Multiplex drop-off digital polymerase chain reaction methods

A method for quantification of mutations at a plurality of microsatellite sequence loci in a sample comprising nucleic acid molecules,

• wherein the nucleic acid molecules are distributed among a plurality of partitions of the sample, and
• wherein substantially all partitions each comprises:
• a plurality of primer sets corresponding to the plurality of microsatellite sequence loci, wherein each primer set of the plurality of primer sets comprises:
• a forward oligonucleotide primer and a reverse oligonucleotide primer suitable for amplifying target fragments from the nucleic acid molecules,
• wherein each target fragment comprises the microsatellite sequence locus corresponding to the primer set and an adjacent reference region upstream or downstream to the microsatellite sequence locus;
• a plurality of probe pairs corresponding to the plurality of microsatellite sequence loci, wherein each probe pair of the plurality of probe pairs comprises:
• a drop-off probe comprising a drop-off label and an oligonucleotide drop-off sequence complementary to a wildtype sequence of a microsatellite sequence locus corresponding to the respective probe pair,
• a reference probe comprising a reference label and an oligonucleotide reference sequence complementary to a wildtype sequence of the reference region corresponding to the respective probe pair,
• wherein a reference label and a drop-off label of each probe pair of the plurality of probe pairs are detectable via different detection channels;
• wherein reference labels of the plurality of probe pairs are detectable via different detection channels with respect to each other;
• wherein drop-off labels of the plurality of probe pairs are detectable via different detection channels with respect to each other;
• wherein at least one reference label of the plurality of probe pairs and at least one drop-off label of the plurality of probe pairs are detectable via the same detection channel;
• wherein the method comprises:
• amplifying the target fragments in the plurality of partitions;
• and
• detecting hybridization of reference probes and drop-off probes of the plurality of probe pairs to amplicons of the target fragments in the plurality of partitions,
• thereby providing quantification of mutations at the plurality of microsatellite sequence loci in the sample.

A method for quantification of unmodified, homology directed repair (HDR)-edited, and/or non-homologous end joining (NHEJ)-edited sequences at a plurality of target regions in nucleic acid molecules from a sample of cells, wherein the cells have been contacted with a site-specific genome-editing reagent and HDR template nucleic acids comprising HDR replacement sequences, wherein the site-specific genome-editing reagent is configured to cleave target sites in the plurality of target regions,

• wherein the nucleic acid molecules are distributed among a plurality of partitions of the sample, and
• wherein substantially all partitions each comprises:
• a plurality of probe sets corresponding to the plurality of target regions,
• wherein each probe set of the plurality of probe sets comprises:
• a HDR probe comprising a HDR label and an oligonucleotide HDR sequence complementary to a HDR replacement sequence inserted at a target region corresponding to the respective probe set,
• an NHEJ drop-off probe comprising an NHEJ drop-off label and an oligonucleotide drop-off sequence complementary to a wildtype sequence of the target region corresponding to the respective probe set, and wherein the drop-off sequence does not hybridize to NHEJ-edited mutant sequences at the target region corresponding to the respective probe set,
• a reference probe comprising a reference label and an oligonucleotide reference sequence complementary to a wildtype sequence at an adjacent reference region upstream or downstream to the target region corresponding to the respective probe set,
• wherein a HDR label, an NHEJ drop-off label, and a reference label of each probe set of the plurality of probe sets are detectable via different detection channels;
• wherein HDR labels of the plurality of probe sets are detectable via different detection channels with respect to each other;
• wherein NHEJ drop-off labels of the plurality of probe sets are detectable via different detection channels with respect to each other;
• wherein reference labels of the plurality of probe sets are detectable via different detection channels with respect to each other;
• wherein at least one reference label of the plurality of probe sets and at least one NHEJ drop-off label of the plurality of probe sets are detectable via the same detection channel, and/or at least one reference label of the plurality of probe sets and at least one HDR label of the plurality of probe sets are detectable via the same detection channel;
• wherein the method comprises:
• detecting hybridization of reference probes of the plurality of probe sets to nucleic acid molecules or amplicons thereof comprising wildtype sequences at the reference regions in the plurality of partitions;
• detecting hybridization of HDR probes of the plurality of probe sets to nucleic acid molecules or amplicons thereof comprising the HDR replacement sequences at the target regions in the plurality of partitions;
• and
• detecting hybridization of NHEJ drop-off probes of the plurality of probe sets to nucleic acid molecules or amplicons thereof comprising wildtype sequences at the target regions in the plurality of partitions;
• thereby providing quantification of unmodified, HDR-edited, and/or NHEJ-edited sequences at the plurality of target regions in the sample.

A method for quantification of wildtype and/or allelic sequences at R number of target regions in a sample comprising nucleic acid molecules,

• wherein the nucleic acid molecules are distributed among a plurality of partitions of the sample, wherein substantially all partitions each comprises R number of probe triplets corresponding to the R number of target regions,
• wherein a first probe triplet of the R number of probe triplets comprises:
• a first reference probe corresponding to a first reference sequence (w1) and a first reference label detectable via a first detection channel (X1),
• a first AS probe of the first probe triplet (“first AS probe 1”) corresponding to a first allelic sequence (r1) and a first AS label of the first probe triplet (“first AS label 1”) detectable via the first detection channel (X1), and
• a second AS probe of the first probe triplet (“second AS probe 1”) corresponding to the first allelic sequence (r1) and a second AS label of the first probe triplet (“second AS label 1”) detectable via the second detection channel (X2);
• wherein a second probe triplet of the R number of probe triplets comprises:
• a second reference probe corresponding to a second reference sequence (w2) and a second reference label detectable via the second detection channel (X2), a first AS probe of the second probe triplet (“first AS probe 2”) corresponding to a second allelic sequence (r2) and a first AS label of the second probe triplet (“AS label 2”) detectable via the second detection channel (X2), and
• a second AS probe of the second probe triplet (“second AS probe 2”) corresponding to the second allelic sequence second allelic sequence (r2) and a second AS label of the second probe triplet (“second AS label 2”) detectable via a third detection channel (X3);
• wherein, if (e.g., when) R is strictly larger than 3, an i-th probe triplet (2<i<R) of the R number of probe triplet comprises:
• an i-th reference probe corresponding to an i-th reference sequence (w1) and an i-th reference label detectable via an i-th detection channel (Xi),
• a first AS probe of the i-th probe triplet (“first AS probe i”) corresponding to an i-th allelic sequence (ri) and a first AS label of the i-th probe triplet (“first AS label i”) detectable via the i-th detection channel (Xi), and
• a second AS probe of the i-th probe triplet (“second AS probe i”) corresponding to an i-th allelic sequence (ri) and a second AS label of the i-th probe triplet (“second AS label i”) detectable via the (i+1)-th detection channel (Xi+1);
• wherein, if (e.g., when) R is strictly larger than 2, a R-th probe triplet of the R number of probe triplets comprises:
• a R-th reference probe corresponding to a R-th reference sequence (wR) and a R-th reference label detectable via a R-th detection channel (XR),
• a first AS probe of the R-th probe triplet (“first AS probe R”) corresponding to an R-th allelic sequence (rR) and a first AS label of the R-th probe triplet (“first AS label R”) detectable via the R-th detection channel (XR), and
• a second AS probe of the R-th probe triplet (“second AS probe R”) corresponding to a R-th allelic sequence (rR) and a second AS label of the R-th probe triplet (“second AS label R”) detectable via the first detection channel (X1);
• 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 complementary sequences thereof at a target region corresponding to the respective probe triplet;
• wherein the reference sequence of each probe triplet is at a reference region corresponding to the respective probe triplet;
• wherein the detection channels X1-XR are different from each other;
• wherein the method comprises detecting hybridization of reference probes of the R number of probe triplets to nucleic acid molecules or amplicons thereof comprising reference sequences or complementary sequences thereof at the reference regions in the plurality of partitions via each of the detection channels X1-XR;
• and detecting hybridization of the first AS probes and the second AS probes of the R number of probe triplets to nucleic acid molecules or amplicons thereof comprising allelic sequences or complementary sequences thereof at the target regions in the plurality of partitions via each of the detection channels X1-XR;
• thereby providing quantification of wildtype and/or allelic sequences at the R number of target regions in the sample.