(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(10) International Publication Number
`
`WO 2014/121239 A2
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ,
`OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM,
`ZW.
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`KM, ML, MR, NE, SN, TD, TG).
`
`gg
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`
`7 August 2014 (07.08.2014)
`
`WI P O | P C T
`
`(51)
`
`International Patent Classification:
`CIZQ 1/68 (2006.01)
`
`(21)
`
`International Application Number:
`
`(81)
`
`PCT/US2014/014514
`
`(22)
`
`International Filing Date:
`
`3 February 2014 (03.02.2014)
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
`
`(74)
`
`Filing Language:
`
`Publication Language:
`
`English
`
`English
`
`Priority Data:
`61/759,930
`61/759,772
`61/759,931
`
`1 February 2013 (01.02.2013)
`1 February 2013 (01.02.2013)
`1 February 2013 (01.02.2013)
`
`(84)
`
`US
`US
`US
`
`Applicant: BIO-RAD LABORATORIES, INC. [i/US];
`1000 Alfred Nobel Drive, Hercules, CA 94547 (US).
`
`Inventors: TZONEV, Svilen, S.; 121 Rocky Creek Place,
`Pleasanton, CA 94566 (US). LITTERST, Claudia; 2562
`Walnut Boulevard #56, Walnut Creek, CA 94596 (US).
`
`Agent: ABNEY, James, R.; Kolisch Hartwell, P.C., 520
`SW. Yamhill Street, Suite 200, Portland, OR 97204 (US).
`
`(54) Title: MULTIPLEXED DIGITAL ASSAY WITH DATA EXCLUSION FOR CALCULATION OF TARGET LEVELS
`
`[Continued on nextpage]
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`(57) Abstract: Digital assay system, including meth-
`ods and apparatus, for calculating a level of one or
`more targets. In an exemplary method, data for amp-
`lification of a plurality of targets including a first
`target may be collected from partitions. A level of
`the first target may be calculated from only a subset
`of the data that excludes partitions according to tar-
`get content.
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`Fig 3
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`”1055“
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`HEGHS as;
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`Lowe {3.) AMPLITUDE
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`EMPTiES
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`Published:
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`— without international search report and to be republished
`upon receipt ofthot report (Rule 48.2(g))
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`WO 2014/121239
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`PCT/US2014/014514
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`MULTIPLEXED DIGITAL ASSAY WITH DATA EXCLUSION
`
`FOR CALCULATION OF TARGET LEVELS
`
`5
`
`This application is based upon and claims the benefit under 35 U.S.C. §
`
`Cross-References to Priority Applications
`
`119(e) of U.S. Provisional Patent Application Serial No. 61/759,772, filed February 1,
`
`2013; U.S. Provisional Patent Application Serial No. 61/759,930, filed February 1,
`
`2013; and U.S. Provisional Patent Application Serial No. 61/759,931, filed February
`
`1, 2013. Each of these priority applications is incorporated herein by reference in its
`
`10
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`entirety for all purposes.
`
`Cross-References to Other Materials
`
`This application incorporates by reference in their entireties for all purposes
`
`the following materials: U.S. Patent No. 7,041,481, issued May 9, 2006; U.S. Patent
`
`Application Publication No. 2010/0173394 A1, published July 8, 2010; U.S. Patent
`
`Application Publication No. 2011/0217712 A1, published September 8, 2011; U.S.
`
`Patent Application Publication No. 2012/0152369 A1, published June 21, 2012; U.S.
`
`Patent Application Publication No. 2013/0040841 A1, published February 14, 2013;
`
`U.S. Patent Application Serial No. 14/099,750, filed December 6, 2013; and Joseph
`
`R. Lakowicz, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY (2nd Ed. 1999).
`
`Introduction
`
`Digital assays generally rely on the ability to detect the presence or activity of
`
`individual copies of an analyte in a sample. In an exemplary digital assay, a sample
`
`is separated into a set of partitions, generally of equal volume, with each containing,
`
`on average, less than about one copy of the analyte. If the copies of the analyte are
`
`distributed randomly among the partitions, some partitions Should contain no copies,
`
`others only one copy, and,
`
`if the number of partitions is large enough, still others
`
`Should contain two copies, three copies, and even higher numbers of copies. The
`
`probability of finding exactly 0, 1, 2, 3, or more copies in a partition, based on a given
`
`average concentration of analyte in the partitions,
`
`is described by a Poisson
`
`distribution. Conversely, the concentration of analyte in the partitions (and thus in the
`
`sample) may be estimated from the probability of finding a given number of copies in
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`a partition.
`
`Estimates of the probability of finding no copies and of finding one or more
`
`copies may be measured in the digital assay. EaCh partition can be tested to
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`determine whether the partition is a positive partition that contains at least one copy
`
`of the analyte, or is a negative partition that contains no copies of the analyte. The
`
`probability of finding no copies in a partition can be approximated by the fraction of
`
`partitions tested that are negative (the “negative fraction”), and the probability of
`
`finding at least one copy by the fraction of partitions tested that are positive (the
`
`“positive fraction”). The positive fraction or the negative fraction then may be utilized
`
`to determine the concentration of the analyte in the partitions, such as with Poisson
`
`statistics.
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`Digital assays frequently rely on amplification of a nucleic acid target
`
`in
`
`partitions to enable detection of a single copy of an analyte. Amplification may be
`
`conducted via the polymerase chain reaction (PCR), to achieve a digital PCR assay.
`
`The target amplified may be the analyte itself or a surrogate for the analyte
`
`generated before or after formation of the partitions. Amplification of the target can
`
`be detected optically from a fluorescent probe included in the reaction. In particular,
`
`the probe can include a fluorophore that provides a fluorescence signal indicating
`
`whether or not the target has been amplified.
`
`A digital PCR assay can be multiplexed to permit detection of two or more
`
`different
`
`targets within
`
`each partition. Amplification of
`
`the targets
`
`can be
`
`distinguished by utilizing target-specific probes. However, in multiplexed assays with
`
`a higher concentration of targets, where two or more targets can be present together
`
`in the same partition, distinct partition populations within the assay often produce
`
`overlapping signal amplitudes. Data interpretation for these assays can be difficult,
`
`with calculated target concentrations being inaccurate.
`
`New approaches are needed for improving multiplexed digital assays, such as
`
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`assays in which the data are not well resolved.
`
`My
`
`The present disclosure provides a multiplexed digital assay system, including
`
`methods and apparatus,
`
`for calculating a level of one or more targets.
`
`In an
`
`exemplary method, data for amplification of a plurality of targets including a first
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`
`target may be collected from partitions. A level of the first target may be calculated
`
`from only a subset of the data that excludes partitions according to target content.
`
`Brief Description of the Drawings
`
`Figure 1
`
`is a flowchart of an exemplary method of performing a multiplexed
`
`digital assay on targets in partitions containing a generic reporter, optionally with at
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`least one of the targets being an obscuring target that at least partially masks the
`
`presence of another target in partitions containing both the obscuring target and the
`
`masked target, in accordance with aspects of the present disclosure.
`
`Figure 2 is a schematic view of an exemplary system for performing the
`
`multiplexed digital assay of Figure 1,
`
`in accordance with aspects of the present
`
`disclosure.
`
`Figure 3 is a schematic graph of multiplexed assay data that may be collected
`
`in a single detection channel from partitions, such as droplets, collectively containing
`
`an obscuring target (H) associated with a higher amplitude signal and a masked
`
`target (L) associated with a lower amplitude signal, in accordance with aspects of the
`
`present disclosure.
`
`Figure 3A is a schematic graph of multiplexed assay data that may be
`
`collected as in Figure 3, but with the population of double-positive partitions (HL,
`
`HLL, etc.) exhibiting a small, detectable increase in signal amplitude over the
`
`population of partitions containing only target H, with the increase in signal amplitude
`
`being insufficient to resolve the two higher amplitude populations from each other,
`
`such that target L is still at least partially masked by the presence of target H,
`
`in
`
`accordance with aspects of the present disclosure.
`
`Figure 4 is another schematic graph of multiplexed assay data that may be
`
`collected generally as in Figure 3, but with the obscuring target (L) having a lower
`
`amplitude signal
`
`than the masked target (H),
`
`in accordance with aspects of the
`
`present disclosure.
`
`Figure 5 is a schematic graph of multiplexed assay data that may be collected
`
`in a single detection channel from partitions, such as droplets, collectively containing
`
`K targets that mask one another serially according to a masking hierarchy,
`
`in
`
`accordance with aspects of the present disclosure.
`
`Figure 6 is a schematic view of a partition from an exemplary multiplexed
`
`digital assay capable of generating the data of Figure 3, with the partition containing
`
`a copy of a masking target (H) and a copy of a masked target (L) of different length,
`
`each amplifiable at a different efficiency with the same pair of forward (F) and
`
`reverse (R) primers and detectable via the same probe (P),
`
`in accordance with
`
`aspects of the present disclosure.
`
`Figure 7 is a schematic view a partition from an exemplary multiplexed digital
`
`assay capable of generating the data of Figure 4, with the partition containing a copy
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`of a masking target (L) and a masked target (H) of different length, each amplifiable
`
`at a different efficiency with the same pair of forward (F) and reverse (R) primers and
`
`detectable via the same generic reporter for amplicons (e.g., an intercalating dye), in
`
`accordance with aspects of the present disclosure.
`
`Figure 8 is a schematic view of a partition from another exemplary multiplexed
`
`digital assay capable of generating the data of Figure 3, with the partition containing
`
`a copy of a masking target (H) and a copy of a masked target (L) of the same length,
`
`each amplifiable at a different efficiency with the same pair of forward (F) and
`
`reverse (R) primers and detectable via the same probe (P),
`
`in accordance with
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`
`aspects of the present disclosure.
`
`Figure 9 is a schematic view of a partition from yet another exemplary
`
`multiplexed digital assay capable of generating the data of Figure 3, with the partition
`
`containing a copy of a masking target (H) and a copy of a masked target (L) of the
`
`same length, each amplifiable at the same efficiency with the same pair of fon/vard
`
`(F) and reverse (R) primers and detectable at different efficiencies via the same
`
`probe (P), in accordance with aspects of the present disclosure.
`
`Figure 10 is a schematic view of a partition from still yet another exemplary
`
`multiplexed digital assay capable of generating the data of Figure 3, with the partition
`
`containing a copy of an obscuring target and a copy of a masked target, each
`
`amplifiable at a different efficiency with a different pair of fon/vard and reverse
`
`primers (FH and RH or FL and RL) and detectable via different probes (PH and PL),
`
`in
`
`accordance with aspects of the present disclosure.
`
`Figure 11 is a schematic view of a partition from an exemplary multiplexed
`
`digital assay performed with a single detection channel, with the partition containing
`
`a copy of target A and target B, each amplifiable with a different pair of forward and
`
`reverse primers (FA and RA or F3 and RB) and detectable via a generic reporter (e.g.,
`
`an intercalating dye), in accordance with aspects of the present disclosure.
`
`Figure 11A is a schematic graph of fluorescence amplitude data that may be
`
`collected from five sets of partitions (lanes 1-5) in a single detection channel for the
`
`multiplexed digital assay of Figure 11, with each set containing the same
`
`concentrations of targets A and B, fon/vard primer A, and reverse primers A and B,
`
`and a variable concentration of forward primer B for target B,
`
`in accordance with
`
`aspects of the present disclosure.
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`Figure 12 is a schematic view of a partition from an exemplary multiplexed
`
`digital assay performed with two detection channels, with the partition containing a
`
`copy of a masked target A and a copy of a masking target B, each amplifiable at a
`
`different efficiency with a different pair of fon/vard and reverse primers (FA and RA or
`
`F3 and “RB”) and detectable via different probes (PA and PB) having different
`
`fluorophore labels, in accordance with aspects of the present disclosure.
`
`Figure 12A is a schematic scatter plot of amplification data that may be
`
`collected from the multiplexed digital assay of Figure 12, with three distinct clusters
`
`of partitions numbered sequentially and identified according to target content,
`
`in
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`10
`
`accordance with aspects of the present disclosure.
`
`Figure 13 is a schematic view of a partition from another exemplary
`
`multiplexed digital assay performed with two detection channels, with the partition
`
`containing a copy of a masked target (A) and a copy of a masking target (B), each
`
`amplifiable with a different pair of forward and reverse primers (FA and RA or F3 and
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`15
`
`RB) and detectable via a generic reporter (e.g., an intercalating dye) and a specific
`
`probe (P3), in accordance with aspects of the present disclosure.
`
`Figure 14 is a schematic scatter plot of amplification data that may be
`
`collected from the multiplexed digital assay of Figure 13, with three distinct clusters
`
`of partitions numbered sequentially and identified according to target content,
`
`in
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`20
`
`accordance with aspects of the present disclosure.
`
`Figure 14A is a schematic view of a partition from an exemplary multiplexed
`
`digital assay performed with a single detection channel, with the partition containing
`
`a copy of a masked target (A) and a copy of a masking target (B), each amplifiable
`
`with a different pair of fon/vard and reverse primers (FA and RA or F3 and RB) and
`
`detectable via a generic reporter (e.g., an intercalating dye) and a specific probe
`
`(P3), in accordance with aspects of the present disclosure.
`
`Figure 14B is a schematic graph of fluorescence intensity data that may be
`
`collected from five sets of partitions (lanes 1-5) in a single detection channel for the
`
`multiplexed digital assay of Figure 14A, with each set containing the same
`
`concentrations of targets A and B and of both pairs of primers, and a variable
`
`concentration of the probe (PB) for target B,
`
`in accordance with aspects of the
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`present disclosure.
`
`Figure 15 is a schematic view of a partition from another exemplary
`
`multiplexed digital assay performed with two detection channels, with the partition
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`containing a copy of a masked target (A), a second target (B), and a masking target
`
`(C), each amplifiable with a different pair of fon/vard and reverse primers (FA and RA,
`
`F3 and R3, or F0 and Rc) and detectable via a generic reporter (e.g., an interca|ating
`
`dye) and specific probes (PB and Pc),
`
`in accordance with aspects of the present
`
`disclosure.
`
`Figure 16 is a schematic scatter plot of amplification data that may be
`
`collected from the multiplexed digital assay of Figure 15 in a first channel
`
`that
`
`detects amplification of targets A and C and in a second channel
`
`that detects
`
`amplification of target B, with five distinct clusters of target-positive partitions
`
`numbered sequentially and identified according to target content, in accordance with
`
`aspects of the present disclosure.
`
`Figure 17 is another schematic scatter plot of amplification data that may be
`
`collected from the multiplexed digital assay of Figure 15 and plotted as in Figure 16,
`
`except that two of the clusters ((A) and (A8» of Figure 16 are no longer resolved and
`
`form a heterogeneous cluster with heterogeneous target content.
`
`Figure 18 is another schematic scatter plot of amplification data that may be
`
`collected from the multiplexed digital assay of Figure 15, but with a first channel that
`
`detects amplification of targets A and C and a second channel
`
`that detection
`
`amplification of targets A and B, with four distinct clusters of target-positive partitions
`
`numbered sequentially and identified according to target content, in accordance with
`
`aspects of the present disclosure.
`
`Figure 19 is a schematic view of a partition from an exemplary multiplexed
`
`digital assay to determine the quality of a library, with the partition containing a copy
`
`of an empty library member (an adapter-adapter inverted repeat with no insert) and a
`
`copy of a library member containing an insert, with each library member being
`
`amplifiable with two copies of the same primer (F) and detectable with an adapter-
`
`specific probe (P), in accordance with aspects of the present disclosure.
`
`Figure 20 is a graph of amplification data collected from the multiplexed assay
`
`of Figure 19 performed in droplets.
`
`Figure 21 is a schematic view of a partition from an exemplary multiplexed
`
`digital assay to quantify spliced and unspliced species in a sample, with the partition
`
`containing a copy of an unspliced species (Exon-lntron-Exon) and a spliced species
`
`(Exon-Exon), with each species being amplifiable with the same pair of forward and
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`reverse primers (F and R) and detectable via a generic reporter, in accordance with
`
`aspects of the present disclosure.
`
`Figure 22 is a graph of amplification data collected from the multiplexed assay
`
`of Figure 21 performed in droplets.
`
`Figure 23 is a schematic view of a partition from another exemplary
`
`multiplexed digital assay to quantify spliced and unspliced species in a sample, with
`
`the partition containing a copy of an unspliced species (Exon-lntron-X-lntron-Exon)
`
`and two distinct spliced species (Exon-X-Exon and Exon-Exon), with each species
`
`being amplifiable with the same pair of forward and reverse primers (F and R) and
`
`detectable via a generic reporter,
`
`in accordance with aspects of the present
`
`disclosure.
`
`Figure 24 is a graph of amplification data collected from the multiplexed assay
`
`of Figure 23.
`
`Figure 25 is a schematic view of a partition from an exemplary multiplexed
`
`digital assay to quantify a pair of targets (targets A and B) in partitions, with the
`
`depicted partition containing a copy of each target and only a single reporter that
`
`binds to only one of the targets (target A),
`
`in accordance with aspects of the present
`
`disclosure.
`
`Figure 26 is a schematic plot of amplification data that may be collected from
`
`the multiplexed digital assay of Figure 25 in a pair of optical channels, with only one
`
`of the channels detecting the single reporter, and with the plot having three distinct
`
`clusters of partitions numbered sequentially (0, 1, and 2) and identified according to
`
`target content, in accordance with aspects of the present disclosure.
`
`Detailed Description
`
`The present disclosure provides a multiplexed digital assay system, including
`
`methods and apparatus,
`
`for calculating a level of one or more targets.
`
`In an
`
`exemplary method, data for amplification of a plurality of targets including a first
`
`target may be collected from partitions. A level of the first target may be calculated
`
`from only a subset of the data that excludes partitions according to target content.
`
`An exemplary method of performing a multiplexed digital assay is provided. In
`
`the method, a mixture may be provided. The mixture may include a plurality of
`
`targets and reagents sufficient for amplification of each of the targets, with the
`
`plurality of targets including a first target. Partitions may be formed, with each
`
`partition including a portion of the mixture. The plurality of targets may be amplified
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`in the partitions. Data for amplification of each of the plurality of targets may be
`
`collected from a plurality of the partitions. A level of the first target may be calculated
`
`from only a portion of the data. The plurality of targets also may include a second
`
`target. The portion of the data may or may not exclude each partition testing positive
`
`for the second target.
`
`In some embodiments, partition populations may be identified in the data, with
`
`each partition population having a different target content of the plurality of targets.
`
`The portion of the data may be collected from only a subset of the plurality of the
`
`partitions, and the subset of partitions may exclude two or more of the partition
`
`populations. The two or more excluded partition populations may include at least a
`
`pair of excluded partition populations that are not resolved from each other in the
`
`data. The at least a pair of excluded populations may be heterogeneous with respect
`
`to the first target.
`
`In some embodiments,
`
`the data may be plottable to form three or more
`
`clusters of data points. Each data point may represent a partition. Each cluster may
`
`have a different content of the plurality of targets. The level of the first target may be
`
`calculated from only a portion of the data that excludes at least one of the clusters of
`
`data points. The at
`
`least one excluded cluster may include a heterogeneous
`
`excluded cluster composed of overlapping sets of data points representing at least
`
`two excluded partition populations. At least one of the excluded partition populations
`
`may be positive for the first
`
`target, and at
`
`least one of the excluded partition
`
`populations may be negative for the first target.
`
`The digital assays of the present disclosure may have numerous advantages,
`
`such as higher levels of multiplexing, multiplexing with a generic reporter alone or in
`
`combination with at least one specific reporter, determination of target levels (e.g.,
`
`concentrations) with partition populations that are indistinguishable and/or not well
`
`resolved in the data, determination of target levels using competing assays within
`
`partitions, or any combination thereof, among others.
`
`Further aspects of the present disclosure are presented in the following
`
`sections:
`
`(l) overview of multiplexed digital assays with a generic reporter,
`
`(ll)
`
`determination
`
`of
`
`concentration for masked
`
`targets,
`
`(lll)
`
`exemplary assay
`
`configurations with target masking, (IV) multiplexed assays with a specific reporter
`
`and a generic reporter, and (V) examples.
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`I.
`
`Overview of Multiplexed Digital Assays with a Generic Reporter
`
`This section provides an overview of multiplexed digital assays performed
`
`with a generic reporter and, optionally, in which the presence of a target in a partition
`
`is at least partially masked by the presence of a different target in the same partition;
`
`see Figures 1 and 2. The digital assays may utilize data exclusion as described
`
`Section II and elsewhere herein to calculate a level of one or more of the targets.
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`Figure 1 shows a flowchart of an exemplary method 50 of performing a
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`multiplexed digital assay with a generic reporter, optionally with target masking. The
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`steps presented for method 50 may be performed in any suitable order and in any
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`suitable combination. Furthermore, the steps may be combined with and/or modified
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`by any other suitable steps, aspects, and/features of the present disclosure,
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`including those described in the patent documents listed above under Cross-
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`References, which are incorporated herein by reference.
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`Sample preparation. A sample may be prepared for the assay,
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`indicated at
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`52. Preparation of the sample may include any suitable manipulation of the sample,
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`such as collection, dilution, concentration, purification,
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`lyophilization,
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`freezing,
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`extraction, combination with one or more assay reagents to form a mixture (also
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`termed a sample-containing mixture,
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`a bulk phase, or a
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`reaction mixture),
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`performance of at least one preliminary reaction to prepare the sample for one or
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`more reactions in the assay, or any combination thereof, among others. The
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`preparation may isolate an analyte, such as nucleic acid that includes copies of one
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`or more nucleic acid targets, and/or may modify and/or fragment
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`the analyte.
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`Preparation of
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`the sample may include rendering the sample competent
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`for
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`subsequent performance of one or more reactions, such as one or more enzyme
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`catalyzed reactions and/or binding reactions.
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`In some embodiments, preparation of the sample may include combining the
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`sample with reagents to produce a sample-containing mixture for performing a
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`reaction (such as an amplification reaction) for each target and for reporting an
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`extent of each reaction (e.g., whether or not the reaction occurred above a threshold
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`level or within a range). Reagents for amplification may include any combination of
`
`primers for targets, dNTPs and/or NTPs, at least one enzyme (e.g., a polymerase, a
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`ligase, a reverse transcriptase, a restriction enzyme, or a combination thereof,
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`among others, each of which may or may not be heat-stable), and/or the like.
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`Accordingly, the mixture may have a complete set of reagents for (Le, may be
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`competent for) amplification of each target under suitable environmental conditions
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`(e.g., incubation at an elevated temperature or modulation of temperature (such as
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`by thermocycling)). The mixture may be capable of amplification of each of one or
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`more targets, if present, in the sample (or a partition thereof). Reagents for reporting
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`may include at least one generic reporter and/or at least one specific reporter. The
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`generic reporter may be sensitive to amplification of each target and the specific
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`reporter may be specifically sensitive to amplification of only a subset of the targets,
`
`such as only one of the targets. The mixture may or may not include a different
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`reporter for each target to be assayed. Preparation of the mixture may render the
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`sample capable of reporting, or being analyzed for, whether or not a reaction, such
`
`as amplification, has occurred, on a target-by-target basis, and optionally the extent
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`of any such reaction.
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`Providing partitions. Partitions for the assay may be provided, indicated at 54.
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`Each partition may include a portion of a same mixture.
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`In some cases, the portion
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`may constitute the entire partition. The mixture may contain each target (e.g.,
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`provided by a same sample), each reporter, and/or one or more amplification
`
`reagents (e.g., a complete set of reagents for amplification of each target).
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`Accordingly, the partitions, collectively, may contain a plurality of targets and each
`
`partition may contain the same generic reporter and optionally the same specific
`
`reporter. The targets may include at least one maskable target (interchangeably
`
`termed a masked target), and at least one obscuring target (interchangeably termed
`
`a masking target or a dominant target). The obscuring target is capable of at least
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`partially masking the presence of one or maskable targets when a copy of each
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`target is present in the same partition.
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`The partitions when provided (e.g., when formed) may contain each target at
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`“partial occupancy,” which means that each partition of only a subset of the partitions
`
`contains at least one copy of each target (and/or template) to be assayed. For
`
`example, with a multiplexed assay performed on a first target and a second target,
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`only a first subset of the partitions contains the first target, and only a second subset
`
`of the partitions contains the second target. The first subset and the second subset
`
`of the partitions may be the same subset, if the first target and the second target are
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`fully associated with and/or linked to each other when the partitions are formed.
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`Alternatively, the first subset and the second subset of the partitions may be distinct
`
`if the first target and the second target are not fully associated with and/or linked to
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`each other when the partitions are formed. In some cases, if the targets are not fully
`
`associated and/or linked, each partition of a distinct third subset of the partitions may
`
`contain at least one copy of each target. Accordingly, with partial occupancy, one or
`
`more (e.g., a plurality) of the partitions contain no copies of the first target, one or
`
`more (e.g., a plurality) of the partitions may contain a single copy (only one copy) of
`
`the first target, and, optionally, yet one or more of the partitions (e.g., the rest of the
`
`partitions) may contain two or more copies of the first target. Similarly, with partial
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`occupancy, one or more (e.g., a plurality) of the partitions contain no copies of the
`
`second target, one or more (e.g., a plurality) of the partitions may contain a single
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`copy (only one copy) of the second target, and, optionally, yet one or more of the
`
`partitions (e.g., the rest of the partitions) may contain two or more copies of the
`
`second target.
`
`The term “partial occupancy” is not restricted to the case where there is no
`
`more than one copy of a particular template/target of interest
`
`in any partition.
`
`Partitions containing a template and/or a target at partial occupancy may,
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`for
`
`example, contain an average of more than, or less than, about one copy, two copies,
`
`or three copies, among others, of the template/target per partition when the partitions
`
`are provided or formed. Copies of a template (and/or target) may have a random
`
`distribution among the partitions, which may be described as a Poisson distribution.
`
`In some cases, a significant number of the partitions (e.g., at least about 1%, 2%,
`
`5%, 10%, or 20%, among others, of the partitions) may contain a copy of each of at
`
`least two targets, and/or a plurality of the partitions each may contain at least one
`
`copy of all targets.
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`Targets may be unlinked, partially linked, or fully linked when partitions are
`
`formed. Linked targets may be attached to each other covalently and/or by base
`
`pairing.
`
`Each target may be an analyte on interest (e.g., a nucleotide sequence of
`
`interest) or a surrogate therefor (e.g., a nucleic acid bound to and/or corresponding
`
`to a nucleotide sequence of interest). The target may be nucleic acid that includes a
`
`sequence of nucleotides. The target,
`
`if nucleic acid, may be single-stranded or
`
`double-stranded, among others. A nucleic acid target may be provided by a
`
`template, with the target forming at least a portion or all of the template. The target
`
`may correspond to an amplicon produced by amplification. The amplicon may be
`
`single-stranded or double-stranded, among others. In some cases, the target may be
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`or correspond to an analyte that is not nucleic acid, such as a small molecule, a
`
`polypeptide, a lipid, an amino acid, an ion, or the like.
`
`The partitions may be provided by distributing or separating portions of a
`
`sample-containing bulk phase into partitions. Any suitable fraction including up to all
`
`of the bulk phase may be distributed to the partitions. Each partition may be and/or
`
`include a fluid volume that is isolated from the fluid volumes of other partitions. The
`
`partitions may be isolated from one another by a fluid phase, such as a continuous
`
`phase of an emulsion, by a solid phase, such as at least one wall of a co