U.S. patent application number 13/245575 was filed with the patent office on 2012-01-26 for controls and calibrators for tests of nucleic acid amplification performed in droplets.
This patent application is currently assigned to QuantaLife, Inc.. Invention is credited to Billy Wayne Colston, JR., Benjamin Joseph Hindson, Donald Arthur Masquelier, Kevin Dean Ness.
Application Number | 20120021423 13/245575 |
Document ID | / |
Family ID | 42311957 |
Filed Date | 2012-01-26 |
United States Patent
Application |
20120021423 |
Kind Code |
A1 |
Colston, JR.; Billy Wayne ;
et al. |
January 26, 2012 |
CONTROLS AND CALIBRATORS FOR TESTS OF NUCLEIC ACID AMPLIFICATION
PERFORMED IN DROPLETS
Abstract
System, including methods and apparatus, for performing
droplet-based tests of nucleic acid amplification that are
controlled and/or calibrated using signals detected from
droplets.
Inventors: |
Colston, JR.; Billy Wayne;
(San Ramon, CA) ; Hindson; Benjamin Joseph;
(Livermore, CA) ; Ness; Kevin Dean; (San Mateo,
CA) ; Masquelier; Donald Arthur; (Tracy, CA) |
Assignee: |
QuantaLife, Inc.
Pleasanton
CA
|
Family ID: |
42311957 |
Appl. No.: |
13/245575 |
Filed: |
September 26, 2011 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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12586626 |
Sep 23, 2009 |
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13245575 |
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61194043 |
Sep 23, 2008 |
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61206975 |
Feb 5, 2009 |
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61271538 |
Jul 21, 2009 |
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61275731 |
Sep 1, 2009 |
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61277200 |
Sep 21, 2009 |
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61277203 |
Sep 21, 2009 |
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61277204 |
Sep 21, 2009 |
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61277216 |
Sep 21, 2009 |
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61277249 |
Sep 21, 2009 |
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61277270 |
Sep 22, 2009 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
B01L 2300/1822 20130101;
Y02A 90/10 20180101; B01L 2300/0858 20130101; G01N 21/6486
20130101; B01F 2003/0834 20130101; B01L 2200/12 20130101; B01F
15/00922 20130101; B01F 2215/0037 20130101; B01L 2200/0673
20130101; B01L 3/502715 20130101; B01L 7/52 20130101; B01L
2300/0819 20130101; G01N 21/3563 20130101; B01F 13/0062 20130101;
G01N 21/6428 20130101; B01L 3/0241 20130101; B29C 45/006 20130101;
B29L 2031/752 20130101; B01L 2300/0867 20130101; B01L 2400/0622
20130101; B01L 2300/0816 20130101; B01L 2400/049 20130101; C12Q
1/686 20130101; Y02A 90/26 20180101; B01L 2400/0478 20130101; G01N
2021/6439 20130101; B01L 2200/0689 20130101; B01L 2200/10 20130101;
B29C 2045/0079 20130101; B01L 2400/0487 20130101; B29C 45/0053
20130101; B01L 2300/0654 20130101; B01L 7/525 20130101; B01L
2300/041 20130101; G01N 21/49 20130101; B01F 3/0807 20130101; B01F
2003/0842 20130101; B01L 3/502784 20130101 |
Class at
Publication: |
435/6.12 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of performing a droplet-based assay, comprising:
detecting a signal from each of a plurality of droplets; comparing
a width of the signal from each droplet to a permitted range;
excluding droplets for which the signal has a width that is not in
the permitted range, to identify a set of included droplets; and
determining a concentration of a target provided by a sample
disposed in the plurality of droplets using data collected from the
included droplets and without any contribution of data collected
from the excluded droplets.
2. The method of claim 1, wherein the step of excluding droplets
includes a step of comparing a width of a peak formed by the signal
from each droplet to a width maximum and a step of excluding each
droplet for which the corresponding peak has a width that is
greater than the width maximum.
3. The method of claim 2, wherein the step of excluding droplets
includes a step of comparing a width of a peak formed by the signal
from each droplet to a width minimum and a step of excluding each
droplet for which the corresponding peak has a width that is less
than the width minimum.
4. The method of claim 1, wherein the width corresponds to a time
interval during which the signal is detected from a droplet.
5. The method of claim 1, wherein the step of determining a
concentration is based on an intensity of the signal from included
droplets.
6. The method of claim 1, further comprising a step of thermally
cycling the plurality of droplets to promote amplification of the
target.
7. The method of claim 1, wherein the step of detecting a signal
includes a step of detecting a first signal and a second signal
from each droplet of the plurality of droplets, and wherein the
data used for determining a concentration is obtained from the
first signal.
8. The method of claim 1, wherein the step of detecting a signal
includes a step of detecting a fluorescence signal.
9. The method of claim 1, wherein the step of detecting a signal
includes a step of detecting a signal from each droplet traveling
through a detection region.
10. The method of claim 1, wherein the signal has an intensity that
varies according to whether or not the target is present in a
droplet.
11. A method of performing a droplet-based assay, comprising:
detecting a signal from at least two types of calibration droplets,
the signal for each type of calibration droplet being of different
intensity; detecting sample data from sample droplets; and
determining if amplification of a target occurred in each of the
sample droplets based on the sample data and the signal of each
different intensity detected from the calibration droplets.
12. The method of claim 11, wherein the at least two types of
calibration droplets include a first type and a second type
configured to provide respective signal intensities corresponding
at least generally to sample droplets that are negative or positive
for amplification of the target.
13. The method of claim 12, wherein each sample droplet contains a
PCR mixture for amplification of the target.
14. The method of claim 11, wherein the step of determining
includes a step of determining a threshold using the signal
detected from the calibration droplets and a step of comparing data
for individual sample droplets to the threshold, to distinguish
sample droplets that are negative from those that are positive for
amplification of the target.
15. The method of claim 11, wherein the step of detecting a signal
includes a step of detecting a signal of different intensity from
at least three distinct types of calibration droplets.
16. The method of claim 11, wherein the step of detecting a signal
and the step of detecting sample data are both performed at a same
wavelength or wavelength range.
17. The method of claim 11, wherein the step of detecting a signal
and the step of detecting sample data are performed with a same
detector.
18. The method of claim 11, wherein the step of detecting a signal
and the step of detecting sample data are performed with the
calibration droplets and the sample droplets arranged in separate
groups.
19. The method of claim 18, further comprising a step of detecting
a signal from the at least two types of calibration droplets with
the at least two types intermixed.
20. The method of claim 11, wherein each type of calibration
droplet contains a different amount of a same dye.
21. The method of claim 11, wherein the step of detecting a signal
and the step of detecting sample data are performed on droplets
flowing through a same detection region.
22. The method of claim 21, further comprising a step of loading
the calibration droplets and the sample droplets into a flow
channel that intersects the detection region, wherein the
calibration droplets are loaded before the sample droplets.
23. The method of claim 11, further comprising a step of thermally
cycling the sample droplets.
24. The method of claim 23, further comprising a step of thermally
cycling the calibration droplets, wherein each different intensity
of the signal detected from the calibration droplets is not
affected substantially by the step of thermally cycling.
25. The method of claim 23, wherein the calibration droplets are
not thermally cycled.
26. The method of claim 11, wherein the step of detecting a signal
includes a step of detecting a fluorescence signal from each type
of calibration droplet, and wherein the step of detecting sample
data includes a step of detecting sample data as fluorescence
intensity.
27. A method of performing a droplet-based assay, comprising:
generating droplets from an aqueous phase including a first dye and
a second dye, the second dye being an internal reference; detecting
sample data from the first dye included in the droplets, the sample
data being related to a reaction performed in the droplets;
detecting reference data from the second dye included in the
droplets; transforming the sample data with the reference data to
reduce variability in the sample data that is independent of the
reaction; and determining if the reaction occurred in each of the
sample droplets based on sample data that has been transformed with
the reference data.
28. The method of claim 27, further comprising a step of amplifying
a nucleic acid target in the droplets, wherein the step of
detecting sample data includes a step of detecting amplification
data from the first dye.
29. The method of claim 27, wherein the second dye is not
conjugated to a nucleic acid.
30. The method of claim 27, wherein the step of generating droplets
includes a step of generating monodisperse droplets.
31. The method of claim 27, wherein the step of transforming the
sample data includes a step of dividing a sample data value by a
reference data value for each droplet.
32. A method of performing a droplet-based assay, comprising:
detecting a signal from each of a plurality of droplets flowing
through a detection region; transforming an intensity of the signal
for each of the plurality of droplets according to a duration of
such signal to obtain transformed signals; and determining whether
amplification of a target occurred in individual droplets based on
the transformed signals.
33. The method of claim 32, wherein the step of detecting a signal
includes a step of detecting a fluorescence signal.
34. The method of claim 32, wherein each signal forms a peak, and
wherein the duration corresponds to a width of the peak.
35. The method of claim 34, wherein the step of transforming an
intensity of each signal includes a step of transforming a value
corresponding to a height or an area of the peak formed by such
signal.
36. The method of claim 32, wherein the step of transforming
includes a step of dividing the intensity of each signal by the
duration of such signal.
37. The method of claim 32, further comprising: comparing a
duration of each signal to a permitted range; and excluding each
signal having a duration that is not in the permitted range.
38. The method of claim 37, wherein the step of comparing includes
a step of comparing a duration of each signal to a duration maximum
and a step of excluding each signal having a duration that is
greater than the duration maximum.
39. The method of claim 32, further comprising a step of thermally
cycling the plurality of droplets to promote amplification of the
target.
40. A method of performing a droplet-based assay, comprising:
generating droplets from an aqueous phase including a sample and
first and second dyes; detecting sample data from the first dye in
the droplets, the sample data being related to amplification of a
test target from the sample; detecting control data from the second
dye in the droplets, the control data being related to
amplification of a control target in individual droplets; analyzing
the sample data and the control data to determine respective
concentrations of the test target and the control target; and
correlating the concentration of the test target with the
concentration of the control target.
41. The method of claim 40, wherein the test target and the control
target are both endogenous to the sample.
42. The method of claim 40, wherein the test target is endogenous
to the sample and the control target is not endogenous to the
sample.
43. The method of claim 40, wherein the step of correlating
includes a step of determining a validity of the test target
concentration based on the control target concentration.
44. A method of performing a droplet-based assay, comprising:
obtaining a first set of droplets configured to amplify a test
target from a sample disposed in the first set of droplets, and a
second set of droplets configured to amplify a control target in
the second set; detecting test amplification data from the first
set of droplets and control amplification data from the second set
of droplets; and analyzing the test amplification data and the
control amplification data to determine a concentration of the test
target and the control target; and correlating the concentration of
the test target with the concentration of the control target.
45. The method of claim 44, wherein the step of detecting includes
a step of detecting data from the first set of droplets as a group
and from the second set of droplets as a separate group.
46. The method of claim 44, wherein the first set and the second
set of droplets each includes a same sample that provides the test
target and the control target.
47. The method of claim 44, wherein test target is provided by a
sample that does not provide the control target.
48. The method of claim 44, wherein the step of correlating
includes a step of determining a validity of the test target
concentration based on the control target concentration.
Description
CROSS-REFERENCES TO PRIORITY APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/586,626, filed Sep. 23, 2010.
[0002] U.S. patent application Ser. No. 12/586,626, in turn, is
based upon and claims the benefit under 35 U.S.C..sctn.119(e) of
the following U.S. provisional patent applications: Ser. No.
61/194,043, filed Sep. 23, 2008; Ser. No. 61/206,975, filed Feb. 5,
2009; Ser. No. 61/271,538, filed Jul. 21, 2009; Ser. No.
61/275,731, filed Sep. 1, 2009; Ser. No. 61/277,200, filed Sep. 21,
2009; Ser. No. 61/277,203, filed Sep. 21, 2009; Ser. No.
61/277,204, filed Sep. 21, 2009; Ser. No. 61/277,216, filed Sep.
21, 2009; Ser. No. 61/277,249, filed Sep. 21, 2009; and Ser. No.
61/277,270, filed Sep. 22, 2009.
[0003] These priority applications are incorporated herein by
reference in their entireties for all purposes.
CROSS-REFERENCES
[0004] This application incorporates by reference in their
entireties for all purposes the following materials: U.S. Pat. No.
7,041,481, issued May 9, 2006; and Joseph R. Lakowicz, PRINCIPLES
OF FLUORESCENCE SPECTROSCOPY (2.sup.nd Ed. 1999).
INTRODUCTION
[0005] Droplet-based tests for amplification generally need to be
accurate. If inaccurate, these tests can generate erroneous
results, that is, false negatives and false positives. Each type of
erroneous result can have detrimental consequences. False negatives
related to detection of a disease could mean that the disease is
not treated early and is permitted to spread. In contrast, false
positives could cause unnecessary alarm, potentially triggering an
unnecessary response that may be costly and disruptive. To avoid
problems associated with false negatives and false positives,
inaccurate amplification tests must be repeated to improve their
reliability, which increases cost and uses more sample and reagent,
each of which may be precious.
[0006] FIG. 1 shows a graph 5710 illustrating an exemplary approach
for using fluorescence to measure amplification of a nucleic acid
target in droplets formed by partitioning a sample. The graph
plots, with respect to time, fluorescence signals that may be
detected from a flow stream containing the droplets. Each droplet
may be detected as a transient change (e.g., a transient increase)
in intensity of the fluorescence signal, such as a peak or spike
5712 (i.e., a wave) formed by the fluorescence signal.
[0007] To improve clarity, the illustrative data shown here and in
other figures of the present disclosure, are presented in a
simplified form: each peak has no width and projects from a
constant background signal 5713 formed by detection of a continuous
phase carrying the droplets. However, a signal peak may have any
suitable shape based on, for example, the frequency of detecting
signals, the shape of each droplet, the size and geometry of a
channel carrying the flow stream, the flow rate, and the like.
Moreover, the signal peaks may have any suitable temporal
distribution, for example, occurring at relatively constant
intervals, as shown here, or at varying intervals. A droplet signal
provided by and/or calculated from the peak (e.g., a signal
corresponding to peak height or peak area, among others) may be
used to determine whether amplification occurred in the
corresponding droplet, and thus whether the droplet received at
least one molecule of the nucleic acid target when the sample was
partitioned.
[0008] Each droplet signal may be compared to a signal threshold
5714, also termed a cutoff. This comparison may provide a
determination of whether each droplet signal represents a positive
signal (target is present) or a negative signal (target is absent
and/or not detected), for amplification in the droplet. For
example, droplet signals greater than (and, optionally, equal to)
the threshold may be considered as representing positive droplets.
Conversely, droplet signals less than (and, optionally, equal to)
the threshold may be considered as representing negative droplets.
(A positive droplet signal above threshold 5714 is indicated at
5716, and a negative droplet signal below threshold 5714 is
indicated at 5718 in FIG. 1.) Comparison to the threshold thus may
transform each droplet signal to a digital value, such as a binary
value (e.g., a "1" for a positive droplet and "0" for a negative
droplet). In any event, the fraction of droplets that are positive
can be determined. For a given droplet size, the fraction of
positive droplets can be used as an input to an algorithm based on
Poisson statistics to determine the number of copies (molecules) of
the nucleic acid target present in the initial sample volume. In
some embodiments, more than one threshold may be used to categorize
results (e.g., negative, positive, or inconclusive).
[0009] FIG. 2 shows an exemplary histogram 5720 of ranges of
droplet signal intensities that may be measured from the flow
stream of FIG. 1. The relative frequency of occurrence of each
range is indicated by bar height. The distribution of positive and
negative signal intensities may be larger than the modest
difference in signal intensity produced by amplification (a
positive droplet) relative to no amplification (a negative
droplet). Thus, the distributions of droplet signals from positive
droplets and negative droplets may produce a problematic overlap
between the amplification-positive and amplification-negative
droplet signals, indicated at 5724. Accordingly, as shown in FIG.
1, some amplification-positive droplets may provide relatively weak
droplet signals, such as false-negative signal 5726, that are less
than threshold 5714, resulting in incorrect identification of these
positive droplets as negative. Conversely, some
amplification-negative droplets may provide relatively strong
droplet signals, such as false-positive negative signal 5728, that
are greater than threshold 5714, resulting in incorrect
identification of these negative droplets as positive. Since either
type of erroneous result may be costly and harmful, it is desirable
to minimize their occurrence.
[0010] There are many factors that can lead to variation in the
fluorescence signal from droplets tested for amplification.
Examples of physical parameters that may affect the fluorescence
signal may include droplet position when detected (e.g., relative
to the "sensed volume" of the detector), droplet volume and shape,
optical alignment of detection optics (including excitation source,
filters, and detector), detector response, temperature, vibration,
and flow rate, among others. Examples of reaction chemistry
parameters that may affect the fluorescence signal include the
number of target molecules and/or the amount of background nucleic
acid present in each droplet, amplification efficiency,
batch-to-batch variations in reagent concentrations, and volumetric
variability in reagent and sample mixing, among others. Variations
in these physical and chemical parameters can increase the overlap
in the distribution of positive and negative droplet signals, which
can complicate data interpretation and affect test performance
(e.g., affect the limit of detection). The variations can occur
within a run and/or between runs, within a test on a target and/or
between tests on different targets, on the same instrument and/or
different instruments, with the same operator and/or different
operators, and so on.
[0011] Thus, there is a need for improved accuracy and reliability
in droplet-based amplification tests. For example, it would be
desirable to have droplet-based controls for these tests,
optionally, droplet-based controls that can be incorporated into
test droplets or incorporated into control droplets that can be
intermixed with test droplets. Such integrated controls may have
the benefit of reducing cost by processing control reactions in
parallel with test reactions, which may speed the analysis. It also
would be useful to have one or more controls that can be used to
verify hardware, reagent, and/or software (e.g., algorithm)
performance.
SUMMARY
[0012] The present disclosure provides a system, including methods
and apparatus, for performing droplet-based tests of nucleic acid
amplification that are controlled and/or calibrated using signals
detected from droplets.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is an exemplary graph of fluorescence signals that
may be measured with respect to time from a flow stream of
droplets, with the graph exhibiting a series of peaks representing
droplet signals, and with the graph indicating a signal threshold
for assigning droplet signals as corresponding to
amplification-positive and amplification-negative droplets, in
accordance with aspects of the present disclosure.
[0014] FIG. 2 is an exemplary histogram of ranges of droplet signal
intensities that may be measured from the flow stream of FIG. 1,
with the relative frequency of occurrence of each range indicated
by bar height, in accordance with aspects of the present
disclosure.
[0015] FIG. 3 is a schematic view of an exemplary system for
performing droplet-based tests of nucleic acid amplification with
the aid of controls and/or calibrators, in accordance with aspects
of the present disclosure.
[0016] FIG. 4 is a schematic view of selected aspects of the system
of FIG. 3, with the system in an exemplary configuration for
detecting amplification of a nucleic acid target using a first dye,
and for controlling for system variation during a test using a
second dye, in accordance with aspects of present disclosure.
[0017] FIG. 5 is a schematic view of exemplary reagents that may be
included in the system configuration of FIG. 4, to permit detection
of amplification signals in a first detection channel and detection
of a passive control signals in a second detection channel, in
accordance with aspects of present disclosure.
[0018] FIG. 6 a flowchart of an exemplary approach to correcting
for system variation using the system configuration of FIG. 4, in
accordance with aspects of the present disclosure.
[0019] FIG. 7 is a schematic view of selected aspects of the system
of FIG. 3, with the system in an exemplary configuration for
detecting amplification of a nucleic acid target using a first dye
in a set of droplets, and for (a) calibrating the system before,
during, and/or after a test or (b) controlling for aspects of
system variation during a test using either the first dye or a
second dye in another set of droplets, in accordance with aspects
of present disclosure.
[0020] FIG. 8 is an exemplary graph of fluorescence signals that
may be detected over time from a flow stream of the system
configuration of FIG. 7 during system calibration and sample
testing performed serially, in accordance with aspects of present
disclosure.
[0021] FIG. 9 is a flowchart of an exemplary method of correcting
for system variation produced during a test using the system
configuration of FIG. 7, in accordance with aspects of the present
disclosure.
[0022] FIG. 10 is a schematic view of selected aspects of the
system of FIG. 3, with the system in an exemplary configuration for
testing amplification of a pair of nucleic acid targets in the same
droplets, in accordance with aspects of present disclosure.
[0023] FIG. 11 is a schematic view of selected aspects of the
system of FIG. 3, with the system in another exemplary
configuration for testing amplification of a pair of nucleic acid
targets in the same droplets, in accordance with aspects of present
disclosure.
[0024] FIG. 12 is a schematic view of exemplary target-specific
reagents that may be included in the system configurations of FIGS.
10 and 11, to permit detection of amplification signals in a
different detection channel (i.e., a different detected wavelength
or wavelength range) for each nucleic acid target, in accordance
with aspects of present disclosure.
[0025] FIG. 13 is a pair of exemplary graphs of fluorescence
signals that may be detected over time from a flow stream of the
system configuration of FIG. 10 or 11 using different detection
channels, with one of the channels detecting successful
amplification of a control target, thereby indicating no inhibition
of amplification, in accordance with aspects of present
disclosure.
[0026] FIG. 14 is a pair of exemplary graphs with fluorescence
signals detected generally as in FIG. 13, but with control signals
indicating that amplification is inhibited, in accordance with
aspects of present disclosure.
[0027] FIG. 15 is a schematic view of selected aspects of the
system of FIG. 3, with the system in an exemplary configuration for
testing amplification of a pair of nucleic acid targets using a
different set of droplets for each target, in accordance with
aspects of present disclosure.
[0028] FIG. 16 is a pair of exemplary graphs of fluorescence
signals that may be detected over time from a flow stream of the
system configuration of FIG. 15 using different detection channels,
with each channel monitoring amplification of a distinct nucleic
acid target, in accordance with aspects of present disclosure.
[0029] FIG. 17 is a pair of graphs illustrating exemplary
absorption and emission spectra of fluorescent dyes that may be
suitable for use in the system of FIG. 3, in accordance with
aspects of the present disclosure.
[0030] FIG. 18 is a schematic diagram illustrating exemplary use of
the fluorescent dyes of FIG. 17 in an exemplary embodiment of the
system of FIG. 3, in accordance with aspects of the present
disclosure.
[0031] FIG. 19 is a flowchart of an exemplary approach to
correcting for system variation within a test by processing a set
of droplet test signals to a more uniform signal intensity, in
accordance with aspects of the present disclosure.
[0032] FIG. 20 is a flowchart of an exemplary approach for
transforming droplet signals based on the width of respective
signal peaks providing the droplet signals, in accordance with
aspects of the present disclosure.
DETAILED DESCRIPTION
[0033] The present disclosure provides a system, including methods
and apparatus, for performing droplet-based tests of nucleic acid
amplification that are controlled and/or calibrated using signals
detected from droplets.
[0034] The present disclosure provides a method of sample
analysis.
[0035] Droplets may be obtained. The droplets may be generated
on-line or at least a subset of the droplets may be pre-formed
off-line. At least a subset or all of the droplets may include a
partition of a sample to be tested and may be capable of
amplification of at least one test nucleic acid target, if present,
in the partition. In some embodiments, the droplets may be capable
of amplification of a test nucleic acid target and a control
nucleic acid target. The droplets collectively or each may include
a dye, or at least a first dye and a second dye. In some
embodiments, the droplets may be of at least two types, such as two
or more types of test droplets, test droplets and calibration
droplets, or test droplets and control droplets, among others. In
some embodiments, the two or more types of droplets may be
distinguishable based on distinct temporal positions of the
droplets types in a flow stream, the presence of respective
distinct dyes in the droplet types, distinguishable signal
intensities of the same dye (or different dyes), or a combination
thereof, among others.
[0036] Signals, such as fluorescence signals, may be detected from
the droplets. The signals may include test signals, calibration
signals, control signals, reference signals, or any combination
thereof. In some embodiments, test signals and control signals may
indicate respectively whether amplification of a test nucleic acid
target and a control nucleic acid target occurred in individual
droplets. In some embodiments, detection may include (a) exciting
first and second dyes with a same wavelength of excitation light
and (b) detecting emitted light from the first and second dyes at
least substantially independently from one another in respective
first and second detection channels.
[0037] The signals detected may be analyzed to determine a test
result related to a presence (number, concentration, etc.), if any,
of a test nucleic acid target in the sample. In some embodiments,
analysis may include transforming test signals based on reference
signals to reduce variation in the test signals. The test signals
and the reference signals may be detected in respective distinct
detection channels or in the same detection channel. In some
embodiments, the reference signals may be provided by a second dye
that is not coupled to an amplification reaction and thus serves as
a passive reference. In some embodiments, the reference signals may
be provided by control signals detected from a control
amplification reaction. The control amplification reaction may
measure amplification of an exogenous or endogenous template. In
some embodiments, analysis may include (a) comparing test signals,
or a transformed set of the test signals, to a signal threshold to
assign individual droplets as positive or negative for a test
nucleic acid target, and (b) estimating a number of molecules of
the test nucleic acid target in the sample based on the comparison.
In some embodiments, analysis may include (a) analyzing control
signals to determine a control value corresponding to a number
and/or fraction of the droplets that are amplification-positive for
a control nucleic acid target, and (b) interpreting a test result,
such as determining its validity, based on the control value.
[0038] The systems disclosed herein may offer improved instrument
calibration and/or substantial improvements in the accuracy and/or
reliability of droplet-based amplification tests. Exemplary
capabilities offered by the present disclosure may include any
combination of (1) correcting/minimizing variations in the
fluorescence signal to increase the accuracy of droplet PCR
results; (2) providing an internal indicator of whether nucleic
acid amplification failed (e.g., PCR inhibition from interfering
components in the sample, incorrect sample and reagent mixing,
incorrect thermal cycling, incorrect droplet formation); (3)
providing measurement of droplet volumes without having to add
additional hardware components; (4) providing measurement of
changes in the baseline fluorescence signal (i.e., baseline drift);
(5) providing calibration of a droplet detector before and/or
during a run; (6) monitoring the performance of quantitative
droplet PCR measurements and data processing algorithms before
and/or during a run; (7) verification of droplet integrity (e.g.,
absence of coalescence); (8) obtaining information on droplet
generation and detection frequency (spatially and temporally) using
an in-line detector; (9) measuring variations and comparing them to
predefined tolerances; (10) processing of raw droplet PCR data to
correct for variations and increase test accuracy and performance;
(11) incorporating control assays preferably using a single
excitation source; and/or (12) quantifying one or more genetic
targets by amplifying more than one genetic target in a single
droplet.
[0039] Further aspects of the present disclosure are presented in
the following sections: (I) definitions, (II) system overview,
(III) exemplary instrument controls and calibrators, (IV) exemplary
amplification controls, (V), exemplary multi-channel detection, and
(VI) exemplary self-normalization of test signals.
I. Definitions
[0040] Technical terms used in this disclosure have the meanings
that are commonly recognized by those skilled in the art. However,
the following terms may have additional meanings, as described
below.
[0041] Signal--detectable and/or detected energy and/or
information. Any of the signals detected, after detection, may be
described as signals and/or data. For example, detected droplet
signals may provide test signals and test data, control signals or
control data, reference signals and reference data, calibration
signals and calibration data, transformed signals and transformed
data, or any combination thereof, among others.
[0042] Transform--to change one or more values, and/or the number,
of signals of a data set using one or more mathematical and/or
logical operations. Transformation of a set of signals may produce
a transformed set of the signals by changing values of one or more
of the signals and/or by deleting/invalidating any suitable subset
of the signals. Signal transformation may include reducing signal
variation, deleting/invalidating outlier signals, subtracting a
baseline value from signals, reducing the frequency of outliers,
reducing the overlap of distributions of positive and negative
droplet signals, modifying signals according to a regression line,
assigning new values to signals based on comparing signal values to
a threshold or range, or any combination thereof, among others.
[0043] Run--an operating period during which a set of droplets,
generally droplets of about the same size and including partitions
a sample, are tested.
[0044] Oligonucleotide--a nucleic acid of less than about
one-hundred nucleotides.
[0045] Exogenous--originating externally. For example, a nucleic
acid exogenous to a sample is external to the sample as originally
isolated. As another example, a nucleic acid exogenous to an
organism or cell is not native to the organism or cell, such as a
nucleic acid introduced into the organism or cell by infection or
transfection.
[0046] Endogenous--originating internally, such as present in a
sample as originally isolated or native to a cell or organism.
[0047] Reporter--a compound or set of compounds that reports the
condition of something else, such as the extent of reaction.
Exemplary reporters comprise at least one dye, such as a
fluorescent dye or an energy transfer pair, and/or at least one
oligonucleotide.
II. System Overview
[0048] FIG. 3 shows an exemplary system 5740 for performing
droplet-based tests of nucleic acid amplification with the aid of
controls and/or calibrators. System 5740 may include any
combination of a sample/reagent storage/preparation assembly 5742,
at least one droplet generator 5744, an amplification assembly,
such as a thermal cycler 5746, a detection assembly 5748, and a
controller 5750 incorporating a data analyzer 5752 and a feedback
and control portion 5754, among others.
[0049] The system may provide at least one flow stream that carries
at least one sample and reagents from one or more upstream
positions and in a downstream direction to detection assembly 5748.
Signals detected from the flow stream, and particularly droplet
signals, may be communicated to data analyzer 5752. The data
analyzer may analyze the signals to determine one or more test
results, control results, calibration results, a quality (e.g.,
validity, reliability, confidence interval, etc.) of any of the
results, or a combination thereof. Any of the results may be
communicated to feedback and control portion 5754, which may
control and/or adjust control of any of storage/preparation
assembly 5742, droplet generator 5744, thermal cycler 5746,
detection assembly 5748, and data analyzer 5752, based on the
results determined.
[0050] Storage/preparation assembly 5742 may contain and/or supply
at least one sample 5756, at least one set of test reagents 5758
(also termed target reagents), one or more control reagents 5760,
one or more calibration reagents 5762, or any combination thereof.
Any of the samples and/or reagents may be stored and/or supplied
separately, may be stored and/or supplied as one or more pre-formed
mixtures, and/or may be mixed selectably before they are supplied
to a downstream region of the system (e.g., droplet generator 5744,
thermal cycler 5746, or detection assembly 5748). Furthermore, any
of the samples and/or reagents may travel sequentially from
storage/preparation assembly 5742 to droplet generator 5744,
thermal cycler 5746, and then detection assembly 5748 for detection
of droplet signals. Alternatively, any of the samples and/or
reagents may reach the detection assembly without travel through
the droplet generator, as indicated at 5764, the thermal cycler, or
both, as indicated at 5766. Accordingly, any of the samples and/or
reagents disclosed herein may be stored and/or supplied in
pre-formed droplets. Droplets may, for example, be pre-formed
off-line, either locally or remotely. Pre-formed droplets may be
intermixed randomly with droplets formed by droplet generator 5744
before reaching detection assembly 5748, or distinct types of
droplets may be detected as spatially and/or temporally separated
sets of droplets.
[0051] Test reagents 5758 are any reagents used to test for
amplification of one or more targets, such as one or more primary
targets, in partitions of a sample. Primary targets generally
comprise any targets that are of primary interest in a test.
Primary targets may be present at an unknown level in a sample,
prior to performing tests on the sample. Test reagents 5758
generally include one or more sets of target reagents conferring
specificity for amplification of one or more particular nucleic
acid targets to be tested in a sample. Thus, the test reagents may
include at least one pair (or two or more pairs) of primers capable
of priming amplification of at least one (or two or more) nucleic
acid target(s). The test reagents also may comprise at least one
reporter to facilitate detecting amplification of each test target,
a polymerase (e.g., a heat stable polymerase), dNTPs, and/or the
like. The test reagents enable detection of test signals from
droplets.
[0052] Control reagents 5760 are any reagents used to control for
test signal variation (generally, variation other than that
produced by differences in amplification) and/or to interpret
results obtained with the test reagents (such as a reliability
and/or validity of the results). The control reagents permit
control signals and/or reference signals to be detected from
droplets, either the same or different droplets from the test
signals. Control reagents may be mixed with test reagents prior to
droplet formation and/or control droplets containing control
reagents may be produced separately from the test droplets and
introduced independently of the sample.
[0053] The control reagents may provide instrument controls, that
is, controls for variation introduced by the system (and/or its
environment). Thus, instrument controls may control for variation
in droplet volume, droplet detection efficiency, detector drift,
and the like. Reference signals may be detected from droplets
containing control reagents that function as instrument
controls.
[0054] The control reagents also or alternatively may provide
amplification controls, that is, controls that test for
secondary/control amplification in droplets. The control reagents
thus may include reagents used to test for amplification of at
least one secondary or control target in droplets. The
secondary/control target may be of secondary interest in a test,
and/or may be present at a known or expected level in the sample,
among others. In any event, the control reagents may include one or
more sets of target reagents conferring specificity for
amplification of one or more control nucleic acid targets to be
tested in droplets. The control reagents may include at least one
pair (or two or more pairs) of primers capable of priming
amplification of at least one (or two or more) control nucleic acid
target(s). The control reagents also may comprise at least one
reporter to facilitate detecting amplification of each control
target, a polymerase (e.g., a heat stable polymerase), dNTPs,
and/or the like, or any suitable combination of these control
reagents may be supplied by the test reagents. Control signals may
be detected from control reagents that function as amplification
controls.
[0055] Calibration reagents 5762 are any reagents used to calibrate
system operation and response. Droplets containing a calibration
reagent (i.e., calibration droplets) may be introduced into a flow
stream of the system, at any position upstream of the detection
assembly, for the purpose of calibrating the system (e.g.,
calibrating flow rates, excitation power, optical alignment,
detector voltage, amplifier gain, droplet size, droplet spacing,
etc.). Calibration droplets may be introduced into a flow stream of
the system before, during, and/or after introduction of test
droplets into the flow stream. In some embodiments, the level of a
dye within control droplets may be used to calibrate and/or
validate detector response, such as by using a pair of dye
concentrations providing calibration signals that bracket an
intended measuring range and/or that are disposed near upper and
lower ends of the measuring range. For example, droplets of known
size and containing one or more known dye concentrations may be
prepared off-line and introduced into the system, and/or may be
generated by the system. In some embodiments, calibration droplets
may comprise fluorescent particles such as quantum dots, polymer
beads, etc.
[0056] System 5740 may used to perform a method of analyzing one or
more samples. The method may include any suitable combination of
the steps disclosed herein, performed in any suitable order.
[0057] Droplets may be obtained. The droplets may be of one type or
two or more types. At least a subset, or all, of the droplets may
be generated by the system or may be pre-formed off-line. At least
a subset of the droplets may include test reagents for testing
amplification of a test nucleic acid target. At least a subset of
the droplets may include control reagents and/or calibration
reagents for testing amplification of a control nucleic acid
target. The droplets may contain one or more dyes.
[0058] The droplets may be introduced into a flow stream upstream
of a detector. All of the droplets may be introduced into the flow
stream at the same position or the droplets, particularly droplets
of different types, may be introduced at two or more distinct
positions.
[0059] The droplets, in the flow stream, may be subjected to
conditions that facilitate amplification. For example, the droplets
may be heated and/or may be heated and cooled repeatedly (thermally
cycled).
[0060] Signals may be detected from the droplets. The signals may
include test signals, control signals, reference signals,
calibration signals, or any combination thereof.
[0061] The signals may be analyzed. Analysis may include
transforming test signals. Analysis also or alternatively may
include comparing test signals and/or transformed test signals to a
signal threshold to assign individual droplets as being positive or
negative for amplification of a nucleic acid target. A number
and/or fraction of target-positive droplets may be determined based
on results of the comparison. Analysis further may include
estimating a presence of a nucleic acid target in the sample. The
estimated presence may be no target in the sample. Estimation of
the presence may (or may not) be performed using Poisson
statistics.
III. Exemplary Instrument Controls and Calibrators
[0062] FIG. 4 shows selected aspects of system 5740 in an exemplary
configuration 5780 for detecting amplification of a nucleic acid
target using a first dye and for controlling for system variation
during a test using a second dye. In FIG. 4 and in other system
configurations presented in succeeding figures of the present
disclosure, the terms "droplet generator," "thermal cycler," and
"detection assembly" are abbreviated "DG," "TC," and "DET."
[0063] Storage/preparation assembly 5742 may supply an
amplification mixture to droplet generator 5744. The amplification
mixture may incorporate a sample 5756, target reagents 5782 (i.e.,
test reagents 5758) including a first dye 5784 (dye 1), and a
second dye 5786 (dye 2). The second dye and the target reagents may
be mixed with one another before introduction into system 5740 or
may be mixed within the system. Target reagents 5782 may provide
primers for amplification of a nucleic acid target, and the first
dye may enable detection of whether amplification occurred. The
first and second dyes may be fluorescent dyes that are
distinguishable optically. The second dye may be a passive
reference or instrument control. In other words, the second dye may
provide a detectable signal having an intensity that is at least
substantially independent of the extent of amplification, if any,
of any nucleic acid target.
[0064] Droplet generator 5744 may form droplets of the
amplification mixture. The droplets may travel through thermal
cycler 5746, to promote amplification of the nucleic acid target,
if any, in each droplet. The droplets then may travel to detection
assembly 5748. Assembly 5748 may detect, for each droplet, a test
signal from the first dye and a reference signal (also termed a
control signal) from the second dye.
[0065] FIG. 5 shows exemplary target reagents 5782 and a control
reagent 5760 that may be included in system configuration 5780 of
FIG. 4. The target and control reagents may permit detection of
test signals in a first detection channel 5788 ("channel 1") and
detection of reference signals in a second detection channel 5790
("channel 2"). The first and second channels may represent distinct
wavelengths and/or at least substantially nonoverlapping wavelength
ranges.
[0066] Target reagents may include a reporter, such as a probe
5792, and target-specific forward and reverse primers 5794. Probe
5792 may be an energy transfer probe (e.g., a TAQMAN probe)
including a nucleic acid, such as an oligonucleotide 5796, that
binds to amplified target, and an energy transfer pair connected to
strand 5796. The energy transfer pair may, for example, be formed
by first dye 5784 and a quencher 5798.
[0067] Control reagent 5760 may include second dye 5786. The second
dye may (or may not) be connected to a nucleic acid, such as an
oligonucleotide 5800. Connection to the oligonucleotide may be
covalent and/or through a binding interaction. Connection of the
second dye to an oligonucleotide or other water-soluble molecule
may improve retention of the second dye in the aqueous phase of a
droplet and/or may facilitate distribution of the dye throughout
the aqueous phase, among others.
[0068] FIG. 6 shows a flowchart illustrating of an exemplary
approach to correcting for system variation using system
configuration 5780 (FIG. 4), and, optionally, the reagents
illustrated in FIG. 5. Test signals (i.e., target signals) and
reference signals may be detected from the same droplets. For
example, test signals may be detected in a first channel and
reference signals may be detected in a second channel. Graphs
illustrating coincident detection of test signals and reference
signals are shown at 5810, 5812, respectively.
[0069] Test signal variation may introduce errors in data
processing. For example, graph 5810 shows substantial variation in
the intensity of the test signals detected. As a result, some of
the test signals may be erroneously classified as positives or
negatives. In the present illustration, two false positives are
marked. However, variation of the test signals may be mirrored by
variation of the reference signals detected from the same droplets.
Accordingly, the test signals may be transformed based on the
reference signals, indicated at 5814, to correct for variation in
the test signals, as shown in a graph 5816, which plots the
transformed test signals. The test signals may be transformed by
any suitable operation or set of operation involving the reference
signals. For example, the test signals may be transformed through
dividing test signals by reference signals, such as dividing each
test signal by its corresponding reference signal, which may be
described as normalizing the test signals. Alternatively, the test
signals may be transformed based on the reference signals by, for
example, baseline subtraction, distance from the regression line,
or the like. A transformation may compensate for variations in the
test channel. This compensation or correction may make the test
signals (i.e., negative test signals and/or positive test signals)
more uniform in value and/or more Gaussian. The transformation also
or alternatively may reduce the frequency of outliers and/or the
overlap of the distributions of positive and negative signals.
[0070] FIG. 7 shows selected aspects of system 5740 in an exemplary
configuration 5830 for (a) detecting amplification of a nucleic
acid target in a set of droplets and (b) system calibration and/or
correction for system variation in another set of droplets.
Configuration 5830 is similar to configuration 5780 of FIG. 4,
except that target reagents 5782 and control reagent 5760 are not
in the same droplets. Accordingly, the target reagents and the
control reagent may be supplied to respective distinct droplet
generators of the system, indicated at 5832, may be supplied to the
sample droplet generator at different times, or the control reagent
may be supplied in pre-formed droplets that do not pass through the
droplet generator, indicated at 5834, 5836. Since the target
reagents and the control reagent are not in the same droplets in
this configuration, the control reagent may include the same dye as
the target reagent (i.e., first dye 5784) or may include a distinct
dye (such as second dye 5786).
[0071] FIG. 8 shows an exemplary graph 5850 of fluorescence signals
that may be detected over time from a flow stream of system
configuration 5830 (FIG. 7) during system calibration, indicated at
5852, and sample testing, indicated at 5854. Calibration and sample
testing may be performed without or with mixing of calibration and
test droplets.
[0072] Calibration and sample testing may be performed serially,
without mixing of droplet types, using the same dye (and/or
detection of the same wavelength(s)). By keeping calibration and
test droplets separate, the distributions of test and calibration
signal intensities may overlap. For example, calibration droplets
and test droplets may be separated temporally in the flow stream,
such that each type of droplet is identifiable based on its time of
arrival at the detection assembly. The time of arrival may be
calculated based on the relative time of introduction of each
droplet type into the flow stream and the velocity of the flow
stream. Thus, the calibration and test droplets may not (or may) be
distinguishable based on signal intensity, but may be
distinguishable temporally. In particular, the test and calibration
droplets may be separated by a temporal (and spatial) gap 5856,
which may identify a transition between droplet types. The use of
temporal gaps also may permit introduction of a set of calibration
droplets within a set of test droplets (i.e., within a test run),
with a gap preceding and following the set of calibration droplets,
to provide identification of each transition to a different droplet
type. Stated differently, calibration may be performed during
sample testing, by inserting calibration droplets into a train of
test droplets, such that the train of test droplets is divided into
two or more discrete groups.
[0073] Calibration droplets may include two or more types of
droplet, which may be introduced separately or intermixed. For
example, FIG. 8 shows a set of stronger calibration signals 5858
followed by a set of weaker calibration signals 5860 produced by
distinct types of calibration droplets. Stronger and weaker
calibration signals 5858, 5860 may correspond generally in
intensity to respective positive test signals 5862 and negative
test signals 5864. In other embodiments, only one type or three or
more types of calibration droplet may be used, and may be
configured respectively to provide one or three or more intensities
of calibration signals.
[0074] Calibration and sample testing alternatively may be
performed with calibration and test droplets randomly intermixed
and thus not distinguishable temporally. Intermixed calibration and
test droplets may be distinguishable by incorporating
distinguishable dyes into the respective droplet types and,
optionally, by detection of the distinguishable dyes at respective
distinct wavelengths. Alternatively, or in addition, calibration
droplets and test droplets may be distinguishable according to
signal intensity detected at the same wavelength(s) and optionally
from the same dye. In particular, calibration droplets may be
designed to have one or more signal intensities outside the signal
range of test droplets (i.e., the signal range provided by the
collective distribution of signal intensities from negative and
positive test droplets (e.g., see FIG. 2)). Thus, calibration
droplets may be identified based on their calibration signals
having signal intensities above and/or below the signal range of
test droplets.
[0075] FIG. 9 shows a flowchart 5880 of an exemplary approach to
correcting for signal variation during an amplification test using
system configuration 5830 of FIG. 7. The approach illustrated in
FIG. 9 distinguishes types of droplet signals, namely, test droplet
signals 5882 and reference droplet signals 5884, based on
differences in signal intensity detected in the same detection
channel, as described above for calibration droplets. In
particular, test droplets may produce a range 5886 of signal
intensities, and reference signals 5884 may have intensities below
(or above) the range. Accordingly, the distinct types of droplets
may be interspersed randomly in the flow stream.
[0076] The reference droplets may be formed with the same amount
(or two or more discrete amounts) of dye. Accordingly, without
signal variation generated by the system, the reference droplets
should produce reference signals of the same intensity. Variation
in reference signal intensity may be mirrored by corresponding
changes in the intensity of test signals. For example, in graph
5888, the intensity of reference signals 5884 and negative test
signals 5890 show a gradual increase with respect to time. As a
result, test signals from amplification-negative droplets may
produce false positives 5892.
[0077] Variation in test signals 5882 may be reduced by
transforming the test signals, indicated at 5894, based on
reference signals 5884, to produce normalized test signals 5896
presented in graph 5898. Transformation may, for example, be
performed by transforming each test signal based on one or more
reference signals temporally proximate to the test signal, a
weighted average of reference signals temporally proximate to the
test signal, a sliding window of averaged reference signals that
overlaps the test signal, or the like. Transformation before
comparing test signals to a threshold may reduce the incidence of
false positives, as shown here, the incidence of false negatives,
or both.
IV. Exemplary Amplification Controls
[0078] FIG. 10 show selected aspects of system 5740 of FIG. 3, with
the system in an exemplary configuration 5910 for testing
amplification of at least a pair of nucleic acid targets in the
same droplets. System configuration 5910 may form an amplification
mixture, which is supplied to droplet generator 5744. The
amplification mixture may incorporate a sample 5756, test
amplification reagents 5858, control amplification reagents 5912,
and at least one control template 5914. Any combination of the
sample, test reagents, control reagents, and control template may
be mixed with one other before introduction into system 5740, or
may be mixed within the system. Test reagents 5758 and control
reagents 5912 may provide primers for respective amplification of
at least one test target and at least one control target.
[0079] Amplification of the test and control targets may, for
example, be detected via a first dye and a second dye,
respectively, which may be included in respective first and second
reporters (e.g., first and second probes). Signals from the first
and second dyes may be detected in distinct (e.g., at least
substantially nonoverlapping) first and second channels (i.e., a
test channel and a control channel) as test signals and control
signals, respectively.
[0080] Control template 5914 may comprise exogenous molecules of
the control target. In contrast, the sample may be tested for a
presence of endogenous molecules of the test target. The control
template 5914 may be present in any suitable amount to provide any
suitable average number of control template molecules per droplet,
to generate a desired fraction of droplets positive for the control
template. For example, the number of template molecules provided by
template 5914 may be substantially less than an average of one per
droplet, such as an average of about 0.1, 0.05, 0.02, or 0.01
molecule per droplet. Accordingly, the number/concentration of
control template molecules may be selected such that the frequency
of amplification of both test and control targets in the same
droplet is low, which may minimize competition that may be caused
by amplification of both test and control targets. For example, the
control template may be present in no more than about one in five
droplets.
[0081] The frequency of amplification of the control target may be
determined by performing an analysis with the system. In some
embodiments, this frequency may be compared with one or more
previously determined frequencies of amplification for the control
target and/or may be compared with an expected value for the
frequency provided by a manufacturer. In any event, a control value
may be determined, with the control value corresponding to a number
and/or fraction of the droplets that are amplification-positive for
the control nucleic acid target.
[0082] Control signals acquired in the control channel may be used
to measure and/or verify the quantitative accuracy of a run and/or
the measurement precision of the system during two or more runs.
The control signals also or alternatively may be used to interpret
a test result, such as the quality of test data measured from a
sample, for example, to verify the quantitative accuracy of the
test data and/or to determine the validity and/or reliability of
the test data. The test result may be interpreted based on control
value determined. For example, the test result may be determined as
being invalid if the control value is less than a threshold value.
Furthermore, data acquired from the control channel, such as
signals from amplification-negative control droplets, may provide
reference signals, as described above in relation to FIG. 6. In
other words, test signals may be transformed using control signals
that functions as reference signals, to normalize the test
signals.
[0083] FIG. 11 shows selected aspects of system 5740 of FIG. 3,
with the system in another exemplary configuration 5920 for testing
amplification of at least a pair of nucleic acid targets in the
same droplets. System configuration 5920 differs from configuration
5910 of FIG. 10 by including a different set of control
amplification reagents 5922 (or a second set of test amplification
reagents) and by the absence of an exogenous control template.
Control reagents 5922 may amplify a control target that is known or
expected to be present in sample 5756, and/or that has a known or
expected representation with respect to a bulk nucleic acid
population present in the sample (e.g., total DNA, total genomic
DNA, genomic DNA from a particular species of organism, total RNA,
total mRNA, etc.). In contrast, target reagents 5758 may amplify a
test target that has an unknown presence in the sample and/or an
unknown presence in with respect to the bulk nucleic acid
population. In any event, amplification of the control target may
be used to determine the quality of test data measured from a
sample, such as to verify the quantitative accuracy of the test
data and/or to determine the reliability of the test data.
Furthermore, an amount of control target determined to be present
in the sample may provide a standard against which an amount of
test target determined to be present in the sample can be compared
and/or normalized. In some embodiments, a control target is
selected that is rare in the sample, such as a target representing
a particular gene mutation. By selecting a rare control target,
amplification of the control target can indicate the limit of
detection of a test target and/or whether amplification of a
low-abundance test target can occur. In some embodiments, the
control target may be replaced by a second test target with an
unknown presence in the sample (before testing).
[0084] FIG. 12 shows exemplary test target reagents 5758 and
control target reagents 5912 (or 5922) that may be included in
system configuration 5910 (or 5920) of FIG. 10 (or 11), to permit
detection of amplification signals in a different detection channel
(i.e., channels 1 and 2, respectively) for each nucleic acid
target. Test target reagents for channel 1 are described above in
relation to FIG. 5. Control target reagents 5912 (or 5922) may be
similar in general structure to the test target reagents, but
different with respect to the nucleic acid sequences of the primers
and probes, to provide test target and control target specificity,
respectively. Also, the test and control probes may include
distinct dyes 5784, 5786 and/or distinct energy transfer partners
5798, 5930 (e.g., distinct quenchers suitable for the respective
dyes). In other embodiments, at least one of the probes may be
replaced by a reporter including an intercalating dye, such as
SYBR.RTM. Green.
[0085] FIGS. 13 and 14 show representative portions of exemplary
data that may be obtained using system configuration 5910 or 5920
and the reagents of FIG. 12. The figures show exemplary graphs
5940-5946 of fluorescence signals that may be detected over time
from a flow stream of the system using different detection
channels, namely, a test channel (channel 1) that detects test data
and a control channel (channel 2) that detects control data. In
FIG. 13, graph 5940 of the test data contains no positive droplet
signals. In contrast, graph 5942 of the control data identifies
positive droplet signals, such as a positive signal 5948, at a
frequency of about one in ten. Thus, the control data demonstrates
that amplification in the droplets is not inhibited substantially
and suggests that the lack of positive signals from the test data
is due to an absence or undetectable level of the test target in
the sample. Accordingly, the control data supports and helps to
validate the negative result in the test data. In contrast, control
graph 5946 of FIG. 14 shows no amplification of the control target
(a substantially larger data set may be analyzed to demonstrate
that the control result holds). The control data of graph 5946 thus
indicates that amplification of the test target also is inhibited
(or the sample is defective, such as too dilute (configuration
5920)), and that the negative test result is not valid.
[0086] FIG. 15 shows selected aspects of system of FIG. 3, with the
system in an exemplary configuration 5960 for testing amplification
of a pair of nucleic acid targets in respective different (i.e.,
nonoverlapping) sets of droplets. Configuration 5960 may be similar
to that of configuration 5910, except that control reagents 5912
and control template 5914 are not mixed with sample 5756 and test
target reagents 5758. Instead, droplets containing the control
reagents and the control template may be formed separately in the
system, indicated at 5962, or may be supplied as pre-formed
droplets that are introduced into the flow stream downstream of
droplet generator 5744, indicated at 5964.
[0087] FIG. 16 shows a pair of exemplary graphs 5980, 5982 of
fluorescence signals that may be detected over time from a flow
stream of system configuration 5960 of FIG. 15 using different
detection channels. Graph 5980 plots fluorescence signals detected
from a first channel, which detects amplification, if any, of a
test target. Graph 5982 plots fluorescence signals detected from a
second channel, which detects amplification, if any, of a control
target. Successful amplification of the control target, as shown
here, may, for example, verify and/or measure aspects of the
system, such as operation of the thermal cycler and/or the
detection assembly, the quality of the reagents, fraction of
amplification-positive droplets, or any combination thereof, among
others.
[0088] In configuration 5960, the test and control reagents are
disposed separately in distinct droplets, so droplet signals in the
first and second channels are not coincident, that is, they are not
detected at the same time. In other embodiments, the control target
may, instead, be a second test target and the control template may,
instead, be another sample (or the same sample). Thus, the use of
at least two detection channels permits droplets for distinct
amplification tests to be interspersed in the flow stream.
V. Exemplary Multi-Channel Detection
[0089] FIG. 17 shows a pair of graphs 5990, 5992 illustrating
exemplary absorption and emission spectra of fluorescent dyes that
may be used in the system of FIG. 3. The dyes are arbitrarily
labeled dye 1 and dye 2, respectively. However, either dye may be
used to detect test signals or control signals in the various
system configurations disclosed herein. Moreover, while illustrated
here for two distinguishable dyes, the system may be used for
detection and analysis with three, four, or more distinguishable
dyes.
[0090] Each graph plots the intensity of absorption ("AB"),
indicated at 5994, 5996, and emission ("EM"), indicated at 5998,
6000, for the corresponding dye. The dyes may have substantially
overlapping absorption spectra, such that the same wavelength of
light may be utilized to excite both dyes. In contrast, the dyes
may exhibit Stokes shifts (i.e., the difference (in wavelength or
frequency units) between the maxima of the absorption and emission
spectra) of different magnitudes. For example, dye 1 may exhibit a
smaller Stokes shift and dye 2 a larger Stokes shift, or vice
versa. Accordingly, the emission spectra of the dyes may be
substantially shifted with respect to one another. As a result,
emission from the two dyes may be detected at least substantially
independently of one another in different detection channels, such
as a detection channel that detects light of a first wavelength or
wavelength range (e.g., .lamda.1) and another detection channel
that detects light of a second wavelength or wavelength range
(e.g., .lamda.2).
[0091] FIG. 18 is a schematic diagram illustrating exemplary use of
the fluorescent dyes of FIG. 17 in an exemplary embodiment 6010 of
system 5740 of FIG. 3. Droplets 6012 containing dyes 1 and 2,
either in the same droplets or different sets of droplets, may be
carried in a flow stream 6014 in a channel 6016. Flow stream 6014
may pass through a detection area 6018 established by an embodiment
6020 of detection assembly 5748.
[0092] Detection assembly 6020 may include a light source 6022 for
exciting the fluorescent dyes in the droplets and at least one
detector 6024 for detecting light emitted from the droplets. Light
source 6022 may, for example, include an LED or laser that emits at
least substantially a single wavelength of excitation light.
Alternatively, or in addition, the light source may include at
least one excitation optical filter that excludes other wavelengths
of light emanating from the light source. Detector 6024 may be
equipped with detection optics 6026, 6028 (e.g., beamsplitters,
emission optical filters, separate detectors) that permit emitted
light from the dyes to be detected separately.
[0093] Exemplary fluorescent dyes that may detected using system
6010 include a fluorescein derivative, such as carboxyfluorescein
(FAM), and a PULSAR 650 dye (a derivative of Ru(bpy).sub.3). FAM
has a relatively small Stokes shift, while PULSAR 650 dye has a
very large Stokes shift. Both FAM and PULSAR 650 dye may be excited
with light of approximately 460-480 nm. FAM emits light with a
maximum of about 520 nm (and not substantially at 650 nm), while
PULSAR 650 dye emits light with a maximum of about 650 nm (and not
substantially at 520 nm). Carboxyfluorescein may be paired in a
probe with, for example, BLACK HOLE Quencher.TM.1 dye, and PULSAR
650 dye may be paired in a probe with, for example, BLACK HOLE
Quencher.TM.2 dye.
VI. Exemplary Self-Normalization of Droplet Signals
[0094] Test signals may be normalized using methods different from
those described above in relation to FIGS. 6 and 9. In particular,
the methods illustrated in FIGS. 6 and 9 involve transformation of
test data with reference data detected (a) in a different detection
channel (FIG. 6) or detected (b) in different droplets (FIG. 9).
This section describes methods that transform test data using
aspects of itself rather than another data set.
[0095] FIG. 19 shows a flowchart 6040 illustrating an exemplary
method of correcting for system fluctuations during a test. The
method involves processing a set of droplet test signals, shown in
a first graph 6042, to produce a transformed set of test signals,
shown in a second graph 6044. Negative test signals 6046 and
positive test signals 6048 each should have respective constant
values over time if there is no system variation. However, system
variation, such as the negative drift over time illustrated in
graph 6042, may produce false negatives, such as a false negative
signal 6050, and/or false positives. Transformation of the test
signals may be performed to correct for system variation before the
test signals are used to estimate a presence of a test target in
sample being tested. In particular, individual test signals may be
transformed differently using the test data, accordingly to the
temporal position of each test signal. For example, each test
signal may be transformed using temporally proximate test data,
such as normalization of each test signal with respect to a sliding
window that averages a subset of the test signals including or
adjacent the test signal. The subset of the test signals used may
be provisionally negative, positive, or negative plus positive test
signals, any of which may be re-assigned as negative/positive after
transformation. For example, graph 6044 shows re-assignment of
false negative signal 6050 as positive after transformation.
[0096] FIG. 20 shows a flowchart 6060 illustrating an exemplary
method of transforming droplet signals based on the width of
respective signal peaks providing the droplet signals. The
flowchart involves graphs 6062, 6064, which represent test data
before and after transformation, respectively.
[0097] Graph 6062 presents test data in which the width and height
of each droplet peak is shown. (Here, each droplet peak is
presented as a square wave to simplify the presentation. However,
in other embodiments, each droplet peak may be detected as having
any suitable shape, such as a wave with sloped leading and trailing
sides.) The width of a droplet fluorescence peak may be used to
determine the size and volume of each droplet, if droplet signals
are detected in a flow stream with known flow rate, generally
within a channel of fixed geometry. Knowing the volume of sample
that is tested for amplification in droplets may be required for
accurately determining the concentration/number of target molecules
in the sample. If droplets of uniform size are desired, peak width
may be used to identify droplets of sizes that are outside the
desired range. For example, in FIG. 20, peaks 6066, 6068 having
widths outside a predefined range are excluded from the data set.
The droplet signals also may be transformed based on width, to
provide transformed test data (i.e., graph 6064), that has been
corrected for volume variation and/or variation in peak width.
[0098] The disclosure set forth above may encompass multiple
distinct inventions with independent utility. Although each of
these inventions has been disclosed in its preferred form(s), the
specific embodiments thereof as disclosed and illustrated herein
are not to be considered in a limiting sense, because numerous
variations are possible. The subject matter of the inventions
includes all novel and nonobvious combinations and subcombinations
of the various elements, features, functions, and/or properties
disclosed herein. The following claims particularly point out
certain combinations and subcombinations regarded as novel and
nonobvious. Inventions embodied in other combinations and
subcombinations of features, functions, elements, and/or properties
may be claimed in applications claiming priority from this or a
related application. Such claims, whether directed to a different
invention or to the same invention, and whether broader, narrower,
equal, or different in scope to the original claims, also are
regarded as included within the subject matter of the inventions of
the present disclosure.
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