DIGITAL ASSAYS WITH COMBINATORIAL
`USE OF SIGNALS
`
`Cross-References
`
`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; and Joseph R.
`
`Lakowicz, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY(24 Ed. 1999).
`
`Introduction
`
`10
`
`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,
`
`15
`
`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
`
`20
`
`be estimated from the probability of finding a given number of copiesin 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 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
`
`
`USE OF SIGNALS
`
`Cross-References
`
`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; and Joseph R.
`
`Lakowicz, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY(24 Ed. 1999).
`
`Introduction
`
`10
`
`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,
`
`15
`
`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
`
`20
`
`be estimated from the probability of finding a given number of copiesin 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 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 in a Poisson equation to determine the
`
`concentration of the analyte in the partitions.
`
`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
`
`10
`
`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 dye
`
`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
`
`15
`
`different targets within each partition. Amplification of the targets can be distinguished
`
`by
`
`utilizing
`
`target-specific probes
`
`labeled with different dyes, which produce
`
`fluorescence detected in different detection channels, namely, at different wavelengths
`
`or wavelength regions
`
`(“colors”).
`
`If a detector
`
`for a digital PCR assay can
`
`distinguishably measure the fluorescence emitted by N different dyes, then the assayis
`
`20
`
`effectively capable of measuring N different targets. However, instruments with more
`
`detection channels, to detect more colors, are more expensive than those with fewer
`
`detection channels. Also, increasing the number of distinguishable dyes is expensive
`
`and becomes impractical beyond a certain number. On the other hand, many
`
`
`
`applications, especially where sample is limited, could benefit greatly from higher
`
`degrees of multiplexing.
`
`A new approachis needed to increase the multiplex levels of digital assays.
`
`Summary
`
`The present disclosure provides a system, including method and apparatus, for
`
`performing a digital assay on a greater number of targets through combinatorial use of
`
`signals.
`
`Brief Description of the Drawings
`
`Figure 1
`
`is a flowchart of an exemplary method of performing a digital assay, in
`
`10
`
`accordancewith aspects of the present disclosure.
`
`Figure 2 is a schematic view of an exemplary system for performing the digital
`
`assay of Figure 1, in accordance with aspects of the present disclosure.
`
`Figure 3 is a schematic view of a pair of targets and corresponding probes
`
`capable of reporting the presence or absence ofthe targets via emitted light that may be
`
`15
`
`detected to create a dedicated signal
`
`for each target
`
`in a digital PCR assay,
`
`in
`
`accordancewith aspects of the present disclosure.
`
`Figure 4 is a pair of exemplary graphs of respective dedicated signals that may
`
`be created by detecting light emitted from the probes of Figure 3 in a digital PCR assay
`
`performed in droplets, with each signal created from light detected over the same time
`
`20
`
`interval from a fluid stream containing the droplets,
`
`in accordance with aspects of the
`
`present disclosure.
`
`
`
`Figure 5 is a schematic representation of how the pair of targets of Figure 3 are
`
`distributed among the droplets from which light
`
`is detected in Figure 4, based on
`
`intensity of the respective dedicated signals of Figure 4,
`
`in accordance with aspects of
`
`present disclosure.
`
`Figure 6 is a schematic view of three targets and corresponding exemplary
`
`probes capable of reporting the presence or absenceof the targets via emitted light that
`
`may be detected to create a pair of composite signals in a digital PCR assay,
`
`in
`
`accordancewith aspects of the present disclosure.
`
`Figure 7 is a pair of exemplary graphs of a pair of composite signals that may be
`
`10
`
`created by detecting fluorescence emission from the three probesof Figure 6 in a digital
`
`PCR assay performed in droplets, with emitted light detected in each graph over the
`
`same time interval from a fluid stream containing the droplets,
`
`in accordance with
`
`aspects of the present disclosure.
`
`Figure 8 is a schematic representation of how the three targets of Figure 6 are
`
`15
`
`distributed among the droplets from which light
`
`is detected in Figure 7, based on
`
`intensity of the respective composite signals of Figure 7, in accordance with aspects of
`
`present disclosure.
`
`Figure 9 is a schematic view ofthe third target of Figure 6 and another exemplary
`
`probe configuration capable of reporting the presence or absenceofthe third target via
`
`20
`
`emitted light, which may be used in conjunction with the other probes of Figure 6 to
`
`create only a pair of composite signals representing the three targets in a digital PCR
`
`assay, in accordance with aspects of the present disclosure.
`
`
`
`Figure 10 is a schematic view of the third target of Figure 6 and yet another
`
`exemplary probe configuration specific for the third target, which may be used in
`
`conjunction with the other probes of Figure 6 to create only a pair of composite signals
`
`representing the three targets in a digital PCR assay, in accordance with aspects of the
`
`present disclosure.
`
`Figure 11 is a schematic view of three targets and corresponding exemplary
`
`primers that enable use of only two probes to report the presence or absence of the
`
`targets in a digital PCR assay, in accordance with aspects of the present disclosure.
`
`Figure 12 is a schematic view of the third target of Figure 6 and another
`
`10
`
`exemplary probe and primer configuration that enables use of only two probesto report
`
`the presence or absence of three targets in a digital PCR assay,
`
`in accordance with
`
`aspects of the present disclosure.
`
`Figure 13 is a schematic view of the three targets of Figure 6 with yet another
`
`exemplary configuration of only two probes that enables use of only two probes to
`
`15
`
`report the presence or absenceof three targets in a digital PCR assay, in accordance
`
`with aspects of the present disclosure.
`
`Detailed Description
`
`The present disclosure provides a system, including method and apparatus, for
`
`performing a digital assay on a greater number of targets through combinatorial use of
`
`20
`
`signals.
`
`
`
`A method of performing a digital assay for more thanNtargets is provided. In the
`
`method, a sample is separated into partitions. N signals are created, with the N signals
`
`being representative of light detected from the partitions. A concentration is estimated of
`
`more than N targets in the partitions based on the N signals.
`
`Further aspects of the present disclosure are presented in the following sections:
`
`(I) system overview,and (Il) examples.
`
`I.
`
`System Overview
`
`Figure 1 shows a flowchart of an exemplary method 40 of performing a digital
`
`assay. The steps presented for method 40 may be performed in any suitable order and
`
`10
`
`in any suitable combination. Furthermore,
`
`the steps may be combined with and/or
`
`modified by any other suitable steps, aspects, and/features of the present disclosure.
`
`A sample may be prepared for the assay,
`
`indicated at 42. Preparation of the
`
`sample may include any suitable manipulation of the sample, such as collection,
`
`dilution, concentration, purification, lyophilization, freezing, extraction, combination with
`
`15
`
`one or more assay reagents, performance of at
`
`least one preliminary reaction to
`
`prepare the sample for one or more reactions in the assay, or any combination thereof,
`
`among others. Preparation of the sample may include rendering the sample competent
`
`for subsequent performance of one or more reactions, such as one or more enzyme
`
`catalyzed reactions and/or binding reactions.
`
`20
`
`In some embodiments, preparation of the sample may include combining the
`
`sample with reagents for amplification and for reporting whether or not amplification
`
`occurred. Such reagents may include any combination of primers for the targets,
`
`reporters (e.g., probes) for the targets, dNTPs and/or NTPs, at least one enzyme(e.g.,
`
`
`
`a polymerase, a ligase, a reverse transcriptase, or a combination thereof, each of which
`
`may or may not be heat-stable), or the like. Accordingly, preparation of the sample may
`
`render the sample (or partitions thereof) capable of amplification of each of one or more
`
`targets, if present, in the sample (or a partition thereof).
`
`The sample may be separated into partitions, indicated at 44. Separation of the
`
`sample mayinvolvedistributing any suitable portion or all of the sample to the partitions.
`
`Each partition may be and/or include a fluid volume that
`
`is isolated from the fluid
`
`volumesof other partitions. The partitions may be isolated from one another byafluid
`
`phase, such as a continuous phase of an emulsion, by a solid phase, such as at least
`
`10
`
`one wall of a container, or a combination thereof, among others. In some embodiments,
`
`the partitions may be droplets disposed in a continuous phase, such that the droplets
`
`and the continuous phasecollectively form an emulsion.
`
`The partitions may be formed by any suitable procedure, in any suitable manner,
`
`and with any suitable properties. For example, the partitions may be formed with a fluid
`
`15
`
`dispenser, such as a pipette, with a droplet generator, by agitation of the sample (e.g.,
`
`shaking, stirring, sonication, etc.), or the like. Accordingly, the partitions may be formed
`
`serially, in parallel, or in batch. The partitions may have any suitable volume or volumes.
`
`The partitions may be of substantially uniform volume or may have different volumes.
`
`Exemplary partitions having substantially the same volume are monodisperse droplets.
`
`20
`
`Exemplary volumesfor the partitions include an average volumeof less than about 100,
`
`10 or 1 uL, less than about 100, 10, or 1 nL, or less than about 100, 10, or 1 pL, among
`
`others.
`
`
`
`The partitions, wnen formed, may be competent for performance of one or more
`
`reactions in the partitions. Alternatively, one or more reagents may be added to the
`
`partitions after they are formed to render them competent for reaction. The reagents
`
`may be added by any suitable mechanism, such as a fluid dispenser, fusion of droplets,
`
`or the like.
`
`One or more reactions may be performed in the partitions, indicated at 46. Each
`
`reaction performed may occur selectively (and/or substantially) in only a subset of the
`
`partitions, such as less than about one-half, one-fourth, or one-tenth of the partitions,
`
`among others. The reaction may involve a target, which may, for example, be a
`
`10
`
`template and/or a reactant (e.g., a substrate), and/or a binding partner, in the reaction.
`
`The reaction may occur selectively (or selectively may not occur) in partitions containing
`
`at least one copyof the target.
`
`The reaction may or may not be an enzyme-catalyzed reaction.
`
`In some
`
`examples, the reaction may be an amplification reaction, such as a polymerase chain
`
`15
`
`reaction and/or ligase chain reaction. Accordingly, a plurality of amplification reactions
`
`for a plurality targets may be performed simultaneously in the partitions.
`
`Performing a reaction may include subjecting the partitions to one or more
`
`conditions that promote occurrence of the reaction. The conditions may include heating
`
`the partitions and/or incubating the partitions at a temperature above room temperature.
`
`20
`
`In some examples,
`
`the conditions may include thermally cycling the partitions to
`
`promote a polymerasechain reaction and/orligase chain reaction.
`
`
`
`N signals may be created that are representative of light detected from the
`
`partitions, indicated at 48. The N signals may be2, 3, 4, or more signals, which may be
`
`created for each of the partitions. In some examples, light corresponding to each signal
`
`may be detected with a distinct sensor and/orlight corresponding to at least two signals
`
`may be detectedat different times with the same sensor. The N signals may correspond
`
`to light detected in respective wavelength ranges (“colors”) that are different from one
`
`another. The light detected may belight emitted from a fluorophore.
`
`Each of
`
`the N signals may be created in a distinct detection channel.
`
`Accordingly, N signals may be created in N detection channels.
`
`10
`
`Each signal may be a composite signal that represents two, three, four, or more
`
`reactions and thus two, three, four, or more targets of the reactions. The composite
`
`signal may include two or more integral signal portions that each represent a different
`
`reaction and target. Analysis of one of the composite signals by itself, without the
`
`benefit of data from the other composite signals, may (or may not) permit estimation of
`
`15
`
`a collective concentration, but not
`
`individual concentrations, for two or more targets
`
`represented by the composite signal. Instead, as described further below, analysis of
`
`the composite signals together permits estimation of the concentration of each target.
`
`The N composite signals (and/or N detection channels) may represent more than
`
`N reactions and/or targets, with the number of reactions and targets depending on
`
`20
`
`configuration. For example, the N signals may be or include two composite signals
`
`(arbitrarily termed a and B) collectively representing three reactions and/or three targets
`
`(arbitrarily termed 1, 2, and 3), with each composite signal representing a different
`
`combination of two reactions/targets (e.g., targets 1 and 2 for a and targets 1 and 3 for
`
`
`
`6). Alternatively, the N signals may be three composite signals (arbitrarily termed a, B,
`
`and y) collectively representing up to seven reactions/targets (1 to 7), if each composite
`
`signal represents a different combination of up to four reactions/targets each (e.g.,
`
`targets 1, 2, 3, and 4 for a; targets 2, 4, 5, and 6 for 6; and targets 3, 4, 6, and 7 for y).
`
`The
`
`N_
`
`signals may be
`
`four
`
`composite
`
`signals
`
`representing
`
`up
`
`to
`
`fifteen
`
`reactions/targets,
`
`if each composite signal represents a different combination of up to
`
`eight reactions/targets each.
`
`More generally, up to 2“-1 targets can be assayed with N composite signals (or
`
`detection channels) collectively representing up to 2" targets. To assay up to 2™-1
`
`10
`
`targets with N detection channels, the targets may be distributed among the detection
`
`channels with each target represented by a different detection channel or combination
`
`of detection channels than every other target. A maximum of 2-4 targets can be
`
`represented and assayed when all of the detection channels have been utilized
`
`individually and in all possible combinations.
`
`15
`
`Each composite signal may be created based on detected light emitted from one
`
`or more reporters in the partitions. The one or more reporters may report whether at
`
`least one of two or more particular reactions represented by the signal has occurred in a
`
`partition and thus whether at least one copy of at least one of two or more particular
`
`targets corresponding to the two or moreparticular reactions is present in the partition.
`
`20
`
`The strength of a composite signal corresponding to the reporters may be analyzed to
`
`determine whether or not at least one of the particular reactions has occurred and at
`
`least one copy of one of the particular targets is present. The strength may vary among
`
`the partitions according to whether at least one of the particular reactions occurred or
`
`10
`
`
`
`did not occur (e.g., above a threshold extent) and at least one of the particular targets is
`
`present or absent in each partition.
`
`The reporters
`
`represented by a composite signal may include different
`
`fluorophores.
`
`In other words, light emitted from different fluorophores may be detected
`
`to create at two different
`
`integral portions of the composite signal for a particular
`
`detection channel. Alternatively, or in addition, the same fluorophore maybeincludedin
`
`one reporter or two or more reporters for at least two targets represented by the
`
`composite signal.
`
`In some cases, the same fluorophore may be included in a reporter
`
`for each target represented by the composite signal.
`
`10
`
`Each reporter may be a probe that
`
`includes a nucleic acid (eg., an
`
`oligonucleotide) and a fluorophore. Different probes may be used to create at least two
`
`different integral portions of the signal. Alternatively, or in addition, the same probe may
`
`be used as a reporter for at least two targets represented by the composite signal.
`
`In
`
`some cases, the same probe may be used as a reporter for each target represented by
`
`15
`
`the composite signal (e.g., see Examples 3-5).
`
`The composite signal detected from each partition, and the partition itself, may
`
`be classified as being positive or negative for the reactions/targets represented by the
`
`signal or corresponding detection channel. Classification may be based on the strength
`
`of the signal. If the signal/partition is classified as positive, at least one of the reactions
`
`20
`
`represented by the signal is deemed to have occurred and at least copy of at least one
`
`of the targets represented by the signal
`
`is deemed to be present in the partition.
`
`In
`
`contrast,
`
`if
`
`the signal/partition is classified as negative, none of
`
`the reactions
`
`11
`
`
`
`represented by the signal is deemed to have occurred and no copy of any of the targets
`
`represented by the signal is deemed to be present in the partition.
`
`The composite signals collectively permit estimation of target concentrations by
`
`representation of a different combination of
`
`targets in each detection channel.
`
`Accordingly, each target, when present without any of the other targets in a partition,
`
`may produce a unique target signature of positive signals among the detection
`
`channels. For example, some of the targets,
`
`if present alone in a partition, may
`
`selectively change the signal strength in only one detection channel. Others of the
`
`targets, if present alone in a partition, may selectively change the signal strength in a
`
`10
`
`unique combination of two of the detection channels, still other targets may selectively
`
`change the signal strength in a unique combination of three of the detection channels,
`
`and so on, optionally up to the number of detection channels. Generally, in a fraction of
`
`the partitions, each partition may contain a copy of each of two or more distinct targets,
`
`which, for a particular partition, collectively may produce a signature that is the same as
`
`15
`
`that of a target not present in the partition. However, the fraction of partitions containing
`
`two or more distinct targets may be keptrelatively low, by working in a dilute regime,
`
`such as with less than about one-half, one-fifth, or one-tenth, among others, of the
`
`partitions containing more than one target molecule. In any event, a suitable estimation
`
`of concentration, as described below, may take into account the occurrence of two or
`
`20
`
`more target molecules,
`
`from the same or different
`
`targets,
`
`in the same partition.
`
`Alternatively,
`
`if working in a sufficiently dilute regime, the occurrence of two or more
`
`target molecules per partition may be sufficiently rare to ignore for a desired accuracy of
`
`concentrations.
`
`12
`
`
`
`A number of partitions that are positive may be determined for each signal,
`
`indicated at 50. For example, a number of partitions that are positive only for each
`
`particular composite signal or corresponding detection channel may be determined
`
`individually (e.g., counted) for each signal or channel(i.e., a number for each channel).
`
`Also, a number of partitions that are positive only for each particular combination (or at
`
`least one combination or each of two or more combinations) of composite signals or
`
`corresponding detection channels may be determined individually (e.g., counted) for
`
`each combination of signals or channels (i.e., a number for each combination, and
`
`particularly each combination corresponding to a particular target).
`
`10
`
`A distinct fraction of the partitions positive for each signal alone and for each
`
`signal combination may be determined. The fraction for each signal or signal
`
`combination may be determined by dividing the number of partitions
`
`for
`
`the
`
`signal/combination, determined at 50, by the total number of partitions from which
`
`signals are detected. The total number ofpartitions may be counted or estimated.
`
`15
`
`A concentration of each target may be estimated, indicated at 52. Generally, if N
`
`signals are detected from the partitions in N detection channels, the concentration of
`
`each of more than N targets (e.g., up to 2%-1
`
`targets) may be estimated. The
`
`concentration of each target may be estimated based on the respective numbers of
`
`partitions positive for each signal alone and signal combination. The calculation may be
`
`20
`
`based on each target having a Poisson distribution among the droplets. The
`
`concentrations may, for example, be estimated by finding solutions to a series of linear
`
`equations. Further aspects of estimating concentrations are described in
`
`the
`
`Appendices referenced in Example 6.
`
`13
`
`
`
`Figure 2 shows an exemplary system 60 for performing the digital assay of
`
`Figure 1. System 60 mayinclude a partitioning assembly, such as a droplet generator
`
`62 (“DG”), a thermal incubation assembly, such as a thermocycler 64 (“TC”), a detection
`
`assembly (a detector) 66 (“DET”), and a data processing assembly (a processor) 68
`
`(“PROC”), or any combination thereof, among others. The data processing assembly
`
`may be, or may be included in, a controller that communicates with and controls
`
`operation of any suitable combination of the assemblies. The arrows between the
`
`assemblies indicate movement or transfer of material, such as fluid (e.g., a continuous
`
`phase of an emulsion) and/or partitions (e.g., droplets) or signals/data, between the
`
`10
`
`assemblies. Any suitable combination of the assemblies may be operatively connected
`
`to one another, and/or one or more of the assemblies may be unconnected to the other
`
`assemblies, such that, for example, material/data is transferred manually.
`
`System 60 may operate as follows. Droplet generator 62 may form droplets
`
`disposed in a continuous phase. The droplets may be cycled thermally with
`
`15
`
`thermocycler 64 to promote amplification of targets in the droplets. Composite signals
`
`may be detected from the droplets with detector 66. The signals may be processed by
`
`processor68 to estimate concentrations of the targets.
`
`ll.
`
`Examples
`
`This section presents selected aspects and embodiments of
`
`the present
`
`20
`
`disclosure related to methods of performing digital assays with combinatorial use of
`
`signals each corresponding to multiple targets.
`
`14
`
`
`
`Example 1. Digital PCR Assays with Dedicated Signals and Composite Signals
`
`This example compares and contrasts exemplary digital PCR assaysutilizing (i)
`
`a pair of dedicated signals for two targets, see Figures 3-5, and(ii) a pair of composite
`
`signals for three targets, see Figures 6-8. The principles explained here may be
`
`extended to N signals for up to 2-1 targets.
`
`Figure 3 shows a pair of targets 80, 82 (“Target 1” and “Target 2”) and
`
`corresponding probes 84, 86 (“Probe 1” and “Probe 2”) that may be used to create a
`
`dedicated signal for each target in a digital PCR assay. Each probe may include an
`
`oligonucleotide 88, 90, a fluorophore 92, 94, and a quencher 96. Each of the
`
`10
`
`fluorophore and the quencher may (or may not) be conjugated to the oligonucleotide by
`
`a covalent bond. The probe also or alternatively may include a binding moiety (a minor
`
`groove binder) for the minor groove of a DNA duplex, which may be conjugated to the
`
`oligonucleotide and may function to permit a shorter oligonucleotide to be used in the
`
`probe.
`
`15
`
`Each
`
`oligonucleotide may
`
`provide
`
`target
`
`specificity
`
`by
`
`hybridization
`
`predominantly or at
`
`least substantially exclusively to only one of the two targets.
`
`Hybridization
`
`of
`
`the oligonucleotide
`
`to
`
`its
`
`corresponding target
`
`is_
`
`illustrated
`
`schematically at 98.
`
`Fluorophores 92, 94 may be optically distinguishable from each other, as
`
`20
`
`illustrated schematically by a distinct hatch pattern for each fluorophore. For example,
`
`the fluorophores may have distinct absorption spectra and/or maxima, and/or distinct
`
`emission spectra and/or emission maxima. Accordingly, proper selection of
`
`the
`
`wavelength or wavelength band of excitation light used for each detection channel
`
`15
`
`
`
`and/or the wavelength or wavelength band of emitted light received and sensed by the
`
`sensor for the detection channel provides selective detection of light from only one of
`
`the fluorophores in the detection channel. Exemplary fluorophores that may be suitable
`
`include FAM, VIC, ROX, TAMRA,JOE, etc.
`
`Quencher 96 is configured to quench the signal produced by fluorophore 92 or
`
`94 in a proximity-dependent fashion. Accordingly,
`
`light detected from the fluorophore
`
`may increase when the associated oligonucleotide 88 or 90 binds to the amplified
`
`target, to increase the separation between the fluorophore and the quencher, or when
`
`the probeis cleaved during target amplification, among others.
`
`10
`
`Figure 4 shows a pair of exemplary graphs 102, 104 of data collected in an
`
`exemplary digital PCR assay for Target 1 and Target 2 performed in droplets. Each
`
`graph plots a dedicated signal 106 (“Signal 1”) or signal 108 (“Signal 2”) that represents
`
`light detected from respective probes 84, 86 (and/or one or more modified (e.g.,
`
`cleavage) products thereof) (see Figure 3). Each dedicated signal is created from light
`
`15
`
`detected over the same time interval from a fluid stream containing the droplets and
`
`flowing through an examination region of a channel. Signal
`
`1 reports whether or not
`
`Target 1
`
`is present in each droplet, and Signal 2 reports whether or not Target 2 is
`
`present in each target. In particular, if strength of Signal 1 (or Signal 2) increases above
`
`a threshold 110, then Target 1 (or Target 2) is deemed to be presentin a corresponding
`
`20
`
`droplet.
`
`In the present illustration, each droplet, whether positive or negative for each
`
`target, produces an increasein signal strength above the baseline signal that forms an
`
`identifiable peak 112. Accordingly, each signal may vary in strength with the presence
`
`or absenceof a droplet and with the presence or absenceof a corresponding target.
`
`16
`
`
`
`Each target is present here at a frequency of about 0.2 in the droplets.
`
`In other
`
`words, each target is detected on average about once every five droplets. Accordingly,
`
`the frequency of droplets containing both targets is the product of the two frequencies,
`
`or about 0.04 (1 out of every 25 droplets). Consistent with this frequency, a droplet that
`
`is positive for both targets is present only once on the twenty droplets analyzed here,
`
`and is indicated by a dashed box at 114 aroundits positive signals in graphs 102, 104.
`
`Figure 5 schematically represents the distribution of Targets 1 and 2 in a set of
`
`droplets 116 corresponding to and in the same order as the droplet signals of Figure 4.
`
`Droplets positive for Signal 1, such as the droplet
`
`indicated at 118, are hatched
`
`10
`
`according to fluorophore 92, and droplets positive for Signal 2, such as the droplet
`
`indicated at 120, are hatched according to fluorophore 94 (see Figure 3). A double-
`
`positive droplet 122 containing both Target1 and Target 2 is double-hatched and
`
`indicated by dashed box 114.
`
`Figure 6 shows three targets 80, 82, and 140 and corresponding exemplary
`
`15
`
`probes 84, 86, and 142, respectively, that may be used to create a pair of composite
`
`signals for the three targets in a digital PCR assay. Two of the targets and probes,
`
`namely, targets 80 and 82 (Target 1 and Target 2) and probes 84 and 86 (Probe 1 and
`
`Probe 2) are the sametargets and probes shown and utilized in Figures 3-5. Target 140
`
`(Target 3) and its corresponding probe 142 (Probe 3) may be introduced into the assay
`
`20
`
`to increase the level of multiplexing and the amount of target information that can be
`
`extracted from the assay without increasing the number of detection channels.
`
`17
`
`
`
`Amplification of Target 3 is
`
`reported by Probe 3. The probe includes an
`
`oligonucleotide 144 that hybridizes specifically to Target 3, relative to Targets 1 and 2.
`
`The probe may be double-labeled with the same fluorophores (92, 94) present
`
`individually in Probe 1 and Probe 2 for reporting respective Target
`
`1 and Target 2
`
`amplification. Figures 9-13, which are described in Examples 2-5,
`
`illustrate other
`
`exemplary probe configurations
`
`that may be suitable to increase the level of
`
`multiplexing.
`
`In any event, the probes for the three targets may be selected to permit
`
`detection of target amplification in only two detection channels, rather than the three
`
`detection channels that would be necessary with the use of a dedicated detection
`
`10
`
`channel for each target.
`
`Figure 7 shows a pair of exemplary graphs 152, 154 of a pair of composite
`
`signals 156, 158 that may be detected in a pair of detection channels. The composite
`
`signals, arbitrarily designated a and f, are representative of light detected from the
`
`three probes of Figure 6 in a digital PCR assay performed in droplets. Each composite
`
`15
`
`signal
`
`is created from light detected over the same time interval from a fluid stream
`
`containing the droplets. To simplify the presentation, Target 1 and Target 2 are present
`
`at the same frequency and in the samedroplets as in Figures 4 and 5.
`
`Each composite signal, a or B (156 or 158), represents a pair of targets. Signal a
`
`(graph 152) has a strength for each droplet that indicates whether the droplet is positive
`
`20
`
`or negative for at least one member of a first pair of targets, namely, Target 1 and
`
`Target 3. Signal 6 (graph 154) has a strength for each droplet that indicates whether the
`
`droplet is positive or negative for at least one member of an overlapping, but different,
`
`second pair of targets, namely, Target 2 and Target 3. Accordingly, each composite
`
`18
`
`
`
`signal analyzed by itself provides no information about how frequently each particular
`
`member of the pair of targets is present in droplets.
`
`The composite signals analyzed in combination provide additional
`
`information
`
`about target frequency that cannot be deduced from the composite signals in isolation
`
`from one another. Each target, when present without other targets in a droplet,
`
`produces a signal signature that is distinct from the signatures of each other target
`
`individually. The signature for Target
`
`1
`
`in a droplet is indicated at 160: positive for
`
`Signal a and negative for Signal 6. The signature for Target 2 in a dropletis indicated at
`
`162: negative for Signal a and positive for Signal 8. Furthermore, the signature for
`
`10
`
`Target 3 in a droplet is outlined by dashed boxes at 164: positive for both Signal a and
`
`Signal 6. Finally, the signature for none of Targets 1, 2, and 3 in a dropletis indicated at
`
`166: nega
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