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EP-4462436-B1 - MULTIPLEX DROP-OFF DIGITAL POLYMERASE CHAIN REACTION METHODS

EP4462436B1EP 4462436 B1EP4462436 B1EP 4462436B1EP-4462436-B1

Inventors

  • ANDRE, Barbara
  • DANGLA, Rémi
  • MADIC, JORDAN
  • MALLORY, ALLISON

Dates

Publication Date
20260513
Application Date
20201222

Claims (16)

  1. A method for quantification of wildtype, mutant and/or allelic sequences "at a plurality 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 a plurality of probe sets corresponding to the plurality of target regions, wherein each probe set of the plurality of probe sets comprises: a drop-off probe comprising a drop-off label and an oligonucleotide drop-off sequence complementary to a wildtype sequence at a 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; an allele-specific (AS) probe comprising an AS label and an oligonucleotide AS sequence complementary to an allelic sequence at the target region corresponding to the respective probe set, wherein a reference label, a drop-off label, and an AS label of each probe set of the plurality of probe sets are detectable via different detection channels; wherein at least one reference label of the plurality of probe sets and at least one 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 AS label of the plurality of probe sets are detectable via the same detection channel wherein the set of reference labels of the plurality of probe sets, the set of drop-off labels of the plurality of probe sets, and the set of AS labels of the plurality of probe sets have overlapping labels; 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; and detecting hybridization of 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; detecting hybridization of AS probes of the plurality of probe sets to nucleic acid molecules or amplicons thereof comprising allelic sequences at the target regions in the plurality of partitions; thereby providing quantification of wildtype sequences, mutants sequences and/or allelic sequences at the plurality of target regions in the sample.
  2. The method of claim 1, wherein the set of reference labels of the plurality of probe sets, the set of drop-off labels of the plurality of probe sets, and the set of AS labels of the plurality of probe sets are permutations with respect to each other.
  3. The method of claim 1 or claim 2, wherein: a) reference labels of the plurality of probe sets are detectable via different detection channels with respect to each other; or b) drop-off labels of the plurality of probe sets are detectable via different detection channels with respect to each other; or c) AS labels of the plurality of probe sets are detectable via different detection channels with respect to each other.
  4. The method of any one of claims 1 to 3, wherein reference labels of the plurality of probe sets are detectable via different detection channels with respect to each other; drop-off labels of the plurality of probe sets are detectable via different detection channels with respect to each other; and wherein AS labels of the plurality of probe sets are detectable via different detection channels with respect to each other.
  5. The method of any one of claims 1 to 4, wherein the total number of detection channels is fewer than the total number of probes.
  6. The method of claim 5, wherein the plurality of probe sets is a plurality of probe triplets, and the total number of detection channels is fewer than three times the total number of probe triplets.
  7. The method of claim 6, wherein the total number of detection channels is equal to the total number of probe triplets plus 1.
  8. The method of any one of claims 1 to 7, wherein the plurality of probe sets are 2 probe triplets, wherein a first probe triplet of the 2 probe triplets comprises: a first reference probe comprising a first reference sequence ( m 1 ) and a first reference label detectable via a first detection channel ( X 1 ); a first drop-off probe comprising a first drop-off sequence ( r 1 ) and a first drop-off label detectable via a second detection channel ( X 2 ); and a first allele-specific (AS) probe comprising a first AS sequence ( w 1 ) and a first AS label detectable via a third channel ( X 3 ); wherein a second probe triplet of the 2 probe triplets comprises: a second reference probe comprising a second reference sequence ( m 2 ) and a second reference label detectable via the second detection channel ( X 2 ); a second drop-off probe comprising a second drop-off sequence ( r 2 ) and a second drop-off label detectable via the third detection channel ( X 3 ); and a second AS probe comprising a second AS sequence ( w 2 ) and a second AS label detectable via a fourth detection channel ( X 4 ); wherein the drop-off sequence of each probe triplet is complementary to a wildtype sequence at a target region corresponding to the respective probe triplet; wherein the reference sequence of each probe triplet is complementary to a wildtype sequence at an adjacent reference region upstream or downstream to the target region corresponding to the respective probe triplet; wherein the AS sequence of each probe triplet is complementary to an allelic sequence at the target region corresponding to the respective probe triplet; wherein the detection channels X 1 , X 2 , X 3 , and X 4 are different from each other; wherein the method comprises detecting hybridization of reference probes of the 2 probe triplets to nucleic acid molecules or amplicons thereof comprising wildtype sequences at the reference regions in the plurality of partitions; detecting hybridization of AS probes of the 2 probe triplets to nucleic acid molecules or amplicons thereof comprising the allelic sequences at the target regions in the plurality of partitions; and detecting hybridization of drop-off probes of the 2 probe triplets to nucleic acid molecules or amplicons thereof comprising wildtype sequences at the target regions in the plurality of partitions; thereby providing quantification of wildtype and allelic sequences at the plurality of target regions in the sample.
  9. The method of any one of claims 1 to 7, wherein the plurality of probe sets are ( R- 1) number of probe triplets, wherein R is an integer of 4 or more, wherein a first probe triplet of the ( R -1) number of probe triplets comprises: a first reference probe comprising a first reference sequence ( m 1 ) and a first reference label detectable via a first detection channel ( X 1 ); a first drop-off probe comprising a first drop-off sequence ( r 1 ) and a first drop-off label detectable via a second detection channel ( X 2 ); and a first allele-specific (AS) probe comprising a first AS sequence ( w 1 ) and a first AS label detectable via a third channel ( X 3 ); wherein a second probe triplet of the (R-1) number of probe triplets comprises: a second reference probe comprising a second reference sequence ( m 2 ) and a second reference label detectable via the second detection channel ( X 2 ); a second drop-off probe comprising a second drop-off sequence ( r 2 ) and a second drop-off label detectable via the third detection channel ( X 3 ); and a second AS probe comprising a second AS sequence ( w 2 ) and a second AS label detectable via a fourth detection channel ( X 4 ); wherein, an i-th probe triplet (2 <i<R- 1) of the (R-1) number of probe triplets comprises: an i-th reference probe comprising an i-th reference sequence ( m i ) and an i-th reference label detectable via an i-th detection channel ( X i ); an i -th drop-off probe comprising an i -th drop-off sequence ( r i ) and an i- th drop-off label detectable via an ( i +1)-th detection channel ( X i +1 ); and an i -th AS probe comprising an i -th AS sequence ( w i ) and an i -th AS label detectable via an ( i+ 2)-th detection channel ( X i +2 ); wherein, a ( R- 1)-th probe triplet of the (R-1) number of probe triplets comprises: a ( R-1) -th reference probe comprising a (R-1) -th reference sequence ( m R -1 ) and a R -th reference label detectable via a R-th detection channel ( X R ); a ( R-1)- th drop-off probe comprising a ( R-1) -th drop-off sequence ( r R -1 ) and a ( R-1) -th drop-off label detectable via a (R-1)-th detection channel ( X R -1 ); and a ( R-1)-th AS probe comprising a ( R-1) -th AS sequence ( w R -1 ) and a ( R-1)-th AS label detectable via the first detection channel ( X 1 ); wherein the drop-off sequence of each probe triplet is complementary to a wildtype sequence at a target region corresponding to the respective probe triplet; wherein the reference sequence of each probe triplet is complementary to a wildtype sequence at an adjacent reference region upstream or downstream to the target region corresponding to the respective probe triplet; wherein the AS sequence of each probe triplet is complementary to an allelic sequence at the target region corresponding to the respective probe triplet; wherein the detection channels X 1 -X R are different from each other; wherein the method comprises detecting hybridization of reference probes of the ( R -1) number of probe triplets to nucleic acid molecules or amplicons thereof comprising wildtype sequences at the reference regions in the plurality of partitions via each of the detection channels X 1 -X R ; detecting hybridization of AS probes of the (R-1) number of probe triplets to nucleic acid molecules or amplicons thereof comprising the allelic sequences at the target regions in the plurality of partitions via each of the detection channels X 1 -X R ; and detecting hybridization of drop-off probes of the ( R -1) number of probe triplets to nucleic acid molecules or amplicons thereof comprising wildtype sequences at the target regions in the plurality of partitions via each of the detection channels X 1 -X R ; thereby providing quantification of wildtype and allelic sequences at the plurality of target regions in the sample.
  10. The method of any one of claims 1 to 9, wherein each of the reference probes, drop-off probes and AS probes has a single detectable label.
  11. The method of any one of claims 1 to 10, wherein the reference labels, drop-off labels, and AS labels are fluorophores.
  12. The method of claim 11, wherein the reference labels, drop-off labels and/or AS labels 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 Quasar705.
  13. The method of any one of claims 1 to 12, wherein one or more different detection channels have different excitation wavelength ranges and/or different emission wavelength ranges.
  14. The method of any one of claims 1 to 13, wherein one or more different detection channels share the same excitation and/or emission wavelength ranges, but are associated with different fluorescence intensities.
  15. The method of any one of claims 1 to 14, wherein at least one reference label of the plurality of probe sets and at least one drop-off label of the plurality of probe sets are detectable via the same detection channel.
  16. The method of any one of claims 1 to 15, wherein at least one reference label of the plurality of probe sets and at least one AS label of the plurality of probe sets are detectable via the same detection channel.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the priority benefits of European Patent Application No. 19306765.9, filed December 23, 2019, and United States Patent Application No. 17/013,222, filed September 4, 2020, FIELD The present application is related to multiplex digital polymerase chain reaction (PCR) assays (such as multiplex drop-off dPCR assays), methods and systems, including methods for assessing microsatellite instability (MSI) and genome-editing products. BACKGROUND Digital polymerase chain reaction (dPCR) is a powerful and sensitive method that can be used to detect rare mutations in nucleic acid samples. Digital PCR assays using allele-specific fluorescent TAQMAN™ probes have been developed to detect somatic mutations in biomarker genes. In a conventional dPCR assay, a wildtype probe recognizing the wildtype allele and a mutant probe recognizing a specific mutant allele are used. Upon hybridization of a wildtype probe or a mutant probe to an amplicon in a dPCR partition, the probe releases its fluorophore through the exonuclease activity of a DNA polymerase. The released fluorophore from a wildtype probe is detected via a fluorescence detection channel that is distinct from the released fluorophore from a mutant probe. Such assays require a dPCR instrument with R detection channels to detect R mutations at one or more genetic loci. In contrast, drop-off assays allow the quantification of any number of mutations occurring at a mutation hotspot by using two probes in a dPCR reaction: a drop-off probe that recognizes a wildtype sequence at the mutation hotspot, and a reference probe that recognizes a sequence at a low-mutation region on the same amplicon. See, Decraene C. et al., Clinical Chemistry 64(2): 317-328 (2017). In a drop-off assay, two detection channels are required to detect any number of mutations at a single genetic locus. WO2019162240 discloses a multiplex drop-off assay for determining microsatellite repeat instability. US2018024061 discloses multiplex digital PCR wherein the digital PCR measurement is performed by four-colour de- tection using ten channels. US8384899 discloses the detection of a plurality of labelled analytes including nucleic acids in a plurality of channels wherein different labels are used. WO2014097888 discloses the detection of DNA fragments labelled with a plurality of fluorescent dyes placed in a plurality of flow channels. Because dPCR instruments have limited fluorescence detection channels, there is a need to increase the multiplex levels of dPCR assays for different mutations and at different genetic loci. Such assays are especially useful in clinical and other applications involving samples that are limited in quantity. Robust methods for quantification of different genetic species based on data from multiplexed dPCR assays are also needed. BRIEF SUMMARY The present application provides methods, apparatus, systems and compositions, for detection and/or quantification of wildtype and mutant sequences at a plurality of target regions in nucleic acid samples using multiplex dPCR assays, such as 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-edited products). One aspect of the present application provides a method for quantification of wildtype and/or mutant sequences at a plurality 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, 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 drop-off probe comprising a drop-off label and an oligonucleotide drop-off sequence complementary to a wildtype sequence at a 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 reference label and a drop-off label of each probe set of the plurality of probe sets are detectable via different detection channels; wherein reference labels of the plurality of probe sets are detectable via different detection channels with respect to each other; wherein drop-off labels of the plurality of probe sets are detectable via different detection channels with respect to each other; whereinat least one reference label of the plurality of probe sets and at least one drop-offlabel 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 a