([2) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(10) International Publication Number
`
`WO 2012/129187 A1
`
`\g2
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`
`27 September 2012 (27.09.2012)
`
`WIPOI PCT
`
`(51)
`
`International Patent Classification:
`
`CIZQ 1/68 (2006.01)
`
`G01N 21/00 (2006.01)
`
`(21)
`
`International Application Number:
`
`PCT/U82012/029712
`
`(22)
`
`International Filing Date:
`
`19 March 2012 (19.03.2012)
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`Filing Language:
`
`Publication Language:
`
`English
`
`English
`
`Priority Data:
`61/454,373
`
`18 March 2011 (18.03.2011)
`
`US
`
`0’01" all designated States except US): BIO-
`Applicant
`RAD LABORATORIES, INC.
`[US/US]; 1000 Alfred
`Nobel Drive, Hercules, CA 94547 (US).
`
`(72)
`(75)
`
`Inventors; and
`Inventors/Applicants for US only): SAXONOV, Serge
`[US/US]; 6676 Heartwood Drive, Oakland, CA 94611
`
`(74)
`
`(81)
`
`(US). DUBE, Simant [US/US]; 5386 Case Avenue, Apt.
`1621, Pleasanton, CA 94566 (US). HINDSON, Benjamin,
`J. [AU/US]; 1039 Bannock Street, Livermore, CA 94551
`(US). MCCOY, Adam, M. [US/US]; 1707 Van Damme
`Drive, Davis, CA 95616 (US).
`
`Agent: ABNEY, James, R.; Kolisch Hartwell, PC, 520
`SW Yamhill Street, Suite 200, Portland, OR 97204 (US).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, 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, IS, JP, KE, KG, KM, 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, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, 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.
`
`(54) Title: MULTIPLEXED DIGITAL ASSAYS WITH COMBINATORIAL USE OF SIGNALS
`
`[Continued on nextpage]
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`€52’M“
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`Signaé a
`(Targets;
`’1. 3}
`
`
`
`3156»
`=
`$2;
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`Miami
`
`’1 54/53
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`m.653'
`Signal g3 m5
`(Targets
`2, 3)
`
`”E20
`
`
`lug-“K170
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`
`
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`Target 1 (Signed on Gniy}
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`Target 2 (Signed (l (firmly)
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`Target: 3 or 1+2 {Signais OWE)
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`
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`
`(84) Designated States (unless otherwise indicated, for every Published:
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, — With international search report (Art. 21(3))
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, — before the expiration of the time limit for amending the
`RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ,
`claims and to be republished in the event of receipt of
`DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT,
`amendments (Rule 482(k))
`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, ML, MR, NE, SN, TD, TG).
`
`(57) Abstract: System, including methods, apparatus, and compositions, for performing a multiplexed digital assay on a greater
`number of targets through combinatorial use of signals.
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`WO 2012/129187
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`PCT/US2012/029712
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`MULTIPLEXED DIGITAL ASSAYS WITH
`
`COMBINATORIAL USE OF SIGNALS
`
`Cross-Reference to Priority Application
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`5
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`This application is based upon and claims the benefit under 35 U.S.C.
`
`§ 119(e) of US. Provisional Patent Application Serial No. 61/454,373, filed
`
`March 18, 2011, which is incorporated herein by reference in its entirety for all
`
`purposes.
`
`Cross-References
`
`This application incorporates by reference in their entireties for all
`
`purposes the following materials: US. Patent No. 7,041,481, issued May 9,
`
`2006; US. Patent Application Publication No. 2010/0173394 A1, published
`
`July 8, 2010; PCT Patent Application Publication No. WO 2011/120006 A1,
`
`published September 29, 2011; PCT Patent Application Publication No. WO
`
`2011/120024 A1, published September 29, 2011; US. Patent Application
`
`Serial No. 13/287,120,
`
`filed November, 1, 2011; US. Provisional Patent
`
`Application Serial No. 61/507,082, filed July 12, 2011; US. Provisional Patent
`
`Application Serial No. 61/510,013,
`
`filed July 20, 2011; 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 average concentration of analyte in the partitions
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`may be estimated from the probability of finding a given number of copies in 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 after formation of the partitions. Amplification
`
`of the target can be detected optically with 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 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”) of emission (and/or
`
`excitation).
`
`If a detector for a digital PCR assay can distinguishably measure
`
`the fluorescence emitted by R different dyes, then the assay is effectively
`
`capable of measuring R 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
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`hand, many applications, especially where sample is limited, could benefit
`
`greatly from higher degrees of multiplexing.
`
`A new approach is needed to increase the multiplex levels of digital
`
`assays.
`
`W!
`
`The present disclosure provides
`
`a
`
`system,
`
`including methods,
`
`apparatus, and compositions, for performing a multiplexed 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 accordance with 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 of target amplification
`
`via emitted light that may be detected to create a dedicated signal for each
`
`target
`
`in a digital amplification assay,
`
`in accordance with 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 amplification assay performed in droplets, with each signal created
`
`from light detected over the same time interval from a fluid stream containing
`
`the droplets, in accordance with aspects of the present disclosure.
`
`Figure 5 is a schematic representation of how copies of the pair of
`
`targets of Figure 3 are distributed among the droplets from which light
`
`is
`
`detected in Figure 4, based on the intensity of the respective dedicated
`
`signals of Figure 4, in accordance with aspects of the present disclosure.
`
`Figure 6 is a schematic view of three targets and corresponding
`
`exemplary probes capable of reporting the presence or absence of target
`
`amplification via emitted light
`
`that may be detected to create a pair of
`
`composite signals in a digital amplification assay, in accordance with aspects
`
`of the present disclosure.
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`Figure 7 is a pair of exemplary graphs of a pair of composite signals
`
`that may be created by detecting fluorescence emission from the three probes
`
`of Figure 6 in a digital amplification assay performed in droplets, with emitted
`
`light detected in two different wavelength regimes 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 copies of the three
`
`targets of Figure 6 are distributed among the droplets from which light
`
`is
`
`detected in Figure 7, based on the intensity of the respective composite
`
`signals of Figure 7, in accordance with aspects of the present disclosure.
`
`Figure 9A is a schematic view of the third target of Figure 6 and
`
`another exemplary probe configuration capable of reporting the presence or
`
`absence of third target amplification via emitted light, which may be used in
`
`conjunction with the first and second target probes of Figure 6 to create only a
`
`pair of composite signals representing amplification of the three targets in a
`
`digital amplification assay,
`
`in accordance with aspects of
`
`the present
`
`disclosure.
`
`Figure QB 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 first and second target probes of Figure 6 to
`
`create only a pair of composite signals representing the three targets in a
`
`digital amplification assay,
`
`in accordance with aspects of
`
`the present
`
`disclosure.
`
`Figure 10 is a schematic view of the third target of Figure 6 and still
`
`another exemplary probe configuration capable of reporting the presence or
`
`absence of third target amplification via emitted light, which may be used in
`
`conjunction with the first and second target probes of Figure 6 to create only a
`
`pair of composite signals representing amplification of the three targets in a
`
`digital amplification assay,
`
`in accordance with aspects of
`
`the present
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`disclosure.
`
`Figure 11
`
`is a schematic view of three targets and corresponding
`
`exemplary primers that enable use of only two probes to report amplification
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`of the three targets in a digital amplification assay, in accordance with aspects
`
`of the present disclosure.
`
`Figure 12 is a schematic view of the third target of Figure 6 and
`
`another exemplary probe and primer configuration that enables use of only
`
`two probes to report amplification of three targets in a digital amplification
`
`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 the
`
`two probes to report amplification of three targets in a digital amplification
`
`assay, in accordance with aspects of the present disclosure.
`
`Figure 14 is a schematic view of a population of fragments containing a
`
`pair of unlinked targets, T1 and T2, in accordance with aspects of the present
`
`disclosure.
`
`Figure 15 is a schematic view of a population of fragments taken as in
`
`Figure 14, but with the pair of targets always linked to each other on the same
`
`individual fragments, in accordance with aspects of the present disclosure.
`
`Figure 16 is a schematic view of a population of fragments taken as in
`
`Figure 14, but with the pair of targets only partially linked to each other within
`
`the population, in accordance with aspects of the present disclosure.
`
`Figure 17 is a schematic representation of a set of exemplary multi-
`
`labeled probes for use in digital amplification assays,
`
`in accordance with
`
`aspects of the present disclosure.
`
`Figure 18 is a schematic illustration of a template molecule being
`
`copied by DNA polymerase during target amplification in the presence of a
`
`multi-labeled probe molecule and depicting probe degradation by the
`
`polymerase to separate a quencher from fluorophores of the probe molecule,
`
`in accordance with aspects of the present disclosure.
`
`Figure 19 is an exemplary two-dimensional histogram of droplet
`
`intensities, showing clusters that may be obtained in a multiplexed digital
`
`amplification assay for three targets performed with a combination of single-
`
`labeled and dual-labeled probes each labeled with FAM, VIC, or both FAM
`
`and VIC, in accordance with aspects of the present disclosure.
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`Figure 20 is another exemplary two-dimensional histogram of droplet
`
`intensities, showing clusters that may be obtained in the assay of Figure 19,
`
`with partial resolution of the cluster for target-1+2-positive droplets from the
`
`cluster for
`
`target-3-positive droplets,
`
`in accordance with aspects of the
`
`present disclosure.
`
`Figure 21
`
`is an exemplary two-dimensional
`
`intensity histogram of
`
`droplet
`
`intensities,
`
`showing clusters that may be obtained in a digital
`
`amplification assay performed with only a multi-labeled FAM, VIC probe,
`
`in
`
`accordance with aspects of the present disclosure.
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`10
`
`Figure 22 is another exemplary two-dimensional intensity histogram of
`
`droplet
`
`intensities,
`
`showing clusters that may be obtained in a digital
`
`amplification assay performed as
`
`in Figure 21, but with
`
`the assay
`
`supplemented with pair of single-labeled FAM or VIC probes in addition to the
`
`multi-labeled FAM, VIC probe,
`
`in accordance with aspects of the present
`
`15
`
`disclosure.
`
`Detailed Description
`
`The present disclosure provides
`
`a
`
`system,
`
`including methods,
`
`apparatus, and compositions, for performing a multiplexed digital assay on a
`
`greater number of targets through combinatorial use of signals. The method
`
`may be described as a color-based approach to multiplexing.
`
`A method of performing a multiplexed digital amplification assay, such
`
`as a PCR assay,
`
`is provided.
`
`In the method, more than R targets may be
`
`amplified in partitions. R signals may be created. The signals may be
`
`representative of light detected in R different wavelength regimes from the
`
`partitions, where R 2 2. An average level of each target in the partitions may
`
`be calculated based on the R signals, with the level calculated accounting for
`
`a coincidence, if any, of different targets in the same individual partitions.
`
`Another method of performing a multiplexed digital amplification assay
`
`is provided. In the method, more than R targets may be amplified in droplets.
`
`R signals may be created, with the signals representative of light detected in
`
`R different wavelength regimes from the droplets, where R 2 2. An average
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`level of each of the more than R targets may be calculated by finding
`
`solutions to a set of simultaneous equations.
`
`Yet another method of performing a multiplexed digital amplification
`
`assay is provided.
`
`In the method, R targets may be amplified in droplets. R
`
`signals may be created, where R 2 2, with the signals representative of light
`
`detected in R different wavelength regimes from the droplets. Each of the
`
`signals may report amplification of a different combination of at least two of
`
`the targets. An average level of each target in the droplets may be calculated
`
`based on the R signals, without determining which of the at least two targets
`
`for each signal amplified in individual amplification-positive droplets for such
`
`signal.
`
`A composition is provided. The composition may comprise a droplet
`
`containing a probe. The probe may include an oligonucleotide, a first
`
`fluorophore, a second fluorophore, and an energy transfer moiety. The energy
`
`transfer moiety may be a quencher and/or an energy transfer partner for one
`
`or both of the first and second fluorophores.
`
`Further aspects of the present disclosure are presented in the following
`
`sections: (l) system overview, and (II) examples.
`
`|.
`
`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 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 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
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`performance of one or more reactions, such as one or more enzyme
`
`catalyzed reactions and/or binding reactions.
`
`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 (e.g., a forward primer and a reverse
`
`primer for each target), reporters (e.g., probes) for detecting amplification of
`
`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 may involve distributing 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 volumes of other partitions. The partitions
`
`may be isolated from one another by a carrier fluid, such as a continuous
`
`phase of an emulsion, by a solid phase, such as at least 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 phase collectively 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 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. Exemplary volumes for the partitions include an average volume of
`
`less than about 100, 10 or 1 uL, less than about 100, 10, or 1 nL, or less than
`
`about 100, 10, or1 pL, among others.
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`The partitions, when 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. Any of the reagents may be
`
`combined with the partitions (or a bulk phase sample) in a macrofluidic or
`
`microfluidic environment.
`
`One or more reactions may be performed in the partitions, indicated at
`
`46. Each reaction performed may occur selectively (and/or substantially) in
`
`10
`
`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 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 copy of the
`
`15
`
`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 reaction and/or
`
`ligase chain reaction. Accordingly, a
`
`plurality of amplification reactions for a plurality of targets may be performed
`
`20
`
`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.
`
`In some examples,
`
`the conditions may include
`
`thermally cycling the partitions to promote a polymerase chain reaction and/or
`
`ligase chain reaction.
`
`R signals may be created that are representative of light detected from
`
`the partitions, indicated at 48. The R signals may be 2, 3, 4, or more signals.
`
`In some examples, light corresponding to each signal may be detected with a
`
`distinct sensor, and/or light corresponding to at least two signals may be
`
`detected at different
`
`times with the same sensor. The R signals may
`
`correspond to light detected in respective wavelength regimes that are
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`different from one another. Each wavelength regime may be characterized by
`
`a wavelength(s) or and/or wavelength range(s) at which the partitions are
`
`illuminated (e.g., with excitation light) and/or a wavelength(s) or and/or
`
`wavelength range(s) at which light from the partitions is detected (e.g.,
`
`emitted light). The light detected may be light emitted from one or more
`
`fluorophores.
`
`Each of the R signals may be created in a distinct detection channel.
`
`Accordingly, R signals may be created in R detection channels.
`
`Each signal may be a composite signal that represents two, three, four,
`
`or more reactions/assays and thus two, three, four, or more targets of the
`
`reactions/assays. The composite signal may include two or more integral
`
`signal portions that each represent a different reaction/assay 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 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 calculation of the
`
`concentration of each target. (The terms “estimation” and “calculation” are
`
`used interchangeably.)
`
`The R composite signals (and/or R detection channels) may represent
`
`more than R reactions and/or targets, with the number of reactions/assays
`
`and targets depending on configuration. For example, the R signals may be or
`
`include two composite signals (arbitrarily termed 0i and [3) collectively
`
`representing three reactions/assays and/or three targets (arbitrarily termed 1,
`
`2, and 3), with each composite signal representing a different combination of
`
`two reactions/assays/targets (e.g., targets 1 and 2 for 0i and targets 1 and 3
`
`for B). Alternatively, the R signals may be three composite signals (arbitrarily
`
`termed
`
`0i,
`
`[3,
`
`and
`
`y)
`
`collectively
`
`representing
`
`up
`
`to
`
`seven
`
`reactions/assays/targets (1
`
`to 7),
`
`if each composite signal
`
`represents a
`
`different combination of up to four reactions/assays/targets each (e.g., targets
`
`1, 2, 3, and 4 for 0i; targets 2, 4, 5, and 6 for [3; and targets 3, 4, 6, and 7 for y).
`
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`
`The R signals may be four composite signals representing up to fifteen
`
`reactions/assays/targets,
`
`if each composite signal
`
`represents a different
`
`combination of up to eight reactions/assays/targets each.
`
`More generally, 2R-1 targets can be assayed with R composite signals
`
`(or wavelength regimes). To assay 2R-1 targets in R wavelength regimes,
`
`each target may be represented by a different wavelength regime or
`
`combination of wavelength regimes than every other target. A set of 2R-1
`
`targets can be represented and assayed when all of the wavelength regimes
`
`have been utilized individually and in all possible combinations.
`
`Each composite signal may be created based on detected light emitted
`
`from one or more probes in the partitions. The one or more probes 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 more
`
`particular reactions is present in the partition. The intensity of a composite
`
`signal corresponding to the probes 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 intensity may vary among the
`
`partitions according to whether at
`
`least one of the particular
`
`reactions
`
`occurred or did not occur (e.g., above a threshold extent) and at least one of
`
`the particular targets is present in or absent from each individual partition.
`
`The probes 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 wavelength regime. Alternatively, or
`
`in addition,
`
`the same
`
`fluorophore may be included in one probe or two or more probes for at least
`
`two targets represented by the composite signal.
`
`In some cases, the same
`
`fluorophore may be included in a probe for each target represented by the
`
`composite signal.
`
`Each probe may include a nucleic acid (e.g., an oligonucleotide) and at
`
`least one fluorophore. Different probes with different oligonucleotide
`
`sequences and/or different fluorophores (or fluorophore combinations) may be
`
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`
`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 different targets represented by the composite signal (e.g., see
`
`Examples 3-5). In some cases, the same probe may be used as a reporter for
`
`each target represented by the composite signal.
`
`The composite signal detected from each partition, and the partition
`
`itself, may
`
`be
`
`classified
`
`as
`
`being
`
`positive
`
`or
`
`negative
`
`for
`
`the
`
`reactions/assays/targets
`
`represented
`
`by
`
`the
`
`signal
`
`or
`
`corresponding
`
`wavelength regime. Classification may be based on the strength of the signal.
`
`If
`
`the
`
`signal/partition is
`
`classified as positive,
`
`at
`
`least one of
`
`the
`
`reactions/assays represented by the signal is deemed to have occurred and
`
`at least one 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/assays 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 among the
`
`wavelength regimes. For example, some of the targets, if present alone in a
`
`partition, may selectively change the signal strength for only one wavelength
`
`regime. Others of the targets, if present alone in a partition, may selectively
`
`change the signal strength for a unique combination of two of the wavelength
`
`regimes, still other targets may selectively change the signal strength for a
`
`unique combination of three of the wavelength regimes, and so on, optionally
`
`up to the number of wavelength regimes/detection channels.
`
`A fraction of the partitions may have a coincidence of different targets,
`
`where each of these partitions contains a copy of each of two or more targets
`
`in the same individual partitions. Moreover, each of these partitions 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 that of a
`
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`
`target not present
`
`in
`
`the partition. However,
`
`the fraction of partitions
`
`containing two or more distinct targets may (or may not) be kept relatively 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 when the partitions are formed. In any event, a suitable estimation of
`
`concentration, as described below, may take into account the occurrence of
`
`two or more target molecules,
`
`representing the same target or different
`
`targets,
`
`in
`
`the same individual partitions. 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
`
`concentration.
`
`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 wavelength
`
`regime/detection channel may be determined individually (e.g., counted) for
`
`each signal or channel (Le, 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 wavelength regimes may be determined individually (e.g.,
`
`counted) for each combination of signals or channels (Le, a number for each
`
`combination, and particularly each combination corresponding to a particular
`
`target).
`
`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 of partitions may be
`
`counted or estimated.
`
`A level of each target may be calculated, indicated at 52. The level may
`
`be an average level, such as an average concentration of molecules of the
`
`target per partition. Generally, if R signals are detected from the partitions in R
`
`wavelengt