL, about 3 nL, about 3.5 nL, about 4 nL, about 4.5 nL, about 5 nL, about 5.5 nL, about 6 nL, about 6.5 nL, about 7 nL, about 7.5 nL, about 8 nL, about 8.5 nL, about 9 nL, about 9.5 nL, about 10 nL, about 11 nL, about 12 nL, about 13 nL, about 14 nL, about 15 nL, about 16 nL, about 17 nL, about 18 nL, about 19 nL, about 20 nL, about 25 nL, about 30 nL, about 35 nL, about 40 nL, about 45 nL, or about 50 nL.

[0339] Sample preparation

[0340] The samples analyzed by the methods in this application contain nucleic acid molecules. In some embodiments, the nucleic acid molecules are DNA molecules, such as genomic DNA, or DNA obtained from reverse transcription of RNA (e.g., cDNA). Genomic DNA can be chromosomal DNA, tumor-derived DNA (i.e., tumor genomic DNA), fetal DNA, or genomic DNA for site-specific genome editing. In some embodiments, the nucleic acid molecules are cell-free DNA (cfDNA), such as circulating DNA, such as circulating tumor DNA, or cell-free fetal DNA.

[0341] In some implementations, the nucleic acid molecule is an RNA molecule. In this case, a reverse transcription step can be performed on the sample.

[0342] The methods described herein may further include one or more sample preparation steps, including but not limited to obtaining biological samples from individuals, extracting nucleic acid molecules from biological samples, fragmenting nucleic acid molecules, and diluting nucleic acid molecules.

[0343] The sample can be prepared from biological samples such as biological fluids or biological tissues. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, pancreatic juice, gastric juice, amniotic fluid, and serous fluids such as pericardial fluid, pleural fluid, or peritoneal fluid.

[0344] Biological tissues are aggregates of cells, typically consisting of a specific type of cells and their intercellular material. This intercellular material forms one of the structural materials of human, animal, plant, bacterial, fungal, or viral structures, including connective tissue, epithelial tissue, muscle tissue, and nervous tissue. Examples of biological tissues also include organs, tumor tissue, lymph nodes, arteries, and diffuse cells. Tissues can be fresh, fresh-frozen, or fixed, such as formalin-fixed paraffin-embedded (FFPE) tissue. Biological samples can be obtained by any means, such as through surgery, like biopsy, or through less invasive methods, including but not limited to ablation or fine-needle aspiration.

[0345] In some embodiments, the biological sample is selected from the group consisting of tumor tissue, diffuse cells, feces, blood cells, plasma, serum, lymph nodes, urine, saliva, semen, sputum, cerebrospinal fluid, tears, mucus, pancreatic juice, gastric juice, amniotic fluid, and serous fluid. In some embodiments, the method further includes extracting nucleic acid molecules from the biological sample.

[0346] In some embodiments, the sample comprises microorganisms, such as bacterial cells, archaea cells, and / or yeast cells, or nucleic acids derived from microorganisms. In some embodiments, the sample comprises viruses or nucleic acids derived from viruses. In some embodiments, the sample comprises one or more pathogens, or nucleic acids derived from pathogens.

[0347] In some embodiments, the sample is derived from animals, such as pets, farm animals, or model animals, such as mammals. In some embodiments, the sample comprises animal cells, such as cells derived from primary cells or cell lines. In some embodiments, the sample is derived from plants, such as crops or model plants, including genetically modified (GM) plants and gene-edited (GE) plants. In some embodiments, the sample comprises genetically engineered cells, such as genome-engineered plant cells or animal cells. In some embodiments, the sample comprises nucleic acids derived from one or more types of cells.

[0348] In some embodiments, the sample is an environmental sample. In some embodiments, the sample is obtained from wastewater.

[0349] In some embodiments, the nucleic acid molecules in the sample have low molecular weights. For example, the length of the nucleic acid molecules may not exceed any one of about 1000, 900, 800, 700, 600, 500, 400, 300, or 200 nucleotides. In some embodiments, the method further includes fragmenting high molecular weight nucleic acid molecules (e.g., chromosomal DNA) into nucleic acid molecules of suitable size, for example, by sonication or restriction digestion.

[0350] In some embodiments, the concentration of nucleic acid molecules in a sample is adjusted, for example, by diluting the sample or by concentrating the sample (e.g., by dialysis or by lyophilization and reconstruction), to provide a suitable concentration for dPCR. In some embodiments, the method is performed with a first sample, and the concentration of nucleic acid molecules in the sample is adjusted based on the counts of each partition that produces a positive signal through three or more detection channels, wherein if (e.g., when) the count is greater than a predetermined value, the adjustment is made by diluting the sample to reduce the concentration of nucleic acid molecules in the sample; or wherein if (e.g., when) the count is less than a predetermined value, the adjustment is made by concentrating the sample to increase the concentration of nucleic acid molecules in the sample. In some embodiments, the dilution factor or concentration factor is based on the counts of each partition that produces a positive signal through three or more detection channels. In some embodiments, the concentration of nucleic acid molecules in the sample is adjusted based on the estimated concentration of wild-type sequences, the estimated concentration of mutant sequences, or the estimated concentration of a specific allele sequence at one or more target regions in the sample. In some embodiments, the method is repeated to dilute the sample to one or more concentrations to provide optimal concentrations for accurately quantifying different genetic species (e.g., wild-type, mutant, and / or allele sequences) at different target regions. In some embodiments, the concentration of the genetic species in the sample is at least about 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 50, 75, 100, 150, 200, 250, 500, 1000, 2000, 5000, 7500, 10000, 15000, 20000, 100000 or more copies per microliter. In some embodiments, the method is designed to detect mutations occurring at comparable frequencies (e.g., frequencies differing by no more than about 100x, 50x, 20x, 10x, 5x, 2x or less) in different target regions.

[0351] In some embodiments, the method includes determining a quality control metric based on the counts of each partition that generates a positive signal through each of the detection channels. In some embodiments, the quality control metric is determined by comparing the counts of each partition that generates a positive signal through each of the detection channels with an estimated count, wherein the estimated count is based on the counts of the partitions rather than on the counts of each partition that generates a positive signal through each of the detection channels X1-X. RThe count is calculated for each partition that generates a positive signal. In some implementations, the count is estimated based on the counts of each partition that generates a positive signal through one of the detection channels and a negative signal through each of the other detection channels. For example, the probability of each partition that generates a positive signal through one of the detection channels can be estimated as the product of the probability of each partition that generates a positive signal through one of the detection channels and the probability of each partition that generates a negative signal through each of the other detection channels.

[0352] In some embodiments, the sample is obtained from an individual. In some embodiments, the individual is a mammal, such as a primate, like a human. In some embodiments, the primate is a monkey or ape. The subject can be male or female and can be of any suitable age, including infants, teenagers, adolescents, adults, and elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[0353] In some implementations, the individual has cancer, is in cancer remission, or is at risk of developing cancer, particularly based on family history. In some implementations, the individual has a familial susceptibility to cancer.

[0354] In some embodiments, the individual has, is in remission, or has a family history of cancer susceptibility. In some embodiments, the individual has a disease caused by a mismatch repair (MMR) gene mutation (e.g., primary mismatch repair deficiency syndrome (CMMRD syndrome) or Lynch syndrome) or is at risk of developing such a disease.

[0355] Cancer can be a solid tumor or a "liquid tumor," such as cancers that affect the blood, bone marrow, and lymphatic system, also known as tumors of the hematopoietic and lymphatic tissues, especially including leukemia and lymphoma. Liquid tumors include, for example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL), including various lymphomas such as mantle cell lymphoma or non-Hodgkin lymphoma (NHL).

[0356] Solid cancers include cancers affecting one of the following organs: colon, rectum, skin, endometrium, lungs (including non-small cell lung cancer), uterus, bones (e.g., osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell tumor, ameloblastoma, and chordoma), liver, kidneys, esophagus, stomach, bladder, pancreas, cervix, brain (e.g., meningioma, glioblastoma, low-grade astrocytoma, oligodendroglioma, pituitary adenoma, schwannoma, and metastatic brain cancer), ovary, breast, head and neck region, testes, prostate, and thyroid.

[0357] An implementation using three (3) drop-off probe pairs

[0358] Figure 7 An exemplary procedure 700 is illustrated according to some embodiments for quantifying wild-type and / or mutant sequences at three (3) target regions in a sample containing nucleic acid molecules. Procedure 700 is performed, for example, by one or more human users or any combination thereof, using an electronic device implementing a software platform. In some instances, procedure 700 is performed using a client-server system, and the frame of procedure 700 is divided in any way between server and client devices. In other instances, the frame of procedure 700 is divided between a server and multiple client devices. Therefore, while portions of procedure 700 are described herein as being performed by a specific device of a client-server system, it should be understood that procedure 700 is not limited thereto. In other instances, procedure 700 is performed using only a client device or on multiple client devices.

[0359] In process 700, some boxes may be combined, some boxes may be rearranged, and some boxes may be omitted. In some instances, process 700 may be combined to perform additional steps. Therefore, the operations shown (and described in more detail below) are exemplary in nature and should not be considered limiting.

[0360] In some embodiments, one or more variables of process 700 can be obtained by a drop-off digital PCR process, wherein three (3) probe pairs corresponding to three target regions are used. Each of the three probe pairs comprises: a drop-off probe containing a drop-off marker and an oligonucleotide drop-off sequence complementary to a wild-type sequence, rather than a mutant sequence, at the target region corresponding to the corresponding probe pair; and a reference probe containing a reference marker and an oligonucleotide reference sequence complementary to a wild-type sequence at an adjacent reference region upstream or downstream of the target region corresponding to the corresponding probe pair.

[0361] In some implementations, the reference marker and drop-off marker of each of the three probe pairs can be detected via different detection channels. In the exemplary implementation shown in Table 2, each of the three probe pairs has a reference marker and drop-off marker that can be detected via different detection channels (i.e., blue to green; green to red; red to blue). In some implementations, the reference markers of the three probe pairs can be detected via different detection channels from each other, and the drop-off markers of the three probe pairs can be detected via different detection channels from each other. In the exemplary implementation shown in Table 2, the reference markers of the three probe pairs can be detected via the blue detection channel, the green detection channel, and the red detection channel, respectively. Furthermore, the drop-off markers of the three probe pairs can be detected via the green detection channel, the red detection channel, and the blue detection channel, respectively.

[0362] Table 2

[0363]

[0364]

[0365] The exemplary schemes in Table 2 are arranged in a cyclical pattern. Specifically, the detection channel of one of the three reference markers is also the detection channel of one of the three drop-off markers. For example, the detection channel corresponding to the drop-off marker of the first probe pair (i.e., the green detection channel) is the same as the detection channel corresponding to the reference marker of the second probe pair. Furthermore, the number of detection channels (i.e., 3) is the same as the number of probe pairs (i.e., 3).

[0366] However, it should be understood that the schemes in Table 2 are merely exemplary, and the cyclic arrangement is not necessary for performing process 700. For example, the drop-off marker corresponding to the third probe pair could be yellow instead of blue. In some embodiments, the total number of detection channels may be the same as or less than twice the number of probe pairs (i.e., 6).

[0367] In digital drop-off PCR, the sample containing nucleic acid molecules is distributed across multiple partitions, and essentially each partition contains three probe pairs. Hybridization of the three probe pairs (reference probes) with nucleic acid molecules or their amplicons containing wild-type sequences at reference regions in the multiple partitions can be detected. Furthermore, hybridization of the three probe pairs (drop-off probes) with nucleic acid molecules or their amplicons containing wild-type sequences at target regions in the multiple partitions can be detected. Procedure 700 can then be performed to provide quantification of wild-type and / or mutant sequences at the three target regions in the sample.

[0368] Process 700 is based on the assumption of independence of partitioned encapsulation of nucleic acid molecules containing target regions 1, 2, and 3. In fact, although the partitioned encapsulation of blue, green, and red fluorophores is not independent in the exemplary schemes in Table 2 due to biomolecular design, the partitioned encapsulation of nucleic acid molecules containing target regions 1, 2, and 3 is independent.

[0369] The following describes process 700 using the following symbols:

[0370] Table 3

[0371]

[0372]

[0373] In box 702, the system (e.g., one or more electronic devices) determines the mutation probability that a given partition contains a mutated sequence at the target region corresponding to the first probe pair.

[0374] In some implementations, block 702 includes blocks 704 and 706. In block 704, the system obtains a first count (n) of one or more partitions. 100 Each partition generates a positive signal through detection channel X1, a negative signal through detection channel X2, and a negative signal through detection channel X3.

[0375] Furthermore, in box 706, the system obtains a second count (n) of one or more partitions. 000 Each of the partitions generates a negative signal on all detection channels X1-X3.

[0376] In some implementations, the system is based on a first count (n) 100 ) and the first count (n) 100 ) and second count (n 000 Calculation of the ratio between the sums of ) As shown below.

[0377]

[0378] In some implementations, the first count and / or the second count is zero. For example, if the mutant sequence is absent, there will be no single positive partition, and the exemplary method will not be able to detect the mutant sequence, but will be able to provide an upper limit for its actual concentration. In some implementations, if the concentration of the mutant (or other target) is too high, there will be no completely negative partition, and the exemplary method will be able to detect its presence and provide a lower limit for its actual concentration.

[0379] The derivation of the above formula is as follows:

[0380]

[0381] In some implementations, the system can calculate in a similar manner. and For example:

[0382]

[0383]

[0384] In some implementations, at block 710, the system is based on the mutation probability in the sample. Determine the estimated concentration of the mutant sequence in the sample corresponding to the target region of the first probe pair. For example, the system can calculate the estimated concentration based on Poisson's law using the following formula:

[0385]

[0386] In some implementations, the system determines the estimated concentration in the sample. The relevant confidence intervals and / or uncertainty measures. For example, the confidence intervals and uncertainty at a 95% confidence level can be calculated as follows:

[0387]

[0388]

[0389] In some implementations, the uncertainty measure refers to the uncertainty of the digital PCR method and can be calculated as provided below. Those skilled in the art will understand that other types of uncertainty (e.g., sampling, sample handling, processing) can be considered.

[0390]

[0391] In some implementations, the system can calculate in a similar manner. and

[0392] In box 712, the system determines the wild-type probability that a given partition contains a wild-type sequence at the target region corresponding to the first probe pair. In some implementations, the wild-type probability is based on In some implementations, the wild-type probability is based on and calculate.

[0393] In some implementations, the system calculates according to the following formula

[0394]

[0395]

[0396] The formula derivation is as follows:

[0397]

[0398]

[0399] therefore, The calculation can be derived accordingly.

[0400] In some implementations, at block 720, the system is based on wild-type probability. Determine the estimated concentration of the wild-type sequence in the sample corresponding to the target region of the first probe pair. For example, the system can calculate the estimated concentration based on Poisson's law using the following formula:

[0401]

[0402] In some implementations, if the wild-type concentration is expected to be the same on each probe pair, a more robust wild-type concentration estimate can be obtained by averaging the three estimates.

[0403] In some implementations, the system can calculate in a similar manner. and and

[0404] In some implementation schemes, it is possible to separately from The variance is used to derive confidence intervals, including and Uncertainty measure at 95% confidence level Where i is 1, 2 or 3.

[0405] The implementation schemes described in this section are applicable to dPCR methods using three probe sets containing dual-labeled AS probes (e.g., each probe set contains a reference probe and an AS probe with two detectable labels, or each probe set contains a reference probe, a first AS probe, and a second AS probe). Exemplary schemes for dual-labeled AS assays capable of detecting six genetic species using three fluorophores are shown in Table 4 below.

[0406] Table 4

[0407]

[0408] Implementation scheme using R drop-off probe pairs

[0409] Figure 8 The illustration depicts an exemplary procedure 800 for quantifying wild-type and / or mutant sequences at R target regions in a sample containing nucleic acid molecules, according to some embodiments. Procedure 800 is performed, for example, by one or more human users or any combination thereof, using an electronic device implementing a software platform. In some instances, procedure 800 is performed using a client-server system, and the frame of procedure 800 is divided in any way between server and client devices. In other instances, the frame of procedure 800 is divided between a server and multiple client devices. Therefore, while portions of procedure 800 are described herein as being performed by a specific device of a client-server system, it should be understood that procedure 800 is not limited thereto. In other instances, procedure 800 is performed using only a client device or multiple client devices.

[0410] In process 800, some boxes may be combined, some boxes may be rearranged, and some boxes may be omitted. In some instances, process 800 may be combined to perform additional steps. Therefore, the operations shown (and described in more detail below) are exemplary in nature and should not be considered limiting.

[0411] In some embodiments, one or more variables of process 800 can be obtained through a drop-off digital PCR process, wherein R probe pairs corresponding to R target regions are used. Each of the R probe pairs comprises: a drop-off probe containing a drop-off marker and an oligonucleotide drop-off sequence complementary to a wild-type sequence, rather than a mutant sequence, at the target region corresponding to the corresponding probe pair; and a reference probe containing a reference marker and an oligonucleotide reference sequence complementary to a wild-type sequence at an adjacent reference region upstream or downstream of the target region corresponding to the corresponding probe pair.

[0412] In some implementations, the reference marker and drop-off marker of each probe pair in a plurality of probe pairs can be detected via different detection channels. In the exemplary implementation shown in Table 5, each probe pair in the plurality of probe pairs has a reference marker and drop-off marker that can be detected via different detection channels (i.e., X). i vX i+1 The reference markers and drop-off markers of the multiple probe pairs are detected through different detection channels. In some embodiments, the reference markers of the multiple probe pairs can be detected through different detection channels, and the drop-off markers of the multiple probe pairs can be detected through different detection channels. In the exemplary embodiment shown in Table 5, the reference markers of the multiple probe pairs can be detected through X1-X1 channels respectively.R Detection. Furthermore, the drop-off markers for the three probe pairs can be detected via X1-X... R Testing.

[0413] Table 5

[0414]

[0415]

[0416] The exemplary schemes in Table 5 are arranged in a circular pattern. Specifically, the detection channel of one of the multiple reference markers is also the detection channel of one of the multiple drop-off markers. For example, corresponding to probe pair i (i.e., X) i+1 The detection channels for the drop-off marker are the same as those for the reference marker corresponding to probe pair i+1. Furthermore, the number of detection channels (i.e., R) is the same as the number of probe pairs (i.e., R).

[0417] However, it should be understood that the schemes in Table 5 are merely exemplary and do not require a cyclic arrangement to perform process 800. In some implementations, the total number of detection channels may be the same as or less than twice the number of probe pairs (i.e., 2R).

[0418] In multiplex drop-off digital PCR, samples containing nucleic acid molecules are distributed across multiple partitions, and essentially each partition contains R probe pairs. These can be detected via channels X1-X. R Each reference probe in the detection array, which detects multiple probe pairs, hybridizes with a nucleic acid molecule or its amplicon containing a wild-type sequence at a reference region in each partition. Furthermore, detection can be performed via channels X1-X. R Each drop-off probe, which detects multiple probe pairs, hybridizes with a nucleic acid molecule or its amplicon containing a wild-type sequence in each partition. Procedure 800 can then be performed to provide quantification of wild-type and / or mutant sequences at R target regions in the sample.

[0419] Process 800 is based on the independence assumption of partitioned encapsulation of nucleic acid molecules containing target regions 1 to R. In fact, although in the exemplary schemes in Table 5, due to biomolecular design, fluorophores X1-X are not present. R The partitioning of nucleic acid molecules containing target regions 1 to R is independent, but the partitioning of nucleic acid molecules containing target regions 1 to R is independent.

[0420] The following describes process 800 using the following symbols:

[0421] Table 6

[0422]

[0423]

[0424] In box 802, the system (e.g., one or more electronic devices) determines the mutation probability that a given partition contains a mutated sequence at the target region corresponding to the i-th probe pair.

[0425] In some implementations, block 802 includes blocks 804 and 806. In block 804, the system obtains a first count of one or more partitions, each of which generates a positive signal through the i-th detection channel and through detection channels X1-X. R Any other detection channel in the array generates a negative signal. Furthermore, in block 806, the system obtains a second count for one or more partitions, each of which passes through all detection channels X1-X. R It generates a negative signal.

[0426] In some implementations, the system is based on a first count (n) i ) and the first count (n) i Calculation of the ratio between the sum of the first and second counts (n0) As shown below.

[0427]

[0428] In some implementations, the first count and / or the second count are zero for the reasons described above.

[0429] The derivation of the above formula is as follows:

[0430]

[0431] In some implementations, at block 810, the system is based on mutation probability. Determine the estimated concentration of the mutation sequence at the target region corresponding to the i-th probe pair in the sample. For example, the system can calculate the estimated concentration based on Poisson's law using the following formula:

[0432]

[0433] In some implementations, the system determines the estimated concentration in the sample. The relevant confidence intervals and / or uncertainty measures. For example, the confidence intervals and uncertainty at a 95% confidence level can be calculated as follows:

[0434]

[0435]

[0436]

[0437] In box 812, the system determines the wild-type probability that a given partition contains a wild-type sequence at the target region corresponding to the i-th probe pair. In some implementations, the wild-type probability is based on In some implementations, the wild-type probability is based on and calculate.

[0438] In some implementations, the system calculates according to the following formula

[0439]

[0440] The formula derivation is as follows:

[0441]

[0442]

[0443] Therefore, by equating the two formulas cited above, we can derive the following: of

[0444] calculate:

[0445] Subsequently:

[0446] In some implementations, at block 820, the system is based on wild-type probability. Determine the estimated concentration of the wild-type sequence in the target region corresponding to the i-th probe pair in the sample. For example, the system can calculate the estimated concentration based on Poisson's law using the following formula:

[0447]

[0448] In some implementations, if the wild-type concentration is expected to be the same, a more robust estimate of the wild-type concentration can be obtained by averaging R estimates.

[0449] In some implementations, the confidence interval and the uncertainty measure at the 95% confidence level can be derived from... The variance is derived, and this variance is denoted as... As shown below:

[0450]

[0451]

[0452]

[0453] in The variance itself can be derived from from The variances of X and A are derived from the variances of X and A, respectively.

[0454] In fact:

[0455] ·

[0456] ·

[0457] ·

[0458] ·

[0459] ·

[0460] ·because and It is independent; we have:

[0461]

[0462] ·

[0463] Given:

[0464] ·

[0465] Where N R R is the sample size, N is the sample size. S The sample size of S

[0466] The sample size of X is N. X =n0+n i +n i+1 +n i,(i+1)

[0467] The sample size of A is N. A =n0+n i +n i+1

[0468] Since the events of A are contained within the events of X, we have: Cov(X,A) = Var(A)

[0469] We deduce:

[0470] ·

[0471] Using this biomolecular design, the higher the wild-type concentration, the greater the uncertainty in mutant concentration.

[0472] In some implementations, R is an integer between 2 and 6. Figure 7 And the corresponding description for the implementation scheme with R equal to 3.

[0473] The implementation schemes described in this section are applicable to dPCR methods using R probe sets containing dual-labeled AS probes (e.g., each probe set contains a reference probe and an AS probe with two detectable labels, or each probe set contains a reference probe, a first AS probe, and a second AS probe). Exemplary schemes for dual-labeled AS assays capable of detecting 2R genetic species using R fluorophores are shown in Table 7 below.

[0474] Table 7

[0475]

[0476]

[0477] An implementation scheme using (R-1) drop-off probe triplets

[0478] Figure 9 The illustration depicts an exemplary procedure 900 for quantifying unmodified, homology-directed repair (HDR) edited, and / or non-homologous end joining (NHEJ) edited sequences at multiple target regions in a nucleic acid molecule from a cell sample, according to some embodiments. The cells have been contacted with a site-specific genome editing reagent and an HDR template nucleic acid containing an HDR substitution sequence, and the site-specific genome editing reagent is configured to cleave target sites in multiple target regions.

[0479] Process 900 may be executed, for example, by one or more electronic devices implementing a software platform, by one or more human users or any combination thereof. In some instances, a client-server system may be used to execute process 900, and the frame of process 900 may be divided in any way between the server and client devices. In other instances, the frame of process 900 may be divided between the server and multiple client devices. Therefore, although parts of process 900 are described herein as being executed by a specific device of a client-server system, it should be understood that process 900 is not limited thereto. In other instances, process 900 may be executed using only client devices or only multiple client devices.

[0480] In process 900, some boxes may be optionally combined, some boxes may be optionally rearranged, and some boxes may optionally be omitted. In some instances, process 900 may be combined to perform additional steps. Therefore, the operations shown (and described in more detail below) are exemplary in nature and should not be considered limiting.

[0481] In some embodiments, one or more variables of process 900 can be obtained through a drop-off digital PCR process, wherein (R-1) probe triplets corresponding to (R-1) target regions are used. Each of the (R-1) probe triplets comprises: an HDR probe containing an HDR marker and an oligonucleotide HDR sequence complementary to an HDR substitution sequence inserted into the target region corresponding to the corresponding probe triplet; an NHEJ drop-off probe containing an NHEJ drop-off marker and an oligonucleotide drop-off sequence complementary to a wild-type sequence corresponding to the target region of the corresponding probe triplet, wherein the drop-off sequence does not hybridize with an NHEJ-edited mutant sequence at the target region corresponding to the corresponding probe triplet; and a reference probe containing a reference marker and an oligonucleotide reference sequence complementary to a wild-type sequence at an adjacent reference region upstream or downstream of the target region corresponding to the corresponding probe triplet.

[0482] In some implementations, the HDR marker, NHEJ drop-off marker, and reference marker of each probe triplet in a plurality of probe triplets can be detected via different detection channels. In the exemplary implementation shown in Table 8, each probe triplet in a plurality of probe triplets has an HDR marker, an NHEJ drop-off marker, and a reference marker, wherein each probe triplet in a plurality of probe triplets can be detected via different detection channels (i.e., X...). i vX i+1 vX i+2 Detection. In some embodiments, the reference markers of multiple probe triplets can be detected through different detection channels, the HDR markers of multiple probe triplets can be detected through different detection channels, and the NHEJ drop-off markers of multiple probe triplets can be detected through different detection channels. In the exemplary embodiment shown in Table 8, the reference markers of (R-1) probe triplets can be detected through X1-X... R-2 and X R Detection. Furthermore, the NHEJ drop-off labeling of the (R-1) probe triplet can be achieved via X2-X... R-1 and X R-1 Detection. HDR labeling of (R-1) probe triplets can be achieved via X1, X3-X... R Testing.

[0483] Table 8

[0484]

[0485]

[0486] The exemplary schemes in Table 8 employ the arrangement of markers in the probe triplet. Figure 6B Exemplary arrangements of four, six, or ten markers in three-probe triplet sets, five-probe triplet sets, or nine-probe triplet sets are shown. For example, for a three-probe triplet, the first triplet includes a first reference probe labeled with fluorophore 1, a first NHEJ drop-off probe labeled with fluorophore 2, and a first HDR probe labeled with fluorophore 3; the second triplet includes a second reference probe labeled with fluorophore 2, a second NHEJ drop-off probe labeled with fluorophore 3, and a second HDR probe labeled with fluorophore 4; the third triplet includes a third reference probe labeled with fluorophore 4, a third NHEJ drop-off probe labeled with fluorophore 3, and a third HDR probe labeled with fluorophore 1. The number of detection channels (R) is one more than the number of probe triplets (R-1).

[0487] However, it should be understood that the schemes in Table 8 are merely exemplary and do not need to be implemented as described above. Figure 6B The arrangement shown is used to perform process 800. In some implementations, the total number of detection channels may be the same as or less than three times the number of probe triplets (i.e., 3(R-1)).

[0488] In multiplex drop-off digital PCR, the sample containing nucleic acid molecules is distributed across multiple partitions, and essentially each partition contains (R-1) probe triplets. These can be detected via channels X1-X. R-2 and X R Each detection (R-1) probe triplet's reference probe hybridizes with a nucleic acid molecule or its amplicons containing a wild-type sequence in the reference region of each partition. This can be achieved through detection channels X2-X. R-1 Each detection (R-1) probe triplet in the NHEJ drop-off probe hybridizes with a nucleic acid molecule or its amplicon containing the wild-type sequence at the target region in each partition. Furthermore, detection can be performed via channels X1 and X3-X. R Each detection (R-1) probe triplet hybridizes with an HDR probe and a nucleic acid molecule or its amplicons containing an HDR-edited sequence (i.e., an HDR replacement sequence) at the target region in each partition. In an alternative setting, detection can be performed via channels X2-X. R-1 Each detection (R-1) probe triplet hybridizes with an HDR probe and a nucleic acid molecule or its amplicons containing an HDR-edited sequence (i.e., an HDR substitution sequence) at the target region in each partition. Furthermore, detection can be performed via channels X1 and X3-X. REach detection (R-1) probe triplet of NHEJ drop-off probes is hybridized with a nucleic acid molecule or its amplicon containing a wild-type sequence at the target region in each partition. Procedure 800 can then be performed to provide quantification of unmodified, NHEJ-edited, and / or HDR-edited sequences at the (R-1) target regions in the sample.

[0489] Process 900 is based on the independence assumption of partitioned encapsulation of all types of sequences at target regions 1 to R-1. In fact, although in the exemplary schemes in Table 8, due to biomolecular design, there are no fluorophores X1-X... R The partitioning of the nucleic acid molecules containing target regions 1 to R-1 is independent, but the partitioning of the nucleic acid molecules containing target regions 1 to R-1 is independent.

[0490] The following describes process 900 using the following symbols:

[0491] Table 9

[0492]

[0493]

[0494]

[0495] Procedure 900 describes the calculation of the NHEJ editing probability corresponding to the i-th probe triplet (if 1 ≤ i ≤ R-2) according to some implementation schemes. Unmodified probability and HDR editing probability An exemplary process.

[0496] In box 902, the system (e.g., one or more electronic devices) calculates the NHEJ editing probability that a given partition contains an NHEJ editing sequence at the target region corresponding to the i-th probe triplet.

[0497] In some implementations, block 902 includes blocks 904 and 906. In block 904, the system obtains a first count (n) of one or more partitions. i Each of the partitions is accessed via X. i The detection channel generates a positive signal and passes through detection channel X1-X. R Any other detection channel in the array generates a negative signal. In block 906, the system obtains a second count (n0) for one or more partitions, each of which passes through all detection channels X1-X2. R A negative signal is generated. In some implementations, the first count and / or the second count is zero.

[0498] In some implementations, the system calculates based on the ratio between the first count and the sum of the first and second counts. As shown below:

[0499]

[0500] The derivation of the above formula is as follows:

[0501]

[0502]

[0503] In some implementations, the system is based on NHEJ edit probabilities. Determine the estimated concentration of the NHEJ edit sequence at target region i in the sample. For example, the system can calculate the estimated concentration based on Poisson's law using the following formula:

[0504]

[0505] In some implementations, the system determines the estimated concentration in the sample. The relevant confidence intervals and / or uncertainty measures. For example, the confidence intervals and uncertainty at a 95% confidence level can be calculated as follows:

[0506]

[0507]

[0508]

[0509] In box 908, the system calculates the probability that a given partition contains an unmodified wild-type sequence at the target region corresponding to the i-th probe triplet.

[0510] In some implementations, block 908 includes blocks 910 and 912. In block 910, the system obtains a third count of one or more partitions, each partition being accessed via X. i The detection channel generates a positive signal, which is transmitted through X. i+1 The detection channel generates a positive signal and passes through detection channel X1-X. R Any other detection channel in the array generates a negative signal. In block 912, the system obtains a fourth count for one or more partitions, each of which passes through a channel other than X. i Detection channels and X i+1 Detection channels X1-X outside of the detection channel R One or more of these generate negative signals. In some implementations, the fourth count is calculated as n0 + n i+n i+1 +n i,(i+1) . In some embodiments, the first count, the second count, the third count, and / or the fourth count is zero.

[0511] In some embodiments, for i < R - 1, the system calculates according to the following formula

[0512] The above formula is derived as follows:

[0513]

[0514]

[0515] By equating the above two formulas, the formula of can be correspondingly derived.

[0516]

[0517] When and are minimized (or maximized), is maximized (or minimized).

[0518] In some embodiments, the system determines the estimated concentration of the unmodified sequence at the target region number i in the sample based on the unmodified probability For example, the system can calculate the estimated concentration according to the following formula based on the Poisson law:

[0519]

[0520] In block 914, the system calculates the HDR editing probability that a given partition contains an HDR editing sequence at the target region corresponding to the i-th probe triplet In some embodiments, block 914 includes blocks 916 and 918.

[0521] In block 916, the system obtains a fifth count of one or more partitions, each of which generates a positive signal through the X i detection channel, generates a positive signal through the X i+2 detection channel and generates a negative signal through any other detection channel in the detection channels X1 - X R . In block 918, the system obtains a sixth count of one or more partitions, each of which generates a negative signal through a detection channel other than the X i detection channel and the X i+2 detection channel among the detection channels X1 - X R In some embodiments, the sixth count is calculated as n0 + ni +n i+2 +n i,(i+2) In some embodiments, the fifth count and / or the sixth count is zero.

[0522] In some embodiments, for i < R - 1, it is calculated according to the following formula

[0523] The above formula is derived as follows:

[0524]

[0525] Furthermore:

[0526]

[0527] In some embodiments, the system determines the estimated concentration of the HDR-edited sequence at the target region number i in the sample based on the HDR editing probability For example, the system can calculate the estimated concentration according to the following formula based on the Poisson law:

[0528]

[0529] As described above, process 900 depicts an exemplary process for calculating the NHEJ editing probability corresponding to the i-th probe triplet (if 1 ≤ i ≤ R - 2) unmodified probability and HDR editing probability of.

[0530] If i = R - 1, the following formula is used. These formulas are derived in a similar manner to those Figure 9 described.

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538] Indeed, by definition "r R-1 " is an impossible event, so P(rR-1 ) = 0

[0539] therefore:

[0540]

[0541]

[0542]

[0543] therefore:

[0544]

[0545]

[0546] Although described in the context of detecting CRISPR-Cas genome-edited sequences, the above analysis is generally applicable to genome editing detection methods tailored to any site-specific genome editing reagent used to edit genomic DNA through repair of cleaved genomic DNA via NHEJ or HDR-mediated splitting. Furthermore, the above-described embodiments for detecting site-specific genome-edited products are also generally applicable to any method described herein for detecting wild-type, mutant, and / or allele sequences at (R-1) target regions using a (R-1) probe triplet, where the wild-type sequence corresponds to the unmodified sequence in the CRISPR embodiment, the mutant sequence corresponds to the NHEJ-edited sequence in the CRISPR embodiment, and the allele sequence corresponds to the HDR-edited sequence in the CRISPR embodiment.

[0547] Multiplex dPCR using double-labeled allele-specific probes

[0548] This application further provides a multiplex dPCR method that, instead of using drop-off probes, utilizes the same concept of cyclical arrangement of labels in a probe set to allow for higher-order multiplexing of dPCR assays. In some embodiments, the method uses multiple probe sets, each containing a reference probe and a dual-labeled allele-specific (AS) probe or AS probe pair, wherein the dual-labeled AS probe or AS probe pair has a first detectable label detectable via the same detection channel as the detectable label of the reference probe, and a second detectable label detectable via a different detection channel than the detectable label of the reference probe. Each probe set and its associated primers allow for the quantification of target species (i.e., allele sequences), such as mutations (e.g., SNPs, insertions, deletions, etc.) or copy number variations (CNVs) relative to the reference species, thereby allowing for the detection and quantification of target species in a sample.

[0549] In some embodiments, a method is provided for quantifying wild-type and / or allele sequences (e.g., rare alleles or CNVs) at multiple target regions in a sample containing nucleic acid molecules, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, wherein substantially all partitions (e.g., all partitions) each contain multiple probe sets corresponding to multiple target regions, wherein each of the multiple probe sets comprises:

[0550] A first allele-specific (AS) probe comprising a first AS marker and an oligonucleotide AS sequence, wherein the oligonucleotide AS sequence is complementary to an allele sequence or a first portion thereof at a target region corresponding to the corresponding probe set.

[0551] The second AS probe comprises a second AS marker and an oligonucleotide AS sequence, wherein the oligonucleotide AS sequence is complementary to the allele sequence, its second part or its complementary sequence at the target region corresponding to the corresponding probe set.

[0552] A reference probe comprising a reference marker and an oligonucleotide sequence complementary to a reference sequence at a reference region corresponding to a respective probe set;

[0553] The reference marker and the first AS marker of each of the multiple probe groups can be detected through the same detection channel;

[0554] The reference marker and second AS marker of each of the multiple probe groups can be detected through different detection channels;

[0555] The reference markers of multiple probe groups can be detected through different detection channels;

[0556] The second AS marker of multiple probe groups can be detected through different detection channels;

[0557] At least one reference marker from multiple probe groups and at least one second AS marker from multiple probe groups can be detected through the same detection channel;

[0558] The method includes: detecting hybridization of reference probes from multiple probe sets with nucleic acid molecules containing reference sequences at reference regions in multiple partitions; and detecting hybridization of first AS probes and second AS probes from multiple probe sets with nucleic acid molecules or their amplicon containing allele sequences or portions thereof at target regions in multiple partitions; thereby providing quantification of wild-type and / or allele sequences at multiple target regions in a sample. In some embodiments, the reference region corresponding to each probe set is adjacent to the target region (e.g., upstream or downstream). In some embodiments, the reference region overlaps with the target region. In some embodiments, the reference region is identical to the target region. In some embodiments, the detection of a signal from a reference marker in a probe set but no signal from a second AS probe indicates the presence of a wild-type sequence in the target region corresponding to the probe set, and the detection of a signal from both the reference marker and the second AS marker in a probe set indicates the presence of an allele sequence in the target region corresponding to the probe set. In some embodiments, the reference marker and the first AS marker of each probe set are identical to each other. In some embodiments, the reference marker set of multiple probe sets and the second AS marker set of multiple probe sets have overlapping markers. In some implementations, the reference marker group of the multiple probe groups and the second AS marker group of the multiple probe groups are arranged in a circular arrangement relative to each other.

[0559] In some embodiments, a method is provided for quantifying wild-type and / or allele sequences (e.g., rare alleles or CNVs) at R target regions in a sample containing nucleic acid molecules, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, wherein substantially all partitions (e.g., all partitions) each contain R probe triplets corresponding to the R target regions.

[0560] The first probe triplet of the R probe triplets includes:

[0561] Corresponding to (e.g., including) a first reference probe of a first reference sequence (w1) and a first reference marker detectable by a first detection channel (X1),

[0562] The first AS probe (“first AS probe 1”) corresponding to (e.g., containing) the first allele sequence (r1) of the first probe triplet and the first AS marker (“first AS marker 1”) of the first probe triplet detectable by the first detection channel (X1), and

[0563] The second AS probe (“second AS probe 1”) of the first probe triplet corresponding to (e.g., containing) the first allele sequence (r1) and the second AS marker (“second AS marker 1”) of the first probe triplet detectable by the second detection channel (X2);

[0564] Where the second probe triplet among the R probe triplets includes:

[0565] A second reference probe corresponding to (e.g., including) a second reference sequence (w2) and a second reference label detectable through a second detection channel (X2),

[0566] A first AS probe of the second probe triplet corresponding to (e.g., including) a second allele sequence (r2) (“first AS probe 2”) and a first AS label of the second probe triplet detectable through the second detection channel (X2) (“AS label 2”), and

[0567] A second AS probe of the second probe triplet corresponding to (e.g., including) the second allele sequence (r2) (“second AS probe 2”) and a second AS label of the second probe triplet detectable through a third detection channel (X3) (“second AS label 2”);

[0568] Where, if (e.g., when) R is strictly greater than 3, the i-th probe triplet among the R probe triplets (2 < i < R) includes:

[0569] An i-th reference probe corresponding to (e.g., including) the i-th reference sequence (w i ) and an i-th reference label detectable through the i-th detection channel (X i ),

[0570] A first AS probe of the i-th probe triplet corresponding to (e.g., including) the i-th allele sequence (r i ) (“first AS probe i”) and a first AS label of the i-th probe triplet detectable through the i-th detection channel (X i ) (“first AS label i”), and

[0571] A second AS probe of the i-th probe triplet corresponding to (e.g., including) the i-th allele sequence (r i ) (“second AS probe i”) and a second AS label of the i-th probe triplet detectable through the (i + 1)-th detection channel (X i+1 ) (“second AS label i”);

[0572] Where, if (e.g., when) R is strictly greater than 2, the R-th probe triplet among the R probe triplets includes:

[0573] An R-th reference probe corresponding to (e.g., including) the R-th reference sequence (w R ) and an R-th reference label detectable through the R-th detection channel (X R ),

[0574] Corresponding to (e.g., containing) the Rth allele sequence (r R The first AS probe (“first AS probe R”) of the Rth probe triplet and the Rth detection channel (X) R The first AS marker (“first AS marker R”) of the Rth probe triplet detected, and

[0575] Corresponding to (e.g., containing) the Rth allele sequence (r R The second AS probe (“second AS probe R”) of the Rth probe triplet and the second AS marker (“second AS marker R”) of the Rth probe triplet that can be detected by the first detection channel (X1);

[0576] In each probe triplet, the first AS probe and the second AS probe hybridize at the target region corresponding to the corresponding probe triplet with the same allele sequence, different parts of the same allele sequence, or their complementary sequence.

[0577] The reference sequence for each probe triplet is located in the reference region corresponding to the corresponding probe triplet.

[0578] Among them, detection channels X1-X R They are different;

[0579] The method includes detecting channels X1-X R Each of the R probes in the detection triad hybridizes with a reference probe and a nucleic acid molecule or its amplicon containing a reference sequence or its complementary sequence at a reference region in multiple partitions; and through detection channels X1-X R Each of the R probe triplet detection methods involves the first and second AS probes hybridizing with a nucleic acid molecule or its amplicons containing an allele sequence or its complementary sequence at a target region in multiple partitions; thereby providing quantification of wild-type and / or allele sequences at R target regions in the sample. In some embodiments, the reference region of the probe triplet is adjacent (e.g., upstream or downstream) to the target region corresponding to the corresponding probe triplet. In some embodiments, the reference region of the probe triplet overlaps with the target region corresponding to the corresponding probe triplet. In some embodiments, the reference region of the probe triplet is the same as the target region corresponding to the corresponding probe triplet. R can be any suitable integer of 2 or greater. In some embodiments, R is between 2 and 6. In some embodiments, the detection of a signal from a reference marker and a signal from a second AS marker in the probe triplet indicates the presence of an allele sequence in the target region corresponding to the probe triplet, and the detection of a signal from a reference marker but no signal from a second AS marker in the probe triplet indicates the presence of a wild-type sequence in the target region corresponding to the probe triplet.

[0580] In some embodiments, a method is provided for quantifying wild-type and / or allele sequences at three target regions in a sample containing nucleic acid molecules, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, wherein substantially all partitions (e.g., all partitions) each contain a triplet of three probes corresponding to the three target regions, wherein the triplet of three probes comprises:

[0581] The first probe triplet includes:

[0582] Corresponding to (e.g., including) a first reference probe of a first reference sequence and a first reference marker detectable by a first detection channel,

[0583] The first AS probe (“first AS probe 1”) corresponding to (e.g., containing) the first allele sequence of the first probe triplet and the first AS marker (“first AS marker 1”) of the first probe triplet detectable by the first detection channel, and

[0584] The second AS probe (“second AS probe 1”) of the first probe triplet corresponding to (e.g., containing) the first allele sequence and the second AS marker (“second AS marker 1”) of the first probe triplet detectable by the second detection channel;

[0585] The second probe triplet includes:

[0586] Corresponding to (e.g., including) a second reference probe of a second reference sequence and a second reference marker detectable by a second detection channel,

[0587] The first AS probe (“first AS probe 2”) of the second probe triplet corresponding to (e.g., containing) the second allele sequence and the first AS marker (“first AS marker 2”) of the second probe triplet detectable by the second detection channel, and

[0588] The second AS probe (“second AS probe 2”) of the second probe triplet corresponding to (e.g., containing) the first allele sequence and the second AS marker (“second AS marker 2”) of the second probe triplet detectable by the third detection channel;

[0589] The third probe triplet includes:

[0590] A third reference probe corresponding to (e.g., containing) a third reference sequence and a third reference marker detectable by a third detection channel,

[0591] The first AS probe (“first AS probe 3”) of the third probe triplet corresponding to (e.g., containing) the second allele sequence and the first AS marker (“first AS marker 3”) of the third probe triplet detectable by the third detection channel, and

[0592] The second AS probe (“second AS probe 3”) of the third probe triplet corresponding to (e.g., containing) the first allele sequence and the second AS marker (“second AS marker 3”) of the third probe triplet detectable by the first detection channel;

[0593] Each probe triplet's first and second AS probes hybridize with the same allele sequence, a different portion of the same allele sequence, or a complementary sequence thereof at the target region corresponding to the corresponding probe triplet; a reference sequence for each probe triplet is located in a reference region corresponding to the corresponding probe triplet; the first, second, and third detection channels are different from each other; the method includes hybridizing the reference probes of the three probe triplets with nucleic acid molecules or their amplicon containing the reference sequence or its complementary sequence at the reference region in multiple partitions through each of the first, second, and third detection channels; and hybridizing the first and second AS probes of the three probe triplets with nucleic acid molecules or their amplicon containing the allele sequence or its complementary sequence at the target region in multiple partitions through each of the first, second, and third detection channels; thereby providing quantification of wild-type and / or allele sequences at the three target regions in the sample. In some embodiments, the reference region of the probe triplet is adjacent to (e.g., upstream or downstream) the target region corresponding to the corresponding probe triplet. In some embodiments, the reference region of the probe triplet overlaps with the target region corresponding to the corresponding probe triplet. In some embodiments, the reference region of the probe triplet is the same as the target region corresponding to the corresponding probe triplet. In some embodiments, the detection of signals from the reference marker and the second AS marker in the probe triplet indicates the presence of an allele sequence in the target region corresponding to the probe triplet, and the detection of a signal from the reference marker but no signal from the second AS marker in the probe triplet indicates the presence of a wild-type sequence in the target region corresponding to the probe triplet. In some embodiments, the target region is a CNV-associated gene locus. In some embodiments, the target region is a gene locus associated with a rare allele.

[0594] In some embodiments, a method is provided for quantifying wild-type and / or allele sequences (e.g., rare alleles or CNVs) at multiple target regions in a sample containing nucleic acid molecules, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, wherein substantially all partitions (e.g., all partitions) each contain multiple probe sets corresponding to multiple target regions, wherein each of the multiple probe sets comprises:

[0595] Allele-specific (AS) probes comprising a first AS marker, a second AS marker, and an oligonucleotide AS sequence, wherein the oligonucleotide AS sequence is complementary to an allele sequence at a target region corresponding to a given probe set; and

[0596] A reference probe comprising a reference marker and an oligonucleotide sequence complementary to a reference sequence at a reference region corresponding to a respective probe set;

[0597] The reference marker and the first AS marker of each of the plurality of probe groups can be detected through the same detection channel;

[0598] The reference mark and second AS mark of each of the plurality of probe groups can be detected through different detection channels;

[0599] The reference markers of the multiple probe groups can be detected through different detection channels.

[0600] The second AS markers of the plurality of probe groups can be detected through different detection channels; at least one reference marker of the plurality of probe groups and at least one second AS marker of the plurality of probe groups can be detected through the same detection channel.

[0601] The method includes: detecting hybridization of reference probes from multiple probe sets with nucleic acid molecules or their amplicones containing reference sequences at reference regions in multiple partitions; and detecting hybridization of AS probes from multiple probe sets with nucleic acid molecules or their amplicones containing allele sequences at target regions in multiple partitions; thereby providing quantification of wild-type and / or allele sequences at multiple target regions in a sample. In some embodiments, the reference region of a probe set is adjacent (e.g., upstream or downstream) to a target region corresponding to the corresponding probe set. In some embodiments, the reference region of a probe set overlaps with the target region corresponding to the corresponding probe set. In some embodiments, the reference region of a probe set is the same as the target region corresponding to the corresponding probe set. In some embodiments, detecting a signal from a reference marker in a probe set but no signal from a second AS probe indicates the presence of a wild-type sequence in the target region corresponding to the probe set, and detecting a signal from both the reference marker and the second AS marker in a probe set indicates the presence of an allele sequence in the target region corresponding to the probe set. In some embodiments, the reference marker and the first AS marker of each probe set are identical to each other. In some embodiments, the reference marker set of multiple probe sets and the second AS marker set of multiple probe sets have overlapping markers. In some implementations, the reference marker group of the multiple probe groups and the second AS marker group of the multiple probe groups are arranged in a circular arrangement relative to each other.

[0602] In some embodiments, a method is provided for quantifying wild-type and / or allele sequences (e.g., rare alleles or CNVs) at R target regions in a sample containing nucleic acid molecules, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, wherein substantially all partitions (e.g., all partitions) each contain R probe pairs corresponding to the R target regions.

[0603] The first probe pair of the R probe pairs includes:

[0604] A first reference probe corresponding to (e.g., containing) a first reference sequence (w1) and a first reference marker detectable by a first detection channel (X1), and

[0605] The first AS probe corresponding to (e.g., containing) the first allele sequence (r1), the first AS marker (“first AS marker 1”) of the first probe pair detectable by the first detection channel (X1), and the second AS marker (“second AS marker 1”) of the first probe pair detectable by the second detection channel (X2);

[0606] The second probe pair among the R probe pairs includes:

[0607] A second reference probe corresponding to (e.g., including) a second reference sequence (w2) and a second reference label detectable through a second detection channel (X2), and

[0608] A second AS probe corresponding to (e.g., including) a second allele sequence (r2), a first AS label (“AS label 2”) of the second probe pair detectable through a second detection channel (X2), and a second AS label (“second AS label 2”) of the second probe pair detectable through a third detection channel (X3);

[0609] Wherein, if (e.g., when) R is strictly greater than 3, the i-th probe pair (2 < i < R) among the R probe pairs includes:

[0610] The i-th reference probe corresponding to (e.g., including) the i-th reference sequence (w i ) and the i-th reference label detectable through the i-th detection channel (X i ), and

[0611] The i-th AS probe corresponding to (e.g., including) the i-th allele sequence (r i ), the first AS label (“first AS label i”) of the i-th probe pair detectable through the i-th detection channel (X i ), and the second AS label (“second AS label i”) of the i-th probe pair detectable through the (i + 1)-th detection channel (X i+1 );

[0612] Wherein, if (e.g., when) R is strictly greater than 2, the R-th probe pair among the R probe pairs includes:

[0613] The R-th reference probe corresponding to (e.g., including) the R-th reference sequence (w R ) and the R-th reference label detectable through the R-th detection channel (X R ), and

[0614] The R-th AS probe corresponding to (e.g., including) the R-th allele sequence (r R ), the first AS label (“first AS label R”) of the R-th probe pair detectable through the R-th detection channel (X R ) and the second AS label (“second AS label R”) of the R-th probe pair detectable through the first detection channel (X1);

[0615] Wherein the reference sequence of each probe pair is located in a reference region corresponding to the corresponding probe group;

[0616] Wherein the detection channels X1 - X R are different from each other;

[0617] The method includes detecting channels X1-X R Each detection of R probe pairs involves the hybridization of a reference probe with a nucleic acid molecule or its amplicons containing the reference sequence or its complementary sequence at a reference region in multiple partitions; and the detection via channels X1-X. R Each AS probe in the detection of R probe pairs hybridizes with a nucleic acid molecule or its amplicons containing an allele sequence or its complementary sequence at a target region in multiple partitions; thereby providing quantification of wild-type and / or allele sequences at R target regions in the sample. In some embodiments, the reference region of the probe pair is adjacent (e.g., upstream or downstream) to the target region corresponding to the corresponding probe pair. In some embodiments, the reference region of the probe pair overlaps with the target region corresponding to the corresponding probe pair. In some embodiments, the reference region of the probe pair is the same as the target region corresponding to the corresponding probe pair. R can be any suitable integer of 2 or greater. In some embodiments, R is between 2 and 6. In some embodiments, the detection of a signal from a reference marker and a signal from a second AS marker in the probe pair indicates the presence of an allele sequence in the target region corresponding to the probe pair, and the detection of a signal from a reference marker but no signal from a second AS marker in the probe pair indicates the presence of a wild-type sequence in the target region corresponding to the probe pair.

[0618] In some embodiments, a method is provided for quantifying wild-type and / or allele sequences at three target regions in a sample containing nucleic acid molecules, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, wherein substantially all partitions (e.g., all partitions) each contain three probe pairs corresponding to the three target regions, wherein the three probe pairs comprise:

[0619] The first probe pair includes:

[0620] A first reference probe corresponding to (e.g., containing) a first reference sequence and a first reference marker detectable by a first detection channel, and

[0621] The first AS probe corresponding to (e.g., containing) a first allele sequence, a first AS marker (“first AS marker 1”) of the first probe pair detectable by a first detection channel, and a second AS marker (“second AS marker 1”) of the first probe pair detectable by a second detection channel;

[0622] The second probe pair includes:

[0623] A second reference probe corresponding to (e.g., containing) a second reference sequence and a second reference marker detectable by a second detection channel, and

[0624] The second AS probe corresponding to (e.g., containing) the second allele sequence, the first AS marker (“first AS marker 2”) of the second probe pair detectable by the second detection channel, and the second AS marker (“second AS marker 2”) of the second probe pair detectable by the third detection channel;

[0625] The third probe pair includes:

[0626] A third reference probe corresponding to (e.g., containing) a third reference sequence and a third reference marker detectable by a third detection channel, and

[0627] The third AS probe corresponding to (e.g., containing) the second allele sequence, the first AS marker (“first AS marker 3”) of the third probe pair detectable by the third detection channel, and the second AS marker (“second AS marker 3”) of the third probe pair detectable by the first detection channel.

[0628] The reference sequence of each probe pair is located in a reference region corresponding to the corresponding probe pair; the first detection channel, the second detection channel, and the third detection channel are different from each other; the method includes detecting hybridization of the reference probe of the three probe pairs with nucleic acid molecules or their amplicon containing the reference sequence or its complementary sequence at the reference region in multiple partitions through each of the first, second, and third detection channels; and detecting hybridization of the AS probe of the three probe pairs with nucleic acid molecules or their amplicon containing the allele sequence or its complementary sequence at the target region in multiple partitions through each of the first, second, and third detection channels; thereby providing quantification of wild-type and / or allele sequences at three target regions in the sample. In some embodiments, the reference region of the probe pair is adjacent to (e.g., upstream or downstream) the target region corresponding to the corresponding probe pair. In some embodiments, the reference region of the probe pair overlaps with the target region corresponding to the corresponding probe pair. In some embodiments, the reference region of the probe pair is the same as the target region corresponding to the corresponding probe pair. In some embodiments, detecting signals from both the reference marker and the second AS marker in the probe pair indicates the presence of an allele sequence in the target region corresponding to the probe pair, and detecting a signal from the reference marker but not from the second AS marker in the probe pair indicates the presence of a wild-type sequence in the target region corresponding to the probe pair. In some embodiments, the target region is a gene locus associated with CNV. In some embodiments, the target region is a gene locus associated with a rare allele.

[0629] Those skilled in the art will recognize the different practical ways of implementing this method, as well as the operating conditions (concentration of each probe, etc.). In particular, Figure 6 (target 3) of, for example, US9,222,128, can be used as an example. Figure 9 A, Figure 9 B Figure 10 The description of the double-labeled probe structure found in Figure 11 and subsequent figures is incorporated herein by reference in its entirety.

[0630] Figure 11A and 11C A schematic diagram of an exemplary probe set is provided, comprising a reference probe and a pair of dual-labeled AS probes. The two AS probes, each using a different detectable label (e.g., a different fluorophore), can target identical sequences, complementary sequences, or can be programmed to bind to nearby regions on a target species carrying them. AS probes from the same probe set detect the same genetic species. When the target genetic species has a unique allele sequence shorter than the AS probes, the two AS probes can hybridize to overlapping sequences, each containing a unique allele sequence. When the target genetic species has a unique allele sequence longer than the AS probes, the two AS probes can hybridize to non-overlapping sequences contained in the unique allele sequences. The two AS probes can target the same strand or different strands. Figure 11B and 11D A schematic diagram of an exemplary probe assembly including a reference probe and a dual-labeled AS probe is provided.

[0631] In some embodiments, the probe set comprises a dual-labeled AS probe and a reference probe that hybridizes to an overlapping region, which includes a 100% identical region in the nucleic acid molecule or its amplicon. The AS probe hybridizes to a mutant sequence at the target region. The reference probe hybridizes to a wild-type sequence at a reference region, which may be identical to the target region, such as... Figure 11E-11F As shown in the image. Figure 11E-11F The “WT-specific probe” corresponds to the reference probe. Figure 11E-11F The “mutation-specific probe” corresponds to the AS probe. In some implementations, multiple probe sets of 11E-11F are used in multiplex dPCR assays for determining multiple allele frequencies (MAF).

[0632] exist Figure 11A and 11B In this method, two primer pairs are used: a pair of forward and reverse primers for amplifying a target fragment containing a target region associated with the target genetic species, and a pair of forward and reverse primers for amplifying a separate reference fragment containing a reference region corresponding to the target region (e.g., a reference region with low mutation or SNP frequencies). Figure 11C-11F In this process, a single primer pair is used, which includes a forward primer and a reverse primer for amplifying the target fragment containing the target region and the reference region.

[0633] The computations of the multiplex drop-off dPCR method proposed in this paper can be directly transferred to a method using probe sets containing dual-labeled AS probes (referred to in this paper as "dual-labeled AS assay"), for example, with the following allocation:

[0634] ·m i (Mutant i in drop-off assay background) = ref i (Reference species i in dual-labeled AS assay). Labeled with a probe (i.e., a reference probe with a detectable label); and

[0635] ·w i (wildtype i in drop-off measurement background) = target i (Target species i in dual-labeled AS assay). Labeled with two probes (i.e., a first AS probe with a first detectable label and a second AS probe with a second detectable label), or labeled with an AS probe with two detectable labels.

[0636] In an exemplary multiplex assay for detecting CNVs in three genes, three sets of probes and primers are designed, one set for each target gene. Each probe set may include a triplet of a reference probe, a first AS probe, and a second AS probe. Each primer set includes a first pair of forward and reverse primers for amplifying a reference fragment containing a reference region, and a second pair of forward and reverse primers for amplifying a target fragment containing a target region. The first AS probe and the second AS probe hybridize with an allelic sequence containing a portion of the corresponding gene. For example, the first AS probe and the second AS probe may hybridize with a portion of a repetitive sequence in a CNV-associated mutant gene. The template nucleic acid may be physically or fractionated during a preparation step, either by restriction enzyme digestion, to cut each repetitive sequence from the next repetitive sequence and separate it into individual droplets. In other instances, the first AS probe and the second AS probe may hybridize with two repetitive sequences in a CNV-associated mutant gene, but not with sequences in the wild-type gene. Other AS probe pairs capable of detecting CNVs may also be used. The reference probe and AS probe may be TAQMAN. TMThe probe, reference probe, and first AS probe are labeled with the same fluorophore, while the second AS probe is labeled with a different fluorophore, which can be detected through different fluorescence detection channels. For nucleic acids containing CNVs of the target gene, the reference probe hybridizes with the amplicon of the reference fragment, and the first and second AS probes hybridize with the amplicon of the target fragment, thereby generating positive signals corresponding to the reference probe and the second AS probe in the fluorescence channel. For nucleic acids containing wild-type sequences of the target gene, only the reference probe hybridizes with the amplicon of the reference fragment, thereby generating a positive signal corresponding only to the reference probe in the fluorescence channel, while there is no signal corresponding to the second AS probe in the fluorescence channel. Signals from dPCR droplets can be plotted in three dimensions. Spatial segments corresponding to different signal clusters are determined, and the number of droplets in each spatial segment is counted. The count is used to estimate the concentration of each wild-type and CNV population.

[0637] Those skilled in the art will readily understand that the features and implementation schemes of the multiplex drop-off dPCR method described herein can be adapted to the above-described multiplex dPCR method with dual-labeled AS probes, with necessary modifications, including but not limited to the features described in the “Probe Sets” and “Digital PCR” sections above, as well as the various applications, systems, kits, and products described in Sections III and IV below.

[0638] In the context of dual-labeled AS assays, two AS probes are used, each with a different detectable label, and the mathematical uncertainty of species labeled with two probes is minimized. In such assays, it is preferred to use a pair of dual-labeled AS probes to detect the target species (e.g., CNV or rare alleles) to minimize uncertainty. In some embodiments, in the context of assays with dual-labeled probes, if the lowest mathematical uncertainty on the reference sequence is desired, a pair of dual-labeled reference probes can be used to detect the reference sequence, and a single-labeled allele-specific probe can be used to detect the target species (e.g., CNV or rare alleles) with a single probe.

[265] The species allocation described above in the paragraph is to utilize the computation presented for multiple drop-off assays and is therefore interchangeable herein, ref i Corresponding to w i target i Corresponding to m i .

[0639] Although dual-labeled probes have the lowest mathematical uncertainty, in some assays, such as MAF assays (e.g., using...), Figure 11E-11F The probe set consists of single-labeled probes for detecting mutant sequences and dual-labeled probes for detecting wild-type sequences. In this case, a single-color droplet can be clearly assigned to the mutant sequence, which may be present in much lower concentrations in the sample than the wild-type sequence.

[0640] Any of the dual-labeled AS probe assays and methods described herein can be used in conjunction with drop-off assays to simultaneously measure one or more dropoffs and calculate one or more alleles (e.g., CNVs), as described above. For example, in some embodiments, the method uses a first plurality of probe sets, each containing a reference probe and a drop-off probe, and a second plurality of probe sets, each containing a reference probe, a first AS probe, and a second AS probe. In some embodiments, the method uses a first plurality of probe sets, each containing a reference probe and a drop-off probe, and a second plurality of probe sets, each containing a reference probe and a dual-labeled AS probe. In some embodiments, the method further uses one or more independent AS probes. The label groups of the plurality of probe sets are rearranged to reduce the number of detection channels required to detect various genetic species.

[0641] In the first embodiment, a multiplex dPCR assay for detecting two CNV sequences and one drop-off sequence was designed to use three fluorescence channels, denoted by 1, 2, and 3, which can detect three corresponding fluorophores: 1, 2, and 3. Two primer sets and two AS probe pairs can be used to detect the two CNVs to quantify their respective targets, and two primer sets and two single probe sets can be used to quantify their respective references. A primer set and a probe pair are used to quantify the drop-off sequence, where one probe in the pair is specific for the drop-off sequence, and the other probe in the pair is specific for the reference sequence. Table 10 below shows an exemplary scheme for this assay. A similar setup can be used to simultaneously quantify two rare allele sequences and one drop-off sequence from two different gene loci (corresponding to CNV1 and CNV2).

[0642] Table 10

[0643]

[0644] In the second embodiment, a multiplex dPCR assay for detecting two drop-off sequences and one CNV sequence was designed to use three fluorescence channels, designated 1, 2, and 3, which can detect three corresponding fluorophores: 1, 2, and 3. Table 11 below shows an exemplary scheme for this assay. A similar setup can be used to simultaneously quantify two drop-off sequences and one rare allele sequence (corresponding to a CNV).

[0645] Table 11

[0646]

[0647] III. Application

[0648] The methods described herein and multiplex dPCR assays (e.g., multiplex drop-off dPCR assays) can be used in a variety of applications, including therapeutics, diagnostics, and genome editing. Due to their sensitivity and multiplexity capabilities, the methods described herein are particularly suitable for detecting predicted mutant biomarkers (e.g., microsatellite instability) in DNA samples containing very low concentrations of target DNA, or for detecting rare NHEJ or HDR-edited sequences at target genomic sites using site-specific genome editing reagents (e.g., CRISPR / Cas).

[0649] Diagnosis and treatment methods

[0650] In some embodiments, a method for diagnosing an individual's disease or condition is provided, wherein the disease or condition is associated with mutations at multiple target gene loci, the method comprising detecting mutated sequences at multiple target gene loci in an individual's sample using any of the methods described in the "Multiplex dPCR Methods" section. In some embodiments, the disease or condition is cancer. In some embodiments, the target gene loci are selected from the group consisting of EGFR, KRAS, NRAS, ESR1, and BRAF. In some embodiments, the target gene loci are microsatellite sequence loci. In some embodiments, the target gene loci are associated with rare alleles or CNVs.

[0651] In some embodiments, a method for prognosticating a disease or condition in an individual is provided, wherein the disease or condition is associated with mutations at multiple target gene loci, the method comprising detecting mutated sequences at multiple target gene loci in an individual's sample using any of the methods described in the "Multiplex dPCR Methods" section. In some embodiments, the disease or condition is cancer. In some embodiments, the target gene loci are selected from the group consisting of EGFR, KRAS, NRAS, ESR1, and BRAF. In some embodiments, the target gene loci are microsatellite sequence loci. In some embodiments, the target gene loci are associated with rare alleles or CNVs.

[0652] In some embodiments, a method is provided for predicting therapeutic efficacy in an individual suffering from a disease or condition, wherein said therapeutic efficacy is associated with mutations at multiple target gene sites, the method comprising detecting mutated sequences at multiple target gene sites in an individual's sample using any of the methods described in the "Multiplex dPCR Methods" section. In some embodiments, the disease or condition is cancer. In some embodiments, the target gene sites are microsatellite sequence sites. In some embodiments, the target gene sites are selected from the group consisting of EGFR, KRAS, NRAS, ESR1, and BRAF. In some embodiments, the treatment is immunotherapy, such as an immune checkpoint modulator. In some embodiments, the target gene sites are associated with rare alleles or CNVs.

[0653] In some embodiments, a method is provided for treating an individual's disease or condition, wherein the disease or condition is associated with mutations at multiple target gene loci, the method comprising detecting mutated sequences at multiple target gene loci in an individual's sample using any of the methods described in the "Multiplex dPCR Methods" section, and if mutated sequences are detected, administering an effective amount of a therapeutic agent to the individual. In some embodiments, the disease or condition is cancer. In some embodiments, the target gene loci are microsatellite sequence loci. In some embodiments, the target gene loci are selected from the group consisting of EGFR, KRAS, NRAS, ESR1, and BRAF. In some embodiments, the therapeutic agent is an immunotherapeutic agent, such as an immune checkpoint modulator. In some embodiments, the target gene loci are associated with rare alleles or CNVs.

[0654] In some embodiments, a method is provided for monitoring an individual diagnosed with a disease or condition associated with multiple target gene loci, the method comprising detecting mutational sequences at multiple target gene loci in a first sample obtained from the individual using any of the methods described in the “Multiplex dPCR Methods” section at a first time point, detecting mutational sequences at multiple target gene loci in a second sample obtained from the individual using the same method at a second time point, and comparing estimated concentrations of mutational sequences at one or more of the multiple target gene loci in the first and second samples. In some embodiments, the disease or condition is cancer. In some embodiments, the target gene loci are microsatellite sequence loci. In some embodiments, the target gene loci are selected from the group consisting of EGFR, KRAS, NRAS, ESR1, and BRAF. In some embodiments, the first time point is before the individual receives treatment. In some embodiments, the second time point is after the individual receives treatment. In some embodiments, the treatment is an immunotherapeutic agent, such as an immune checkpoint modulator. In some embodiments, the target gene loci are associated with rare alleles or CNVs.

[0655] The method described in this article can be used to detect mutations occurring at mutation hotspots in the genome. Because drop-off probes are used to detect mutations, any mutated sequence at a specific target region can be detected, thus allowing the detection of low-frequency mutations that have similar functional effects on gene products.

[0656] The method described in this article can be used to detect microsatellite instability (MSI) in samples from individuals who have cancer or are at risk of developing cancer.

[0657] In some embodiments, a method is provided for quantifying mutations at multiple microsatellite sequence sites in a sample containing nucleic acid molecules, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, and wherein substantially all partitions (e.g., all partitions) each comprise:

[0658] Multiple primer sets corresponding to multiple microsatellite sequence sites, wherein each of the multiple primer sets includes:

[0659] Forward and reverse oligonucleotide primers suitable for amplifying target fragments from nucleic acid molecules, wherein each target fragment contains microsatellite sequence sites corresponding to the primer set and adjacent reference regions upstream or downstream of the microsatellite sequence sites;

[0660] Multiple probe pairs corresponding to multiple microsatellite sequence sites, wherein each of the multiple probe pairs includes:

[0661] Drop-off probes comprise drop-off tags and oligonucleotide drop-off sequences, the oligonucleotide drop-off sequences being complementary to the wild-type sequences at microsatellite sequence sites corresponding to the respective probe pairs.

[0662] A reference probe comprises a reference marker and an oligonucleotide reference sequence, wherein the oligonucleotide reference sequence is complementary to the wild-type sequence corresponding to the reference region of the respective probe pair.

[0663] The reference marker and drop-off marker of each of the plurality of probe pairs can be detected through different detection channels; the reference markers of the plurality of probe pairs can be detected through different detection channels; the drop-off markers of the plurality of probe pairs can be detected through different detection channels; at least one reference marker and at least one drop-off marker of the plurality of probe pairs can be detected through the same detection channel.

[0664] The method includes amplifying target fragments in multiple partitions; and detecting hybridization of reference probes and drop-off probes of multiple probe pairs with the amplicon of the target fragments in the multiple partitions, thereby providing quantification of mutations at multiple microsatellite sequence sites in the sample. In some embodiments, the reference marker set and the drop-off marker set of the multiple probe pairs are cyclically arranged relative to each other. In some embodiments, the method is performed in the form of dPCR, such as DROPLET DIGITAL. TM PCR or CRYSTAL DIGITAL TM PCR. In some implementations, the nucleic acid molecule is genomic DNA, such as chromosomal DNA, genomic tumor DNA, or circulating tumor DNA.

[0665] Microsatellite instability (MSI) is a genetically hypermutagenic (mutational predisposition) condition resulting from impaired DNA mismatch repair (MMR). The presence of MSI represents phenotypic evidence of abnormal MMR function. Mutations at microsatellite loci typically involve the deletion, addition, or substitution of at least one repeat unit at the microsatellite locus. Usually, due to addition, or most commonly deletion, MSI results in changes in the length of the microsatellite locus.

[0666] Many microsatellite sequence sites are known. The ability to detect microsatellite amplification mutations at multiple microsatellite sequence sites in a single assay improves the sensitivity of MSI detection. Exemplary microsatellite sequence sites have been described in Bacher et al., 2004 Disease Markers 20, 237-250 and Hause et al., 2016 Nat Medicine Nov 22(1 1):1342-1350. In some embodiments, target microsatellite sequence sites (or microsatellite markers) are selected from microsatellites found to be highly associated with MSI-positive tumors, based on their instability frequency in colon, endometrial, rectal, and gastric adenocarcinomas. In some embodiments, target microsatellite sequence sites are located in regions of tumor that are frequently amplified (e.g., the chr8q region of the human genome). In some embodiments, the target microsatellite sequence sites are selected from the group comprising BAT-25, BAT-26, BAT-34c4, BAT-40, NR21, NR24, MONO-27, D2S123, D5S346, D17S250, ACVR2A, DEFB105A, DEFB105B, RNF43, DOCK3, GTF2IP1, LOC100093631, PIP5K1A, MSH3, TRIM43B, PPFIA1, and TDRD1. In some embodiments, the target microsatellite sequence sites are selected from the Bethesda group, which includes BAT-25, BAT-26, D2S123, D5S346, and D17S250.

[0667] Single nucleotide repeat sites have been shown to be highly sensitive to alterations in tumors with a dysfunctional DNA mismatch repair system (Parsons, 1995, ibid.), making such sites particularly useful for detecting cancers and other diseases associated with a dysfunctional DNA mismatch repair system. Therefore, single nucleotide MSI biomarkers may be preferred.

[0668] In some implementations, the microsatellite sequence sites are short microsatellite sequences (typically containing 8 to 30, 8 to 25, 8 to 20, 8 to 15, or 8 to 12 nucleotides), such as target microsatellite sequence sites exemplified in the group consisting of D2S123, D5S346, D17S250, ACVR2A, DEFB105A, DEFB105B, RNF43, DOCK3, GTF2IP1, LOC100093631, PIP5K1A, MSH3, TRIM43B, PFIA1, and TDRD1.

[0669] MSI detection methods can be routinely performed on biological samples (or nucleic acid samples derived from biological samples) such as blood, plasma, urine, or fecal samples. The mutational allele frequencies determined using any of the methods described herein can be compared with control mutational allele frequencies obtained from control DNA samples. Control DNA samples can be wild-type samples or samples derived from cell lines of subjects diagnosed with MSI-positive tumors or diseases associated with mutations in DNA mismatch repair at previous time points during the course of disease and / or during treatment.

[0670] In some implementations, cancers (or tumors) associated with MSI are also referred to as MSI-positive cancers (or MSI-positive tumors) and involve cancers (or tumors) in which the genomic tumor DNA has at least one mutation at a microsatellite sequence site. Therefore, MSI is associated with a variety of cancers, such as, but not limited to, colorectal cancer, gastric cancer, endometrial cancer, ovarian cancer, urinary tract cancer, brain cancer, and breast cancer. MSI is the most common outcome of colon cancer. Furthermore, MSI is associated with primary mismatch repair deficiency syndrome (CMMRD syndrome) or Lynch syndrome. Therefore, detecting mutated sequences at one or more microsatellite sequence sites according to the methods described herein can be used to diagnose cancers associated with impaired DNA mismatch repair, such as MSI-positive cancers, or to diagnose an individual's familial susceptibility to cancer.

[0671] The MSI phenotype (i.e., positive or negative) of cancer is of great significance for cancer prognosis and rational treatment planning (Boland and Goel, Gastroenterology 2010). Therefore, even in cancers with a low prevalence of MSI positivity, identifying whether a patient has an MSI-positive or MSI-negative tumor remains highly relevant. The method of this invention can be used for the prognosis of various cancers. Identification of positive MSI cancers is generally associated with better prognosis.

[0672] This application also relates to a method for predicting treatment efficacy. For example, reports have shown that patients with MMR-deficient colorectal cancer respond better to immunotherapy via PD-1 immune checkpoint blockade and exhibit improved progression-free survival. Therefore, identifying patients with MSI-associated cancer (i.e., MSI-positive cancer or tumor) is of high clinical relevance for selecting an appropriate treatment strategy. In some embodiments, the treatment is immunotherapy. Immunotherapy includes, but is not limited to, immune checkpoint modulators (i.e., inhibitors and / or agonists), monoclonal antibodies, and cancer vaccines. In some embodiments, the treatment includes administration of immune checkpoint modulators such as anti-PD-1 and / or anti-PDL-1 inhibitors. In some embodiments, immunotherapy is administered to the subject if a mutated sequence is detected at one or more microsatellite sequence sites in a nucleic acid molecule from a sample.

[0673] The method for detecting microsatellite instability can also be used to monitor individuals diagnosed with tumors associated with impaired DNA mismatch repair. In some embodiments, the monitoring is performed during treatment. The method can also be used to monitor cancer recurrence in subjects with tumors associated with impaired DNA mismatch repair. In individuals with tumors associated with impaired DNA mismatch repair, the detection of microsatellite instability in circulating tumor DNA may indicate recurrence.

[0674] Detection of genome editing

[0675] The method described in this paper can be used to detect unmodified (i.e., wild-type) and mutated (e.g., NHEJ or HDR-edited) sequences at multiple target genomic regions in cells that have undergone site-specific genome editing.

[0676] In some embodiments, a method is provided for quantifying unmodified and / or NHEJ-edited sequences of multiple target regions in a sample containing nucleic acid molecules from cells, wherein the cells have been contacted with a site-specific genome editing reagent, wherein the site-specific genome editing reagent is configured to cleave target sites in multiple target regions, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, and wherein substantially all partitions (e.g., all partitions) each comprise:

[0677] The NHEJ drop-off probe comprises an NHEJ drop-off tag and an oligonucleotide drop-off sequence, wherein the oligonucleotide drop-off sequence is complementary to the wild-type sequence corresponding to the target region of the corresponding probe set.

[0678] A reference probe comprises a reference marker and an oligonucleotide reference sequence, wherein the oligonucleotide reference sequence is complementary to a wild-type sequence in an adjacent reference region upstream or downstream of the target region corresponding to the respective probe set.

[0679] The NHEJ drop-off marker and reference marker of each probe group in the plurality of probe groups can be detected through different detection channels; the NHEJ drop-off markers of the plurality of probe groups can be detected through different detection channels from each other; the reference markers of the plurality of probe groups can be detected through different detection channels from each other; at least one reference marker of the plurality of probe pairs and at least one NHEJ drop-off marker of the plurality of probe pairs can be detected through the same detection channel.

[0680] The method includes detecting hybridization of reference probes from multiple probe sets with nucleic acid molecules or their amplicons containing wild-type sequences at reference regions in multiple partitions; and detecting hybridization of NHEJ drop-off probes from multiple probe sets with nucleic acid molecules or their amplicons containing wild-type sequences at target regions in multiple partitions; thereby providing quantification of unmodified and / or NHEJ-edited sequences at multiple target regions in a sample. In some embodiments, the reference label sets of multiple probe pairs and the NHEJ drop-off label sets of multiple probe pairs are cyclically arranged relative to each other.

[0681] In some embodiments, a method is provided for quantifying unmodified, homology-directed repair (HDR) edited and / or non-homologous end joining (NHEJ) edited sequences at multiple target regions in a sample containing nucleic acid molecules from cells, wherein the cells have been contacted with a site-specific genome editing reagent and an HDR template nucleic acid containing an HDR substitution sequence, wherein the site-specific genome editing reagent is configured to cleave target sites in multiple target regions, wherein the nucleic acid molecules are distributed in multiple partitions of the sample, and wherein substantially all partitions (e.g., all partitions) each contain: multiple probe sets corresponding to multiple target regions, wherein each of the multiple probe sets comprises:

[0682] An HDR probe comprising an HDR marker and an oligonucleotide HDR sequence, the oligonucleotide HDR sequence being complementary to an HDR substitution sequence inserted at a target region corresponding to a respective probe set.

[0683] An NHEJ drop-off probe comprises an NHEJ drop-off tag and an oligonucleotide drop-off sequence, wherein the oligonucleotide drop-off sequence is complementary to a wild-type sequence corresponding to a target region of the corresponding probe set, and wherein the drop-off sequence does not hybridize with an NHEJ-edited mutant sequence at the target region corresponding to the corresponding probe set.

[0684] A reference probe comprises a reference marker and an oligonucleotide reference sequence, wherein the oligonucleotide reference sequence is complementary to a wild-type sequence located upstream or downstream of the target region corresponding to the respective probe set.

[0685] In the plurality of probe groups, the HDR marker, NHEJ drop-off marker, and reference marker of each probe group can be detected through different detection channels; the HDR markers of the plurality of probe groups can be detected through different detection channels from each other; the NHEJ drop-off markers of the plurality of probe groups can be detected through different detection channels from each other; the reference markers of the plurality of probe groups can be detected through different detection channels from each other; at least one reference marker of the plurality of probe groups and at least one NHEJ drop-off marker of the plurality of probe groups can be detected through the same detection channel, and / or at least one reference marker of the plurality of probe groups and / or at least one HDR marker of the plurality of probe groups can be detected through the same detection channel;

[0686] The method includes detecting hybridization of reference probes from multiple probe sets with nucleic acid molecules or their amplicon containing wild-type sequences at reference regions in multiple partitions; detecting hybridization of HDR probes from multiple probe sets with nucleic acid molecules or their amplicon containing HDR substitution sequences at target regions in multiple partitions; and detecting hybridization of NHEJ drop-off probes from multiple probe sets with nucleic acid molecules or their amplicon containing wild-type sequences at target regions in multiple partitions; thereby providing quantification of unmodified, HDR-edited, and / or NHEJ-edited sequences at multiple target regions in a sample. In some embodiments, the reference label set, the HDR label set, and the NHEJ drop-off label set of the multiple probe sets are arranged relative to each other (e.g., as shown in the diagram). Figure 6B (As shown).

[0687] In some embodiments, a method is provided for quantifying unmodified, homology-directed repair (HDR)-edited, and / or non-homologous end joining (NHEJ)-edited sequences at (R-1) target regions in a sample comprising nucleic acid molecules from cells, wherein the cells have been contacted with a site-specific genome editing reagent and an HDR template nucleic acid comprising an HDR replacement sequence, wherein the site-specific genome editing reagent is configured to cleave a target site in a plurality of target regions, wherein the nucleic acid molecules are distributed in a plurality of partitions of the sample, and wherein substantially all partitions (e.g., all partitions) each comprise (R-1) probe triplets corresponding to the (R-1) target regions,

[0688] wherein a first probe triplet of the plurality of probe triplets comprises:

[0689] a first reference probe comprising a first reference sequence (m1) and a first reference label detectable by a first detection channel (X1);

[0690] a first NHEJ drop-off probe comprising a first NHEJ drop-off sequence (r1) and a first NHEJ drop-off label detectable by a second detection channel (X2); and

[0691] a first HDR probe comprising a first HDR sequence (w1) and a first HDR label detectable by a third channel (X3);

[0692] wherein a second probe triplet of the plurality of probe triplets comprises:

[0693] a second reference probe comprising a second reference sequence (m2) and a second reference label detectable by the second detection channel (X2);

[0694] a second NHEJ drop-off probe comprising a second drop-off sequence (r2) and a second NHEJ drop-off label detectable by a third detection channel (X3); and

[0695] a second HDR probe comprising a second HDR sequence (w2) and a second HDR label detectable by a fourth detection channel (X4);

[0696] wherein, if (e.g., when) R is strictly greater than 3, the i-th probe triplet (2 < i < R-1) of the plurality of probe triplets comprises:

[0697] an i-th reference probe comprising an i-th reference sequence (m i ) and an i-th reference label detectable by an i-th detection channel (X i );

[0698] The i-th NHEJ drop-off probe contains the i-th drop-off sequence (r i ) and can be detected through the (i+1)th detection channel (X) i+1 The i-th NHEJ drop-off marker detected; and

[0699] The i-th HDR probe contains the i-th HDR sequence (w i ) and can be detected through the (i+2)th detection channel (X) i+2 The i-th HDR marker detected;

[0700] Wherein, if (for example, when) R is strictly greater than 3, then the (R-1)th probe triplet in the plurality of probe triplets includes:

[0701] The (R-1)th reference probe contains the (R-1)th reference sequence (m R-1 ) and can be detected through the Rth detection channel (X) R The R-th reference marker detected;

[0702] The (R-1)th NHEJ drop-off probe contains the (R-1)th drop-off sequence (r R-1 ) and can be detected through the (R-1)th detection channel (X) R-1 The (R-1)th NHEJ drop-off marker detected; and

[0703] The (R-1)th HDR probe contains the (R-1)th HDR sequence (w R-1 ) and the (R-1)th HDR marker that can be detected by the first detection channel (X1);

[0704] The NHEJ drop-off sequence of each probe triplet is complementary to the wild-type sequence at the target region corresponding to the corresponding probe triplet; the reference sequence of each probe triplet is complementary to the wild-type sequence at the adjacent reference region upstream or downstream of the target region corresponding to the corresponding probe triplet; the HDR sequence of each probe triplet is complementary to the HDR permutation sequence at the target region corresponding to the corresponding probe pair; and the detection channels X1-X R They are different;

[0705] The method includes: detecting channels X1-X R Each of the multiple probe triads in the detection array hybridizes with a nucleic acid molecule or its amplicon containing a wild-type sequence at a reference region in the multiple partitions; through detection channels X1-X REach of the multiple probes in the detection triad hybridizes with a nucleic acid molecule or its amplicons containing an HDR substitution sequence at a target region in multiple partitions; and through detection channels X1-X R Each of the NHEJ drop-off probes in the detection of the multiple probe triad hybridizes with a nucleic acid molecule or its amplicon containing a wild-type sequence at a target region in multiple partitions; thereby providing quantification of unmodified, HDR-edited and / or NHEJ-edited sequences at multiple target regions in the sample.

[0706] In some implementations, the methods described herein are used in dPCR, such as CRYSTAL DIGITAL. TM The assay is performed using PCR. In some embodiments, the site-specific genome editing reagent comprises a Cas nuclease, TALEN, or a zinc finger nuclease. In some embodiments, the method further includes contacting the cells with the site-specific genome editing reagent.

[0707] In some embodiments, a method is provided for identifying optimal conditions for genome editing in cells, comprising: a) performing site-specific genome editing on multiple cells under a first set of conditions to provide a first sample containing nucleic acids from the genome-edited cells; b) performing site-specific genome editing on multiple cells under a second set of conditions to provide a second sample containing nucleic acids from the genome-edited cells; c) quantifying NHEJ-edited sequences and / or HDR-edited sequences at multiple target genomic regions in the first and second samples using any of the methods described in the “Detection of Genome Editing” section to determine the genome editing efficiency of the first and second sets of conditions; and d) comparing the genome editing efficiency of the first and second sets of conditions to identify an optimal set of conditions that provides higher genome editing efficiency. In some embodiments, different sets of conditions include different site-specific genome editing reagents (e.g., different Cas and / or different gRNAs), different target genomic sites, different delivery methods, and / or different concentrations of site-specific genome editing reagents. In some embodiments, editing is performed under multiple conditions, such as a third, fourth, fifth, and sixth. In some cases, higher efficiency of HDR editing is identified as an optimal condition for genome editing. In some cases, higher NHEJ editing efficiency was identified as an optimal condition for genome editing. In other cases, a higher ratio of NHEJ editing efficiency to HDR editing efficiency was identified as an optimal condition for genome editing.

[0708] Figure 6AAn exemplary method is described for detecting unmodified, NHEJ-edited, and HDR-edited sequences at three target genomic sites in cells that have undergone site-specific genome editing using CRISPR / Cas. Reference markers in the CRISPR 1, CRISPR 2, and CRISPR 3 probe triads have fluorophores 1, 2, and 4, respectively, which hybridize with all amplicones in the dPCR assay. NHEJ drop-off probes in the CRISPR 1, CRISPR 2, and CRISPR 3 probe triads have fluorophores 2, 3, and 3, respectively, and do not hybridize with the NHEJ-edited sequences at their respective target genomic sites. HDR probes in the CRISPR 1, CRISPR 2, and CRISPR 3 probe triads have fluorophores 3, 4, and 1, respectively, and hybridize with the HDR substitution sequences at their respective target genomic sites. Each genetic species has a unique fluorescence signal signature. A total of four detection channels are required to detect all nine genetic species in the sample. For an exemplary method for detecting unmodified (including uncut sequences), NHEJ-edited, and HDR-edited sequences at (R-1) target genomic sites in cells undergoing site-specific genome editing, see also “Implementation Scheme Using (R-1) Probe Triads”. Figure 6B The arrangement of four, six, or ten markers in three-probe triplet sets, five-probe triplet sets, or nine-probe triplet sets is shown respectively.

[0709] The method can be used to determine the efficacy of genome editing at multiple genomic sites in a sample, to identify optimal conditions for genome editing, or to guide the enrichment of cellular populations of genome editing products (e.g., through subselection). For example, genome editing conditions can be optimized to reduce or increase the type or number of NHEJ mutations compared to HDR mutations in multiple target genomic regions. As another example, genome editing conditions can be optimized to improve editing efficiency, thereby allowing the use of low concentrations or low-activity genome editing reagents without unduly reducing the amount of editing achieved. Using low concentrations or low-activity genome editing reagents may help reduce off-target editing events.

[0710] In some embodiments, the sample is a cell sample, a genome sample extracted from a cell sample, or a fragment thereof. The cells or genome extracted from cells may be contacted with a site-specific genome editing agent under conditions suitable for genome editing of multiple target genomic regions. In some cases, the cells or genome are contacted with multiple HDR-substituted nucleic acids to introduce a predetermined HDR mutation into the genome. The genome editing agent may contain one or more nucleases that introduce double-strand breaks into DNA.

[0711] Site-specific genome editing reagents are known in the art. Typically, such reagents target genomic regions and induce double-strand cuts into the DNA within the target region. Repair of the cut can occur via two alternative pathways. In non-homologous end joining (NHEJ), the cut ends of the DNA strand are directly joined without the need for homologous template nucleic acids. NHEJ can result in the addition, deletion, substitution, or a combination thereof of one or more nucleotides at the repair site. In homologous directed repair, the cut ends of the DNA strand are repaired by the polymerization of homologous template nucleic acids. Thus, the original sequence is replaced by the template sequence. The homologous template nucleic acid can be provided by homologous sequences elsewhere in the genome (sister chromatids, homologous chromosomes, or repetitive regions on the same or different chromosomes). Alternatively, exogenous template nucleic acids can be introduced to obtain specific HDR mutations.

[0712] As another example, genome editing reagents containing specific heterodimeric nucleases can be used to reduce off-target mutations. Such genome editing reagents can be programmed to produce double-strand breaks only when a specific heterodimeric nuclease is formed in the target genome region by specifically recruiting each monomeric component site to an adjacent target half-site. Exemplary specific heterodimeric nucleases include, but are not limited to, those described in U.S. Patent Application Serial No. 13 / 812,857. Targeting functionality can be provided by a nuclease-deficient Cas9 (dCas9) and a suitable guide RNA, a pair of TALENs, or any other nucleic acid sequence-specific targeting method.

[0713] NHEJ drop-off probes are designed based on the type of site-specific genome editing reagent used. For example, if the genome editing reagent is a Cas9 nuclease and guide RNA, the cleavage site is typically located 3–5 base pairs upstream of the adjacent motif (PAM) in the prespacer region. The PAM is usually composed of the NGG sequence, although some other PAM sequences, such as NGA or NAG, can be used. Therefore, the cleavage site can be, for example, [5'-20nt target-NGG-3'] or [5'-CCN-20nt target-3']. When the target site is 5'-20nt target-NGG, the predicted cleavage site is approximately 3–5 base pairs upstream of the 5' end of the NGG PAM. In this case, the NHEJ drop-off probe can be designed to hybridize with a subregion containing this predicted cleavage site.

[0714] As another example, a genome editing reagent can be a pair of guide RNAs that target a site adjacent to a PAM sequence on the opposite strand of the target genome region. Each guide RNA is complexed with a Cas9 nuclease that is defective or dead (dCas9), which is fused with a monomer of a specific heterodimer of an IIS-type restriction nuclease (e.g., FokI). In this case, the cleavage site is typically located 12 to 21 base pairs between adjacent PAM sequences on the opposite strand of the target genome region. Therefore, an NHEJ drop-off probe can be designed to hybridize with a subregion containing a predicted cleavage site of 12 to 21 base pairs between adjacent PAM sequences. Similar rules can be used to design NHEJ drop-off probes for other genome editing reagents.

[0715] In some embodiments, the NHEJ drop-off probe is sensitive to both HDR and NHEJ mutations. For example, the genome editing agent may include an exogenous HDR template nucleic acid. This template nucleic acid can be used as a template to repair regions containing or located within double-strand breaks introduced by the genome editing agent. Thus, any mutations present in the HDR template nucleic acid associated with the wild-type genome will be introduced. When the HDR site is near or located at the target cleavage site, the NHEJ drop-off probe can hybridize with both potential NHEJ editing sites and potential HDR editing sites. In this case, the NHEJ drop-off probe can detect both HDR and NHEJ mutations by failing to hybridize with target genomic regions containing such mutations. In some embodiments, NHEJ and HDR mutations are distinguished by including an HDR probe in each probe set. If the NHEJ drop-off probe detects a mutation (HDR or NHEJ) while the HDR probe does not detect it, the mutation is classified as NHEJ. Conversely, if the NHEJ probe detects a mutation while the HDR probe also detects it, the mutation is classified as HDR.

[0716] In some embodiments, the site-specific genome editing agent induces double-strand breaks in intracellular DNA. In some embodiments, the site-specific genome editing agent comprises a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas protein, a Cre recombinase, a Hin recombinase, or a Flp recombinase. In some embodiments, the site-specific genome editing agent comprises a fusion protein that binds a homing endonuclease to the modular DNA-binding domain (megaTAL) of a TALEN. For example, megaTAL can be delivered as a protein, or mRNA encoding a megaTAL protein can be delivered to the cell. In some embodiments, the site-specific genome editing agent comprises one or more RNA molecules, such as sgRNA, crRNA, or crRNA and tracrRNA. In some embodiments, the site-specific genome editing agent is a ribonucleoprotein (RNP), and the RNP comprises a Cas protein and sgRNA or crRNA and tracrRNA.

[0717] Non-limiting descriptions relating to gene editing using the CRISPR-Cas system (including HDR repair templates) are discussed in Ran et al. (2013) Nat Protoc. 2013 Nov; 8(11):2281-2308, the entire contents of which are incorporated herein by reference. Implementations involving repair templates are not limited to those involving the CRISPR-Cas system. Various aspects of the CRISPR-Cas system are known in the art. Non-limiting aspects of the system are described, for example, in U.S. Patent No. 9,023,649, issued May 5, 2015; U.S. Patent No. 9,074,199, issued July 7, 2015; U.S. Patent No. 8,697,359, issued April 15, 2014; U.S. Patent No. 8,932,814, issued January 13, 2015; PCT International Patent Application Publication No. WO 2015 / 071474, published August 27, 2015; Cho et al., (2013) Nature Biotechnology Vol. 31 No. 3pp 230-232 (including supplemental information); and Jinek et al., (2012) Science Vol. 337 No. 6096pp 816-821, the entire contents of which are incorporated herein by reference.

[0718] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csxl2), Cas1O, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, their homologs or modified forms thereof. These enzymes are known; for example, the amino acid sequence of the *Streptococcus pyogenes* Cas9 protein can be found in the SwissProt database under accession number Q99ZW2 and in the NCBI database under accession number Q99ZW2.1. UniProt database accession numbers A0A0G4DEU5 and CDJ55032 provide another example of the Cas9 protein amino acid sequence. Another non-limiting example is the *Streptococcus thermophilus* Cas9 protein, whose amino acid sequence can be found in the UniProt database under accession number Q03JI6.1. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments, the CRISPR enzyme is Cas9, and may be Cas9 derived from *Streptococcus pyogenes* or *Streptococcus pneumoniae*. In various embodiments, the CRISPR enzyme directs the cleavage of the two strands at the target sequence location.

[0719] Typically, the guide sequence is any polynucleotide sequence that is sufficiently complementary to the target polynucleotide sequence to hybridize with the target sequence and guide the CRISPR complex to bind sequence-specifically to the target sequence. In some embodiments, when optimal alignment is performed using a suitable alignment algorithm, the complementarity between the guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. In some embodiments, the complementarity is 100%.

[0720] Gene editing nucleases, including ZFN, have been described in Bhakta, M. et al., Genome Research 23:530-538; 2013 and Beerli, R. et al., Proc. Natl. Acad. Sci v.95pp 14628-14633; 1998. TAL has been described in Cermak, T. et al., Nucleic Acids Research 2011, v.39, no.12; Miller, J. et al., Nature Biotechnology vol.29 no.2; 2011; Christian, M. et al., Genetics 186:757-761; 2010; Deng, D. et al., Science 2012: v.335p.720 and Boch, J. et al., Science 2009: v.326p.1509. The full contents of each of these references are incorporated herein by reference. In addition, Cre has been described in Chevalier, B. et al., Nucleic Acids Research 2001, v.29 no.18, the entire contents of which are incorporated herein by reference. MegaTal has been described in Sather, B. et al., Sci Transl Med 7(307)2015, Ibarra, G. et al., Molecular Therapy-Nucleic Acids (2016) 5, e352, Osborn, M. et al., Molecular Therapy v.24 no.3, 570-581 (2016); Wang, Y. et al., Nucleic Acid Research 2014; v.42, 6463-6475; and Gaj, T. et al., Cold Spring Harbor Perspectives in Biology 2015, each of which is incorporated herein by reference.

[0721] In some embodiments, the cells are primary cells, cell lines, or immortalized cells. For example, cells may include mesenchymal stem cells, lung cells, neurons, fibroblasts, human umbilical vein (HUVEC) cells and human embryonic kidney (HEK) cells, primary or immortalized hematopoietic stem cells (HSCs), T cells, natural killer (NK) cells, cytokine-induced killer (CIK) cells, human cord blood CD34+ cells, and B cells. Non-limiting examples of T cells may include CD8+ or CD4+ T cells. In some aspects, a CD8+ subset of CD3+ T cells is used. CD8+ T cells can be purified from a PBMC population by positive isolation using anti-CD8 beads. In some embodiments, primary NK cells are isolated from PBMCs, or an NK cell line, such as NK92, may be used. Cell types also include cells previously modified to enhance their therapeutic efficacy, such as T cells, NK cells, and MSCs. For example: T cells or NK cells expressing chimeric antigen receptors (CAR T cells and CAR NK cells, respectively); T cells expressing modified T cell receptors (TCRs); or engineered MSCs.

[0722] IV. Systems, reagent kits, and products

[0723] Apparatus, devices, systems, compositions, kits, and articles thereof are also provided for use with any of the quantitative methods, multiplex dPCR assays (e.g., multiplex drop-off dPCR assays), therapeutic methods, and diagnostic methods described herein.

[0724] Figure 10 The illustration shows an example of a computing device according to one implementation scheme. Device 1000 can be a host connected to a network. Device 1000 can be a client computer or a server. Figure 10 As shown, device 1000 can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server, or handheld computing device (portable electronic device), such as a telephone or tablet. The device may include one or more of, for example, a processor 1010, an input device 1020, an output device 1030, a memory 1040, and a communication device 1060. Input device 1020 and output device 1030 may generally correspond to components of device 1000 described above and may be connected to or integrated with a computer.

[0725] Input device 1020 can be any suitable device that provides input, such as a touchscreen, keyboard or keypad, mouse, or voice recognition device. Output device 1030 can be any suitable device that provides output, such as a touchscreen, haptic device, or speaker.

[0726] Memory 1040 can be any suitable device providing storage, such as electrical, magnetic, or optical memory, including RAM, cache, hard disk drive, or removable storage disk. Communication device 1060 can include any suitable device capable of sending and receiving signals over a network, such as a network interface chip or device. Computer components can be connected in any suitable manner, such as via a physical bus or wireless connection.

[0727] The software 1050, which can be stored in memory 1040 and executed by processor 1010, may include, for example, programs embodying the functions of this disclosure (e.g., embodied in the device described above).

[0728] Software 1050 may also be stored and / or transmitted in any non-transitory computer-readable storage medium for use by or in connection with an instruction execution system, apparatus, or device, such as those described above, which may retrieve and execute instructions relating to the software from and execute such instructions. In the context of this disclosure, a computer-readable storage medium may be any medium, such as memory 1040, which may contain or store programs for use by or in connection with an instruction execution system, apparatus, or device.

[0729] The software 1050 can also be propagated in any transmission medium for use by or in conjunction with an instruction execution system, apparatus, or device, such as those described above, which can retrieve and execute instructions related to the software from and execute such instructions. In the context of this disclosure, the transmission medium can be any medium capable of communicating, propagating, or transmitting a program for use by or in conjunction with an instruction execution system, apparatus, or device. Transmission readable media can include, but are not limited to, electrical, magnetic, optical, electromagnetic, or infrared wired or wireless transmission media.

[0730] Device 1000 can be connected to a network, which can be any suitable type of interconnected communication system. The network can execute any suitable communication protocol and can be protected by any suitable security protocol. The network can include any suitable network link capable of transmitting and receiving network signals, such as a wireless network connection, T1 or T3 lines, cable networks, DSL, or telephone lines.

[0731] Device 1000 can run any operating system suitable for operation on a network. Software 1050 can be written in any suitable programming language such as C, C++, Java, or Python. In various implementations, application software embodying the functions of this disclosure can be deployed in different configurations, such as in a client / server setup or via a web browser as a web-based application or web service.

[0732] The following exemplary embodiments and examples are intended purely as examples of the invention and should not be construed as limiting the invention in any way. The following exemplary embodiments, examples, and detailed descriptions are provided by way of illustration rather than limitation.

[0733] Exemplary Implementation

[0734] The present invention provides the following implementation schemes:

[0735] 1. A method for quantifying wild-type and / or mutant sequences at multiple target regions in a sample containing nucleic acid molecules.

[0736] The nucleic acid molecules are distributed in multiple regions of the sample, and

[0737] Each of these partitions essentially includes:

[0738] Multiple probe groups corresponding to the plurality of target regions, wherein each of the plurality of probe groups includes:

[0739] A drop-off probe comprising a drop-off tag and an oligonucleotide drop-off sequence, wherein the oligonucleotide drop-off sequence is complementary to a wild-type sequence at a target region corresponding to a respective probe set;

[0740] A reference probe comprising a reference marker and an oligonucleotide reference sequence, the oligonucleotide reference sequence being complementary to a wild-type sequence located upstream or downstream of a target region corresponding to a particular probe set;

[0741] The reference marker and drop-off marker of each of the plurality of probe groups can be detected through different detection channels;

[0742] The reference markers of the multiple probe groups can be detected through different detection channels.

[0743] The drop-off markers of the multiple probe groups can be detected through different detection channels.

[0744] At least one reference marker of the plurality of probe groups and at least one drop-off marker of the plurality of probe groups can be detected through the same detection channel;

[0745] The method includes:

[0746] The hybridization of the reference probes of the plurality of probe sets with nucleic acid molecules or their amplicons containing wild-type sequences at reference regions in the plurality of partitions; and

[0747] Detecting the hybridization of the drop-off probes of the plurality of probe sets with nucleic acid molecules or their amplicons containing wild-type sequences at target regions in the plurality of partitions;

[0748] Thereby providing quantification of wild-type and / or mutant sequences at multiple target regions in the sample.

[0749] 2. The method according to embodiment 1, wherein each of the plurality of probe sets is a probe pair, and wherein the total number of detection channels is less than twice the total number of probe sets.

[0750] 3. The method according to embodiment 2, wherein the total number of detection channels is equal to the total number of probe sets.

[0751] 4. The method according to embodiment 3,

[0752] wherein the plurality of probe sets are R probe pairs,

[0753] wherein the first probe pair among the R probe pairs comprises:

[0754] A first reference probe, which comprises a first reference sequence (r1) and a first reference label detectable through a first detection channel (X1), and

[0755] A first drop-off probe, which comprises a first drop-off sequence (w1) and a first drop-off label detectable through a second detection channel (X2);

[0756] wherein the second probe pair among the R probe pairs comprises:

[0757] A second reference probe, which comprises a second reference sequence (r2) and a second reference label detectable through the second detection channel (X2), and

[0758] A second drop-off probe, which comprises a second drop-off sequence (w2) and a second drop-off label detectable through a third detection channel (X3);

[0759] wherein, if (e.g., when) R is strictly greater than 3, the i-th probe pair (2 < i < R) among the R probe pairs comprises:

[0760] An i-th reference probe, which comprises an i-th reference sequence (r i ) and an i-th reference label detectable through the i-th detection channel (X i ), and

[0761] An i-th drop-off probe, which comprises an i-th drop-off sequence (w i) and can be detected through the (i+1)th detection channel (X) i+1 The i-th drop-off marker detected;

[0762] Wherein, if (for example, when) R is strictly greater than 2, then the Rth probe pair of the R probe pairs includes:

[0763] The R-th reference probe contains the R-th reference sequence (r R ) and with the Rth detection channel (X R The related R-th reference marker, and

[0764] The R-th drop-off probe contains the R-th drop-off sequence (w R ) and the Rth drop-off tag that can be detected by the first detection channel (X1);

[0765] The method includes:

[0766] Through detection channel X1-X R Each of the R probe pairs in the assay detects hybridization between a reference probe and a nucleic acid molecule or its amplicons containing a wild-type sequence at a reference region in the plurality of partitions; and

[0767] Through detection channel X1-X R Each of the R probe pairs is detected by hybridization of the drop-off probe with a nucleic acid molecule or its amplicon containing a wild-type sequence at the target region in the plurality of partitions.

[0768] 5. The method according to embodiment 4 further includes:

[0769] Obtain the first count of one or more partitions, each of which generates a positive signal through the i-th detection channel and passes through detection channels X1-X. R Any other detection channel in the process generates a negative signal;

[0770] Obtain a second count for one or more partitions, each of which passes through all detection channels X1-X. R Generate negative signals; and

[0771] Calculate the mutation probability of a given partition containing a mutated sequence in the target region corresponding to the i-th probe pair. The mutation probability is based on the ratio between the first count and the sum of the first and second counts.

[0772] 6. The method according to embodiment 5 further includes determining the estimated concentration of the mutation sequence at the target region corresponding to the i-th probe pair in the sample based on the mutation probability.

[0773] 7. The method according to embodiment 6, wherein the estimated concentration of the mutant sequence at the target region corresponding to the i-th probe pair in the sample is determined according to the following:

[0774]

[0775] in This represents the estimated concentration of the mutant sequence in the sample corresponding to the target region of the i-th probe pair.

[0776] Where v represents the volume of the partition, and

[0777] in This represents the mutation probability that a given partition contains a mutated sequence at the target region corresponding to the i-th probe pair in the sample.

[0778] 8. The method according to any one of embodiments 4-7 further includes determining a confidence interval and / or uncertainty measure related to the estimated concentration of the mutation sequence at the target region corresponding to the i-th probe pair in the sample.

[0779] 9. The method according to any one of embodiments 4-8 further includes calculating the wild-type probability that a given partition contains a wild-type sequence at a target region corresponding to the i-th probe pair, wherein the wild-type probability is based on the mutation probability corresponding to the i-th probe pair and the mutation probability corresponding to the (i+1)-th probe pair, wherein the (i+1)-th probe pair refers to the first probe pair if (e.g., when) i = R.

[0780] 10. The method according to embodiment 9, wherein the wild-type probability is calculated according to the following:

[0781]

[0782] in This represents the wild-type probability that a given partition contains a wild-type sequence at the target region corresponding to the i-th probe pair.

[0783] Where n i,(i+1) This represents the count of one or more partitions, each represented by X. i The detection channel generates a positive signal, which is transmitted through X. i+1 The detection channel generates a positive signal and passes through detection channel X1-X. R Any other detection channel in the process generates a negative signal;

[0784] Where, if (for example, when) i = R, then n i,(i+1) It refers to n R,1 ;

[0785] Where n0 represents the count of one or more partitions, each of which passes through all detection channels X1-X. R Generates a negative signal;

[0786] Where n i This represents the count of one or more partitions, each of which generates a positive signal through the Xi detection channel and through the detection channels X1-X. R Any other detection channel in the process generates a negative signal;

[0787] Where n i+1 This represents the count of one or more partitions, each represented by X. i+1 The detection channel generates a positive signal and passes through detection channel X1-X. R Any other detection channel in the process generates a negative signal;

[0788] Where, if (for example, when) i = R, then n i+1 It refers to n1.

[0789] in This represents the mutation probability that a given partition contains a mutated sequence at the target region corresponding to the i-th probe pair.

[0790] in This represents the mutation probability that a given partition contains a mutated sequence at the target region corresponding to the (i+1)th probe pair, and

[0791] Wherein, if (for example, when) i = R, then It means

[0792] 11. The method according to embodiment 9 or 10, further comprising determining, based on the wild-type probability, an estimated concentration of the wild-type sequence at the target region corresponding to the i-th probe pair in the sample.

[0793] 12. The method according to embodiment 11, wherein the estimated concentration of the wild-type sequence in the sample corresponding to the target region of the i-th probe pair is determined according to the following:

[0794]

[0795] in This represents the estimated concentration of the wild-type sequence in the target region corresponding to the i-th probe pair in the sample.

[0796] Where v represents the volume of the partition, and

[0797] in This represents the wild-type probability that a given partition contains a wild-type sequence at the target region corresponding to the i-th probe pair in the sample.

[0798] 13. The method according to any one of embodiments 9-12 further includes determining a confidence interval and / or uncertainty measure related to the estimated concentration of the wild-type sequence at the target region corresponding to the i-th probe pair in the sample.

[0799] 14. The method according to any one of embodiments 4-13, further comprising, based on the respective detection channels X1-X R The concentration of nucleic acid molecules in the sample is adjusted by counting three or more partitions that generate positive signals, wherein:

[0800] (i) If (for example, when) the count is greater than a predetermined value, the adjustment is made by reducing the concentration of nucleic acid molecules in the sample through dilution; or

[0801] (ii) If (for example, when) the count is less than a predetermined value, the adjustment is to increase the concentration of nucleic acid molecules in the sample by concentrating the sample.

[0802] 15. The method according to any one of embodiments 4-14, further comprising passing each through detection channels X1-X R The quality control metric is determined by comparing the count of each partition that generates a positive signal with an estimated count, wherein the estimated count is based on the count of each partition except for the count generated by the detection channels X1-X. R The partition count is the count of each partition that generates a positive signal, in addition to the partition count of the partitions in the table.

[0803] 16. The method according to any one of embodiments 4-15, wherein R is between 2 and 6.

[0804] 17. The method according to implementation scheme 16, wherein R is 3.

[0805] 18. The method according to embodiment 17 further includes:

[0806] Get the first count (n) of one or more partitions 100 Each of the partitions generates a positive signal through detection channel X1, a negative signal through detection channel X2, and a negative signal through detection channel X3.

[0807] Obtain the second count (n) of one or more partitions. 000 Each of the partitions generates a negative signal on all detection channels X1-X3, and

[0808] Calculate the mutation probability that a given partition contains a mutated sequence at the target region corresponding to the first probe pair. The mutation probability is based on a first count (n) 100 ) and the first count (n) 100) and second count (n 000 The ratio between the sum of ( ).

[0809] 19. The method according to embodiment 18, further comprising, based on the mutation probability Determine the estimated concentration of the mutant sequence in the sample corresponding to the target region of the first probe pair.

[0810] 20. The method according to embodiment 18 or 19, further comprising determining the concentration in the sample relative to the estimated concentration. The relevant confidence intervals and / or uncertainty measures.

[0811] 21. The method according to any one of embodiments 18-20, further comprising calculating the wild-type probability that a given partition contains a wild-type sequence at a target region in the sample corresponding to the first probe pair. The wild-type probability mentioned above is based on Calculated.

[0812] 22. The method according to embodiment 21, wherein the wild-type probability Based on Definitely.

[0813] Where n 110 This represents the count of one or more partitions, each of which generates a positive signal through a first detection channel, a positive signal through a second detection channel, and a negative signal through a third detection channel.

[0814] Where n 010 This represents the count of one or more partitions, each of which generates a negative signal through a first detection channel, a positive signal through a second detection channel, and a negative signal through a third detection channel.

[0815] in This indicates the probability that a given partition contains a mutated sequence at the target region corresponding to the second probe pair.

[0816] 23. The method according to any one of embodiments 1-22, wherein substantially all partitions further include:

[0817] Allele-specific (AS) probes comprising an AS marker and an oligonucleotide AS sequence complementary to the allele sequence at the target region.

[0818] The AS mark can be detected through a detection channel that is different from the detection channel of the reference probe and drop-off probe corresponding to the plurality of probe groups; and

[0819] The method further includes:

[0820] The hybridization of the AS probe with a nucleic acid molecule or its amplicon containing an allele sequence at a target region in the sample is detected.

[0821] This provides quantification of allele sequences at the target region in the sample.

[0822] 24. The method according to any one of embodiments 1-23, wherein each of the reference probe and the drop-off probe has a single detectable marker.

[0823] 25. The method according to any one of embodiments 1-24...

Claims

1. A method for quantifying wild-type and / or mutant sequences at multiple target regions in a sample containing nucleic acid molecules, where the nucleic acid molecules are distributed in multiple partitions of the sample, and where substantially all partitions each include: multiple probe pairs corresponding to the multiple target regions, wherein each probe pair of the multiple probe pairs includes: a drop-off probe, which comprises a drop-off label and an oligonucleotide drop-off sequence, and the oligonucleotide drop-off sequence is complementary to the wild-type sequence at the target region corresponding to the respective probe pair; a reference probe, which comprises a reference label and an oligonucleotide reference sequence, and the oligonucleotide reference sequence is complementary to the wild-type sequence at an adjacent reference region upstream or downstream of the target region corresponding to the respective probe pair; where the reference label and the drop-off label of each probe pair of the multiple probe pairs can be detected through different detection channels; where the reference labels of the multiple probe pairs can be detected through detection channels different from each other; where the drop-off labels of the multiple probe pairs can be detected through detection channels different from each other; where at least one reference label of the multiple probe pairs and at least one drop-off label of the multiple probe pairs can be detected through the same detection channel; where the group of drop-off labels and the group of reference labels used in the probe pairs are in a cyclic permutation relative to each other; where one or more different detection channels have different excitation wavelength ranges and / or different emission wavelength ranges; where the method includes: detecting the hybridization of the reference probes of the multiple probe groups with nucleic acid molecules or their amplicons containing wild-type sequences at the reference regions in the multiple partitions; and detecting the hybridization of the drop-off probes of the multiple probe groups with nucleic acid molecules or their amplicons containing wild-type sequences at the target regions in the multiple partitions; thereby providing quantification of wild-type and / or mutant sequences at multiple target regions in the sample, where the multiple probe pairs are R probe pairs, where the first probe pair of the R probe pairs includes: The first reference probe contains a first reference sequence. and can pass through the first detection channel The first reference marker for detection, and The first drop-off probe contains the first drop-off sequence. and can be detected through the second detection channel The first drop-off marker detected; where the second probe pair of the R probe pairs includes: The second reference probe contains a second reference sequence. and can be detected through the second detection channel The second reference marker for detection, and The second drop-off probe contains the second drop-off sequence. and can be detected through the third detection channel The second drop-off marker detected; where, if R is strictly greater than 3, the i-th probe pair of the R probe pairs where 2 < i < R includes: The i-th reference probe contains the i-th reference sequence. And can be detected through the i-th detection channel The i-th reference marker detected, and The i-th drop-off probe contains the i-th drop-off sequence. And can be detected through the (i+1)th detection channel The i-th drop-off marker was detected; where, if R is strictly greater than 2, the R-th probe pair of the R probe pairs includes: The R-th reference probe contains the R-th reference sequence. and the Rth detection channel The relevant R-th reference marker, and The R-th drop-off probe contains the R-th drop-off sequence. and can pass through the first detection channel The R-th drop-off marker was detected; where the method includes: Through the detection channel - Each of the R probe pairs in the assay detects hybridization between a reference probe and a nucleic acid molecule or its amplicons containing a wild-type sequence at a reference region in the plurality of partitions; and Through the detection channel - Each of the R probe pairs is detected by hybridization of the drop-off probe with a nucleic acid molecule or its amplicon containing a wild-type sequence at the target region in the plurality of partitions.

2. The method according to claim 1, further comprising: Obtain the first count of one or more partitions, each of which generates a positive signal through the i-th detection channel and passes through the detection channel. - Any other detection channel in the process generates a negative signal; Obtain a second count for one or more partitions, each of which passes through all detection channels. - Generate negative signals; and Calculate the mutation probability of a given partition containing a mutated sequence in the target region corresponding to the i-th probe pair. The mutation probability is based on the ratio between the first count and the sum of the first and second counts.

3. The method according to claim 2, further comprising determining an estimated concentration of mutant sequences at the target region corresponding to the i-th probe pair in the sample based on the mutation probability.

4. The method according to claim 3, where the estimated concentration of mutant sequences at the target region corresponding to the i-th probe pair in the sample is determined according to: in This represents the estimated concentration of the mutant sequence in the sample corresponding to the target region of the i-th probe pair. where v represents the volume of the partition, and in This represents the mutation probability that a given partition contains a mutated sequence at the target region corresponding to the i-th probe pair in the sample.

5. The method according to claim 1, further comprising determining a confidence interval and / or uncertainty measure associated with the estimated concentration of mutant sequences at the target region corresponding to the i-th probe pair in the sample.

6. The method of claim 1, further comprising calculating the wild-type probability that a given partition contains a wild-type sequence at a target region corresponding to the i-th probe pair, wherein the wild-type probability is based on the mutation probability corresponding to the i-th probe pair and the mutation probability corresponding to the (i+1)-th probe pair, wherein the (i+1)-th probe pair refers to the first probe pair if i=R.

7. The method of claim 6, wherein the wild-type probability is calculated according to the following: in This represents the wild-type probability that a given partition contains a wild-type sequence at the target region corresponding to the i-th probe pair. in This represents a count of one or more partitions, each of which is accessed via... The detection channel generates a positive signal, which is transmitted through the channel. The detection channel generates a positive signal and passes through the detection channel. - Any other detection channel in the process generates a negative signal; in, If i = R, then It means ; in This represents the count of one or more partitions, each of which passes through all detection channels. - Generates a negative signal; in This represents a count of one or more partitions, each of which is accessed via... The detection channel generates a positive signal and passes through the detection channel. - Any other detection channel in the process generates a negative signal; in This represents a count of one or more partitions, each of which is accessed via... The detection channel generates a positive signal and passes through the detection channel. - Any other detection channel in the process generates a negative signal; Where, if i = R, then It means , in This represents the mutation probability that a given partition contains a mutated sequence at the target region corresponding to the i-th probe pair. in This represents the mutation probability that a given partition contains a mutated sequence at the target region corresponding to the (i+1)th probe pair, and Where, if i = R, then It means .

8. The method according to claim 6 or 7, further comprising determining, based on the wild-type probability, an estimated concentration of the wild-type sequence at the target region corresponding to the i-th probe pair in the sample.

9. The method of claim 8, wherein the estimated concentration of the wild-type sequence in the sample corresponding to the target region of the i-th probe pair is determined according to the following: in This represents the estimated concentration of the wild-type sequence in the target region corresponding to the i-th probe pair in the sample. Where v represents the volume of the partition, and in This represents the wild-type probability that a given partition contains a wild-type sequence at the target region corresponding to the i-th probe pair in the sample.

10. The method of claim 6 or 7, further comprising determining a confidence interval and / or uncertainty measure related to the estimated concentration of the wild-type sequence at the target region corresponding to the i-th probe pair in the sample.

11. The method of claim 1, further comprising, based on each of the respective detection channels - The concentration of nucleic acid molecules in the sample is adjusted by counting three or more partitions that produce positive signals, wherein: (i) If the count is greater than a predetermined value, the adjustment is made by reducing the concentration of nucleic acid molecules in the sample through dilution; or (ii) If the count is less than a predetermined value, the adjustment is to increase the concentration of nucleic acid molecules in the sample by concentrating the sample.

12. The method of claim 1, further comprising passing each through a detection channel - The quality control metric is determined by comparing the count of each partition that generates a positive signal with an estimated count, wherein the estimated count is based on, in addition to, the counts generated by each partition through the detection channel. - The partition count is the count of each partition that generates a positive signal, in addition to the partition count of the partitions in the table.

13. The method of claim 1, wherein R is between 2 and 6.

14. The method of claim 13, wherein R is 3.

15. The method of claim 14, further comprising: Obtain the first count n of one or more partitions 100 Each of the partitions is detected through a separate channel. A positive signal is generated and passed through the detection channel. Generate a negative signal and pass through the detection channel Generates a negative signal; Obtain the second count n of one or more partitions 000 Each of the partitions is present in all detection channels. - The above generates a negative signal, and Calculate the mutation probability that a given partition contains a mutated sequence at the target region corresponding to the first probe pair. The mutation probability is based on a first count n. 100 With the first count n 100 Second count n 000 The ratio between the sums.

16. The method of claim 14, further comprising, based on the mutation probability Determine the estimated concentration of the mutation sequence in the sample corresponding to the target region of the first probe pair. .

17. The method of claim 15 or 16, further comprising determining the concentration in the sample relative to the estimated concentration. The relevant confidence intervals and / or uncertainty measures.

18. The method of claim 15 or 16, further comprising calculating the wild-type probability that a given partition contains a wild-type sequence at a target region in the sample corresponding to the first probe pair. The wild-type probability is based on Calculated.

19. The method of claim 18, wherein the wild-type probability Based on Definitely. Where n 110 This represents the count of one or more partitions, each of which generates a positive signal through a first detection channel, a positive signal through a second detection channel, and a negative signal through a third detection channel. Where n 010 This represents the count of one or more partitions, each of which generates a negative signal through a first detection channel, a positive signal through a second detection channel, and a negative signal through a third detection channel. in This indicates the probability that a given partition contains a mutated sequence at the target region corresponding to the second probe pair.

20. The method of claim 1, wherein substantially all partitions further comprise: An allele-specific AS probe comprising an AS marker and an oligonucleotide AS sequence complementary to the allele sequence at the target region. The AS mark can be detected through a detection channel that is different from the detection channel of the reference probe and drop-off probe corresponding to the plurality of probe pairs; and The method further includes: The hybridization of the AS probe with a nucleic acid molecule or its amplicon containing an allele sequence at a target region in the sample is detected. This provides quantification of allele sequences at the target region in the sample.

21. The method of claim 1, wherein each of the reference probe and the drop-off probe has a single detectable marker.

22. The method of claim 1, wherein the reference marker and drop-off marker are fluorophores.

23. The method of claim 1, wherein one or more different detection channels share the same excitation and / or emission wavelength range, but are associated with different fluorescence intensities.

24. The method of claim 23, wherein probe pairs corresponding to different target regions within the target gene comprise drop-off probes having drop-off tags associated with different detection channels sharing the same excitation and / or emission wavelength ranges, wherein the drop-off probes are detected at different fluorescence intensities relative to each other.

25. The method of claim 22, wherein the reference marker and drop-off marker are selected from the group consisting of fluorescein, FAM, Yakima Yellow, Cy3, HEX, VIC, ROX, CY5, CY5.5, Alexa Fluor 647, Alexa Fluor 448, and Quasar 705.

26. The method of claim 14, wherein the first reference mark, the second reference mark and the third reference mark are selected from the group consisting of Cy3, FAM and Cy5, or wherein the first reference mark, the second reference mark and the third reference mark are selected from the group consisting of FAM, HEX and Cy5.

27. The method of claim 1, wherein the target region is a mutation hotspot region selected from one or more genes comprising the group consisting of EGFR, NRAS, KRAS, ESR1 and BRAF.

28. The method of claim 1, wherein each partition further comprises: (a) Multiple primer sets corresponding to the multiple target regions, and (b) DNA-dependent DNA polymerase; Each of the plurality of primer sets includes a forward oligonucleotide primer and a reverse oligonucleotide primer, which are suitable for amplifying a target fragment containing a target region corresponding to the primer set and a reference region corresponding to the target region; The method includes amplifying target fragments from nucleic acid molecules in the plurality of partitions; and The detection includes detecting the hybridization of the reference probe and drop-off probe with the amplicon of the target fragment.

29. The method of claim 28, wherein the DNA-dependent DNA polymerase comprises 5' to 3' exonuclease activity, and the detection comprises detecting an increase in fluorescence in the plurality of partitions caused by 5' to 3' exonuclease digestion by a reference label from a hybridization reference probe and / or a drop-off label from a hybridization drop-off probe.

30. The method of claim 1, wherein the length of the amplicon is 100 to 200 nucleotides.

31. The method of claim 1, wherein the reference region is not related to single nucleotide polymorphisms.

32. The method of claim 1, further comprising forming a plurality of partitions having a predetermined volume.

33. The method of claim 1, wherein the nucleic acid molecule is a genomic DNA molecule, tumor DNA, or cDNA.

34. The method of claim 1, further comprising extracting the nucleic acid molecule from a biological sample.

35. The method of claim 34, wherein the nucleic acid molecule is obtained from a formalin-fixed paraffin-embedded FFPE sample or a liquid biopsy sample.

36. The method of claim 34 or 35, further comprising fragmenting nucleic acid molecules in the biological sample to provide a sample containing nucleic acid molecules.

37. The method of claim 1, wherein the plurality of target regions are microsatellite sequence sites.

38. The method of claim 1, wherein the nucleic acid molecule is genomic DNA in a cell sample, wherein the cell has been contacted with a site-specific genome editing reagent configured to cleave target sites in the plurality of target regions, wherein the mutated sequence is a non-homologous end-linked NHEJ editing sequence at the plurality of target regions.

39. A method for quantifying unmodified, homology-directed repair HDR-edited and / or non-homologous-terminus-linked NHEJ-edited sequences at multiple R-1 target regions in nucleic acid molecules from cell samples, wherein the cells have been contacted with a site-specific genome editing reagent and an HDR template nucleic acid containing an HDR substitution sequence, wherein the site-specific genome editing reagent is configured to cleave target sites in the multiple target regions. The nucleic acid molecules are distributed in multiple regions of the sample, and Each of these partitions essentially includes: Multiple probe triplets corresponding to the plurality of R-1 target regions, wherein each of the plurality of R-1 probe triplets includes: An HDR probe comprising an HDR marker and an oligonucleotide HDR sequence, wherein the oligonucleotide HDR sequence is complementary to an HDR substitution sequence inserted at a target region corresponding to the respective probe triplet. An NHEJ drop-off probe comprises an NHEJ drop-off tag and an oligonucleotide drop-off sequence, wherein the oligonucleotide drop-off sequence is complementary to the wild-type sequence corresponding to the target region of the corresponding probe triplet, and wherein the drop-off sequence does not hybridize with the NHEJ-edited mutant sequence corresponding to the target region of the corresponding probe triplet. A reference probe comprises a reference marker and an oligonucleotide reference sequence, the oligonucleotide reference sequence being complementary to a wild-type sequence located upstream or downstream of an adjacent reference region corresponding to the target region of the corresponding probe triplet. The HDR marker, NHEJ drop-off marker, and reference marker of each of the multiple probe triplets can be detected through different detection channels; The HDR markers of the multiple probe triplets can be detected through different detection channels. The NHEJ drop-off markers of the multiple probe triplets can be detected through their respective detection channels; The reference markers of the multiple probe triplets can be detected through their respective detection channels; At least one reference label of the plurality of probe triplets and at least one NHEJ drop-off label of the plurality of probe triplets can be detected through the same detection channel, and / or at least one reference label of the plurality of probe triplets and at least one HDR label of the plurality of probe triplets can be detected through the same detection channel; One or more different detection channels have different excitation wavelength ranges and / or different emission wavelength ranges; The first probe triplet of the R - 1 probe triplets includes: The first reference probe contains a first reference sequence. and can pass through the first detection channel The first reference marker for detection; The first NHEJ drop-off probe contains the first NHEJ drop-off sequence. and can be detected through the second detection channel The first NHEJ drop-off marker detected; and The first HDR probe contains the first HDR sequence. and can be accessed through the third channel The first HDR marker detected; The second probe triplet of the R - 1 probe triplets includes: The second reference probe contains a second reference sequence. and can be detected through the second detection channel The second reference marker for detection; The second NHEJ drop-off probe contains the second drop-off sequence. and can be detected through the third detection channel The second NHEJ drop-off marker detected; and The second HDR probe contains a second HDR sequence. And can be detected through the fourth detection channel The second HDR marker detected; If R is strictly greater than 3, the i-th probe triplet of the R - 1 probe triplets, where 2 < i < R - 1, includes: The i-th reference probe contains the i-th reference sequence. And can be detected through the i-th detection channel The i-th reference marker being detected; The i-th NHEJ drop-off probe contains the i-th drop-off sequence. And can be detected through the (i+1)th detection channel The i-th NHEJ drop-off marker detected; and The i-th HDR probe contains the i-th HDR sequence. And can be detected through the (i+2)th detection channel The i-th HDR marker was detected; If R is strictly greater than 3, the (R - 1)-th probe triplet of the R - 1 probe triplets includes: The (R-1)th reference probe contains the (R-1)th reference sequence. And can be detected through the Rth detection channel The R-th reference marker was detected; The (R-1)th NHEJ drop-off probe contains the (R-1)th drop-off sequence. and can be detected through the (R-1)th detection channel The R-1th NHEJ drop-off marker detected; and The (R-1)th HDR probe contains the (R-1)th HDR sequence. and can pass through the first detection channel The R-1th HDR marker was detected; The method includes: Through the detection channel - and Each of the reference probes used to detect the R-1 probe triplet hybridizes with a nucleic acid molecule or its amplicon containing a wild-type sequence at a reference region in the plurality of partitions; Through the detection channel - Each of the NHEJ drop-off probes in the R-1 probe triplet is used to detect hybridization with a nucleic acid molecule or its amplicons containing a wild-type sequence at the target region in the plurality of partitions; and Through the detection channel and - Each of the HDR probes in the R-1 probe triplet hybridizes with a nucleic acid molecule or its amplicon containing an HDR substitution sequence at a target region in the plurality of partitions; Thereby providing quantification of unmodified, HDR-edited, and / or NHEJ-edited sequences at multiple target regions in the sample.

40. The method according to claim 39, wherein the site-specific genome editing reagent comprises a Cas nuclease, a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease.

41. The method according to claim 39 or 40, further comprising contacting the cell with the site-specific genome editing reagent.

42. The method according to claim 39, further comprising: If 1 ≤ i ≤ R - 2: Obtain a first count of one or more partitions, each of which is obtained through... The detection channel generates a positive signal and passes through the detection channel. - Any other detection channel in the process generates a negative signal; Obtain a second count for one or more partitions, each of which passes through all detection channels. - Generates a negative signal; Or if i is R - 1: Obtain a first count of one or more partitions, each of which is obtained through... The detection channel generates a positive signal and passes through the detection channel. - Any other channel in the signal generates a negative signal; Obtain a second count for one or more partitions, each of which passes through all detection channels. - Generate negative signals; and Calculate the NHEJ editing probability of a given partition containing an NHEJ-edited sequence at the target region corresponding to the i-th probe triplet. The NHEJ editing probability is based on the ratio between the first count and the sum of the first and second counts.

43. The method according to claim 39 or 42, further comprising: If 1 ≤ i ≤ R - 2: Obtain a first count of one or more partitions, each of which is obtained through... The detection channel generates a positive signal, which is transmitted through the channel. The detection channel generates a positive signal and passes through the detection channel. - Any other detection channel in the process generates a negative signal; Obtain a second count for one or more partitions, each of which passes through a detection channel. - Each of them generates a negative signal and passes through the detection channel. Each of them generates a negative signal; And calculate the probability that a given partition contains an unmodified wild-type sequence in the target region corresponding to the i-th probe triplet. The unmodified probability is based on , The ratio between the first count and the sum of the first and second counts; Or if i is R - 1: Obtain a first count of one or more partitions, each of which is obtained through... The detection channel generates a positive signal, in A positive signal is generated at the detection channel and passes through the detection channel. - Any other detection channel in the process generates a negative signal; Obtain a second count for one or more partitions, each of which passes through a detection channel. - Each of them generates a negative signal; and Calculate the probability that a given partition contains an unmodified wild-type sequence at the target region corresponding to the (R-1)th probe triplet. The unmodified probability is based on the ratio between the first count and the sum of the first and second counts.

44. The method according to claim 39, further comprising: If 1 ≤ i ≤ R - 2: Obtain a first count of one or more partitions, each of which is obtained through... The detection channel generates a positive signal, which is transmitted through the channel. The detection channel generates a positive signal and passes through the detection channel. - Any other detection channel in the process generates a negative signal; Obtain a second count for one or more partitions, each of which passes through a detection channel. - Each of them generates a negative signal, in A negative signal is generated and passes through the detection channel. Each of them generates a negative signal; and Calculate the HDR editing probability that a given partition contains an HDR permutation sequence at the target region corresponding to the i-th probe triplet. The HDR editing probability is based on , And the ratio between the first count and the sum of the first and second counts; Or if i is R - 1: Obtain a first count of one or more partitions, each of which is obtained through... The detection channel generates a positive signal, in A positive signal is generated at the detection channel and passes through the detection channel. - Any other detection channel in the process generates a negative signal; Obtain a second count for one or more partitions, each of which passes through a detection channel. - Each of them generates a negative signal; and Calculate the HDR editing probability of a given partition containing a wild-type sequence at the target region corresponding to the (R-1)th probe triplet. The HDR editing probability is based on , And the ratio between the first count and the sum of the first and second counts.

45. A method for quantifying wild-type and / or allelic sequences at R target regions in a sample comprising a nucleic acid molecule, wherein the nucleic acid molecule is distributed in multiple partitions of the sample, and substantially all partitions each contain R probe triplets corresponding to the R target regions, wherein the first probe triplet of the R probe triplets includes: Corresponding to the first reference sequence The first reference probe and the first detection channel The first reference marker for detection, Corresponding to the first allele sequence The first AS probe of the first probe triplet, "first AS probe 1", and the first detection channel The first AS marker "first AS marker 1" of the first probe triplet detected, and Corresponding to the first allele sequence The first probe triplet includes the second AS probe "second AS probe 1" and can be detected through the second detection channel. The second AS marker "Second AS Mark 1" of the first probe triplet was detected; wherein the second probe triplet of the R probe triplets includes: Corresponding to the second reference sequence The second reference probe and the second detection channel The second reference marker for detection, Corresponding to the second allele sequence The second probe triplet contains the first AS probe "first AS probe 2" and can be detected through the second detection channel. The first AS marker "AS marker 2" of the second probe triplet was detected, and Corresponding to the second allele sequence The second AS probe "second AS probe 2" of the second probe triplet and the third detection channel The second AS marker "Second AS Mark 2" of the second probe triplet was detected; If R is strictly greater than 3, the i-th probe triplet of the R probe triplets, where 2 < i < R, includes: Corresponding to the i-th reference sequence The i-th reference probe and the i-th detection channel The i-th reference marker detected, Corresponding to the i-th allele sequence The first AS probe "first AS probe i" of the i-th probe triplet and the probe that can be detected through the i-th detection channel The first AS marker "first AS marker i" of the i-th probe triplet detected, and Corresponding to the i-th allele sequence The second AS probe "second AS probe i" of the i-th probe triplet and the detection channel i+1 The second AS label "second AS label i" for the detected i-th probe triplet; If R is strictly greater than 2, the R-th probe triplet of the R probe triplets includes: Corresponding to the R-th reference sequence The Rth reference probe and the Rth detection channel The R-th reference marker detected, Corresponding to the Rth allele sequence The first AS probe of the Rth probe triplet, "first AS probe R", can be detected through the Rth detection channel. The first AS marker "first AS marker R" of the detected R-th probe triplet, and Corresponding to the Rth allele sequence The second AS probe of the Rth probe triplet, "second AS probe R", can be detected through the first detection channel. The second AS marker "second AS marker R" of the detected R-th probe triplet; wherein the first AS probe and the second AS probe of each probe triplet hybridize to the same allelic sequence, different portions within the same allelic sequence, or their complementary sequences at the target region corresponding to the respective probe triplet; wherein the reference sequence of each probe triplet is located in the reference region corresponding to the respective probe triplet; Among them, the detection channel - They are different; One or more different detection channels have different excitation wavelength ranges and / or different emission wavelength ranges; The method includes detecting channels - Each of the R probes in the detection system is a reference probe that hybridizes with a nucleic acid molecule or its amplicon containing a reference sequence or its complementary sequence at a reference region in the plurality of partitions; and the detection is performed via a detection channel. - Each of the R probes in the sample is used to detect the hybridization of the first AS probe and the second AS probe of the R probe triplet with a nucleic acid molecule or its amplicon containing an allele sequence or its complementary sequence at a target region in the plurality of partitions; thereby providing quantification of wild-type and / or allele sequences at the R target regions in the sample.

46. The method according to claim 45, wherein the allelic sequence is associated with a copy number variation (CNV).

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