U.S. patent application number 16/741299 was filed with the patent office on 2020-07-16 for system and method for determining copies-per-unit-volume using pcr and flow control of droplets.
The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to Mark ANDERSEN, Jian GONG, Gordon A. JANAWAY, Ferrier N. LE, Casey R. McFARLAND, Gothami Padmabandu, Michael C. PALLAS, Marcin SIKORA, Haopeng Wang, Kornelija ZGONC.
Application Number | 20200224280 16/741299 |
Document ID | / |
Family ID | 45929638 |
Filed Date | 2020-07-16 |
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United States Patent
Application |
20200224280 |
Kind Code |
A1 |
JANAWAY; Gordon A. ; et
al. |
July 16, 2020 |
SYSTEM AND METHOD FOR DETERMINING COPIES-PER-UNIT-VOLUME USING PCR
AND FLOW CONTROL OF DROPLETS
Abstract
Methods and systems for quantification of a target nucleic acid
in a sample are provided. The method includes forming a plurality
of discrete sample portions. Each of the plurality of discrete
sample portions comprising a portion of the sample, and a reaction
mixture. The method further includes amplifying the plurality of
discrete sample portions to form a plurality of discrete processed
sample portions. At least one discrete processed sample portion
containing nucleic acid amplification reaction products.
Fluorescence signals are detected from the at least one of the
plurality of discrete processed sample portions to determine a
presence of the at least one target nucleic acid. The method also
includes determining the respective volumes of the plurality of the
plurality of discrete processed sample portions, and estimating the
number of copies-per-unit-volume of the at least one target nucleic
acid in the sample. Estimating the number of copies-per-unit-volume
is based on the number of discrete processed sample portions
determined to contain the at least one target nucleic acid
therein.
Inventors: |
JANAWAY; Gordon A.; (Castro
Valley, CA) ; ANDERSEN; Mark; (Carlsbad, CA) ;
ZGONC; Kornelija; (Carlsbad, CA) ; PALLAS; Michael
C.; (San Bruno, CA) ; SIKORA; Marcin;
(Burlingame, CA) ; McFARLAND; Casey R.; (San
Francisco, CA) ; LE; Ferrier N.; (San Jose, CA)
; Wang; Haopeng; (Arlington, VA) ; GONG; Jian;
(San Marcos, CA) ; Padmabandu; Gothami; (San
Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Family ID: |
45929638 |
Appl. No.: |
16/741299 |
Filed: |
January 13, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15080801 |
Mar 25, 2016 |
10557174 |
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16741299 |
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14009304 |
Oct 1, 2013 |
9322055 |
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PCT/US12/31533 |
Mar 30, 2012 |
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15080801 |
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61481085 |
Apr 29, 2011 |
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61470713 |
Apr 1, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/158 20130101;
G01N 21/6486 20130101; C12Q 1/686 20130101; C12Q 1/6886 20130101;
C12Q 2600/178 20130101; C12Q 1/686 20130101; C12Q 2537/16 20130101;
C12Q 2563/143 20130101; C12Q 2563/159 20130101; C12Q 2565/626
20130101 |
International
Class: |
C12Q 1/6886 20060101
C12Q001/6886; C12Q 1/686 20060101 C12Q001/686; G01N 21/64 20060101
G01N021/64 |
Claims
1-22. (canceled)
23. A method, comprising: forming a plurality of discrete sample
portions of a sample comprising a target nucleic acid, each of the
plurality of discrete sample portions comprising a portion of the
sample; amplifying the plurality of discrete sample portions to
form at least one discrete processed sample portion containing a
nucleic acid amplification reaction product of the target nucleic
acid; detecting a fluorescence signal from the at least one of the
plurality of discrete processed sample portions; using an optical
imager, determining the respective volumes of the plurality of the
plurality of discrete processed sample portions by analyzing a
reference dye signal to determine a dimension of each discrete
processed sample portion; and determining an amount of the target
nucleic acid based on the number of discrete processed sample
portions containing the target nucleic acid therein.
24. The method of claim 23, using the optical imager to determine
the respective volumes of the plurality of discrete processed
sample portions by analyzing a time-of-flight of the processed
sample portions.
25. The method of claim 23, further comprising estimating a portion
size of the discrete sample portions to provide a predetermined
average number of copies a target nucleic acid within the discrete
sample portions.
26. The method of claim 23, wherein the plurality of discrete
sample portions comprises discrete sample portions of substantially
two different sizes and/or of substantially a plurality of
predetermined sizes.
27. The method of claim 23, wherein each of the plurality of sample
portions is at least partially surrounded by a medium that is at
least substantially immiscible with the plurality of discrete
sample portions and wherein the medium that is substantially
immiscible with the plurality of discrete sample portions
optionally comprises at least one of a mineral oil, a silicone oil,
a paraffin oil, a fluorinated fluid, or a perfluorinated
polyether.
28. The method of claim 23, wherein the plurality of discrete
sample portions comprises at least one of porous beads or magnetic
beads.
29. The method of claim 23, wherein determining the respective
volumes comprises imaging the plurality of discrete processed
sample portions.
30. The method of claim 23, further comprising estimating the
number of copies-per-unit-volume of the target nucleic acid in the
sample based on the number of discrete processed sample portions
determined to contain the at least one target nucleic acid
therein.
31. A system, comprising: a droplet apparatus, in a first
configuration, capable of forming a plurality of discrete sample
portions of a sample comprising a target nucleic acid, each of the
plurality of discrete sample portions comprising a portion of the
sample; an amplification apparatus configured to amplify the
plurality of discrete sample portions to form at least one discrete
processed sample portion containing nucleic acid amplification
reaction product of the target nucleic acid; a detection apparatus
configured to detect a fluorescence signal from the at least one of
the plurality of discrete processed sample portions in response to
a presence of the at least one target nucleic acid; an optical
imager configured to determine the respective volumes of the
plurality of discrete processed sample portions by analyzing a
reference dye signal to determine a dimension of each discrete
processed sample portion; and a processor configured to determine
an amount of the target nucleic acid based on the number of
discrete processed sample portions containing the target nucleic
acid therein.
32. The system of claim 31, wherein the optical imager is
configured to determine the respective volumes of the plurality of
discrete processed sample portions by analyzing a time-of-flight of
the processed sample portions.
33. The system of claim 31, further comprising a processor
configured to estimate a portion size of the discrete sample
portions to provide a predetermined average number of copies a
target nucleic acid within the discrete sample portions.
34. The system of claim 31, further comprising an optical imager
configured to determine the respective volumes of the plurality of
the plurality of discrete processed sample portions.
35. The system of claim 31, wherein the droplet apparatus is
configured to form the plurality of discrete sample portions of at
least one of a plurality of sizes, substantially two different
sizes, or substantially a plurality of predetermined sizes.
36. The system of claim 31, wherein each of the plurality of
discrete sample portions is at least partially surrounded by a
medium that is at least substantially immiscible with the plurality
of discrete sample portions.
37. The system of claim 36, wherein the medium that is
substantially immiscible with the plurality of sample portions
comprises at least one of a mineral oil, a silicone oil, a paraffin
oil, a fluorinated fluid, or a perfluorinated polyether.
38. The system of claim 31, wherein the plurality of discrete
sample portions comprises at least one or porous beads or magnetic
beads.
39. The system of claim 31, wherein determining the respective
volumes comprises imaging the plurality of discrete processed
sample portions.
40. The system of claim 31, further comprising a processor
configured to estimate the number of copies-per-unit-volume of the
target nucleic acid in the sample based on the number of discrete
processed sample portions determined to contain the at least one
target nucleic acid therein.
41. A method, comprising: forming a plurality of discrete sample
portions of a sample comprising a target nucleic acid, each of the
plurality of discrete sample portions comprising a portion of the
sample; amplifying the plurality of discrete sample portions to
form at least one discrete processed sample portion containing a
nucleic acid amplification reaction product of the target nucleic
acid; detecting a fluorescence signal from the at least one of the
plurality of discrete processed sample portions; using an optical
imager, determining the respective volumes of the plurality of the
plurality of discrete processed sample portions by imaging the
plurality of discrete processed sample portions; and determining an
amount of the target nucleic acid based on the number of discrete
processed sample portions containing the target nucleic acid
therein.
42. A system, comprising: a droplet apparatus, in a first
configuration, capable of forming a plurality of discrete sample
portions of a sample comprising a target nucleic acid, each of the
plurality of discrete sample portions comprising a portion of the
sample; an amplification apparatus configured to amplify the
plurality of discrete sample portions to form at least one discrete
processed sample portion containing nucleic acid amplification
reaction product of the target nucleic acid; a detection apparatus
configured to detect a fluorescence signal from the at least one of
the plurality of discrete processed sample portions in response to
a presence of the at least one target nucleic acid; an optical
imager configured to determine the respective volumes of the
plurality of discrete processed sample portions by imaging the
plurality of discrete processed sample portions; and a processor
configured to determine an amount of the target nucleic acid based
on the number of discrete processed sample portions containing the
target nucleic acid therein.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
15/080,801 filed Mar. 25, 2016, which is a continuation of
application Ser. No. 14/009,304 filed Oct. 1, 2013, now U.S. Pat.
No. 9,322,055, and a National Stage entry from PCT Application No.
PCT/US2012/031533 filed Mar. 30, 2012, which claims a benefit under
35 USC .sctn. 119(e) from U.S. Provisional Application Nos.
61/470,713, filed Apr. 1, 2011, and 61/481,085, filed Apr. 29,
2011; all applications are incorporated herein by reference in
their entirety.
BACKGROUND
[0002] The present disclosure generally relates to systems and
methods for carrying out digital polymerase chain reaction (dPCR)
assays. The disclosure further relates to controlling the flow of
droplets dispersed in a carrier fluid through a conduit.
[0003] Digital Polymerase Chain Reaction (dPCR) is a method that
has been described, for example, in U.S. Pat. No. 6,143,496 to
Brown et al. Results from dPCR can be used to detect and quantify
the concentration of rare alleles, to provide absolute quantitation
of nucleic acid samples, and to measure low fold-changes in nucleic
acid concentration.
[0004] dPCR is often performed using apparatus adapted from
conventional qPCR, in which replicates are arrayed in a two
dimensional array format including m rows by n columns, i.e., an
m.times.n format. PCR cycling and read-out (end-point or real-time)
generally occurs within the same array. A maximum of m.times.n
replicates can be processed in a single batch run. Generally,
increasing the number of replicates increases the accuracy and
reproducibility of dPCR results.
[0005] The (m.times.n) format in most quantitative polymerase chain
reaction (qPCR) platforms is designed for sample-by-assay
experiments, in which PCR results need to be addressable for
post-run analysis. For dPCR, however, the specific position or well
of each PCR result may be immaterial and only the number of
positive and negative replicates per sample may be analyzed.
[0006] The read-out of dPCR, that is, the number of positive
reactions and the number of negative reactions, is linearly
proportional to the template concentration, while the read-out of
qPCR (signal vs. cycle) is proportional to the log of the template
concentration. For this reason, dPCR typically is constrained to a
narrow dynamic range of template input.
SUMMARY
[0007] According to various embodiments, the present teachings
provide methods and systems for quantification of a target nucleic
acid in a sample. The method includes forming a plurality of
discrete sample portions. Each of the plurality of discrete sample
portions comprising a portion of the sample, and a reaction
mixture. The method further includes amplifying the plurality of
discrete sample portions to form a plurality of discrete processed
sample portions. At least one discrete processed sample portion
containing nucleic acid amplification reaction products.
Fluorescence signals are detected from the at least one of the
plurality of discrete processed sample portions to determine a
presence of the at least one target nucleic acid. The method also
includes determining the respective volumes of the plurality of the
plurality of discrete processed sample portions, and estimating the
number of copies-per-unit-volume of the at least one target nucleic
acid in the sample. Estimating the number of copies-per-unit-volume
is based on the number of discrete processed sample portions
determined to contain the at least one target nucleic acid
therein.
[0008] In some embodiments, the plurality of discrete sample
portions may comprise sample portions of different sizes or all of
the same size. Each of the plurality of discrete sample portions
may comprise a volume of an aqueous reaction medium at least
partially surrounded by a medium that is at least substantially
immiscible with the plurality of discrete sample portions. As used
herein, sample portions may be referred to as droplets, sample
volumes, or reactions volumes, for example. The medium that is
substantially immiscible with the plurality of discrete sample
portions may comprise one or more of a mineral oil, a silicone oil,
a paraffin oil, a fluorinated fluid, a perfluorinated polyether,
and a combination thereof. In some embodiments, the plurality of
discrete sample portions comprise magnetic beads and the method may
further comprise magnetically focusing the magnetic beads within a
flow stream in a flow cytometer. In some embodiments, the plurality
of sample portions may comprise non-magnetic beads, including
porous or hollow beads, for example. The porous or hollow beads may
be spherical or cylindrical. One or more of the plurality of sample
portions may comprise a passive reference dye, a light-scattering
enhancing material, q-dots, a protein, a colloidal metal, colloidal
gold, a reporter dye, a reaction-independent flow marker, or a
combination thereof. In some embodiments, one or more sample
discrete portions of the plurality of sample portions comprises a
primer pair, a nucleotide probe, and a Taq polymerase.
[0009] In another embodiment, a system for quantification of a
target nucleic acid in a sample is provided. The system includes an
emulsion apparatus configured to form a plurality of discrete
sample portions. Each of the plurality of discrete sample portions
includes a portion of the sample, and a reaction mixture. The
system further includes an amplification apparatus configured to
amplify the plurality of discrete sample portions to form a
plurality of discrete processed sample portions, including at least
one discrete processed sample portion containing nucleic acid
amplification reaction products. The system also includes
excitation detection apparatus configured to detect fluorescence
signals from the at least one of the plurality of discrete
processed sample portions to determine a presence of the at least
one target nucleic acid. Furthermore, the system includes a
processor configured to estimate the number of
copies-per-unit-volume of the at least one target nucleic acid in
the sample based on the number of discrete processed sample
portions determined to contain the at least one target nucleic acid
therein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] A better understanding of the features and advantages of the
present disclosure will be obtained by reference to the
accompanying drawings, which are intended to illustrate, not limit,
the present teachings.
[0011] FIG. 1 is a flow diagram of an exemplary method according to
various embodiments of the present teachings.
[0012] FIG. 2 is a flow diagram of another exemplary method
according to various embodiments of the present teachings.
[0013] FIG. 3 is a schematic diagram of an exemplary system and
method according to various embodiments of the present
teachings.
[0014] FIG. 4 is a block diagram of an exemplary system according
to various embodiments of the present teachings.
[0015] FIG. 5A depicts a plurality of discrete sample portions
according to various embodiments of the present teachings.
[0016] FIG. 5B depicts a plurality of discrete processed sample
portions according to various embodiments of the present
teachings.
[0017] FIG. 6 illustrates an exemplary optical setup using flow
cytometry to analyze a plurality of discrete sample portions
according to various embodiments of the present teachings.
[0018] FIG. 7A illustrates flow data of samples that included no
template control according to various embodiments of the present
teachings.
[0019] FIG. 7B illustrates flow data of samples including a
template control according to various embodiments of the present
teachings.
[0020] FIGS. 8A and 8B illustrate a three layered emulsion
according to various embodiments of the present teachings.
[0021] FIG. 9 illustrates a method of preparing a sample according
to various embodiments of the present teachings.
[0022] FIGS. 10-15 illustrate exemplary plots showing a
relationship between C.sub.q and copy number according to various
embodiments of the present teachings.
[0023] FIGS. 16-21 illustrate exemplary modeled C.sub.q plots
versus actual C.sub.q plots.
[0024] FIGS. 22-33 illustrate exemplary C.sub.q distributions
according to various embodiments of the present teachings.
[0025] FIG. 34 illustrates an exemplary histogram for determining
PCR efficiency according to various embodiments of the present
teachings.
DETAILED DESCRIPTION
[0026] The systems and methods described herein provides a dPCR
system and method that does not rely on the fixed (m.times.n)
format and instead uses droplet flow control to greatly increases
the number of dPCR replicates that can be obtained while also
obtaining a tally of positive and negative results useful for dPCR.
Unlike fixed format approaches, the accuracy and precision of the
dPCR result can be tuned as desired, simply by varying the
observation time and flow rate to obtain the required number of
replicates.
[0027] In various embodiments, the methods and systems described
herein may be used to detect other biological components of
interest. These biological components of interest may be any
suitable biological target including, but are not limited to, DNA
sequences (including cell-free DNA), RNA sequences, genes,
oligonucleotides, molecules, proteins, biomarkers, cells (e.g.,
circulating tumor cells), or any other suitable target
biomolecule.
[0028] Furthermore, in addition to dPCR, the methods and systems in
various embodiments may be used in applications, such as fetal
diagnostics, PCR, qPCR, dPCR, allele-specific PCR, asymmetric PCR,
ligation-mediated PCR, multiplex dPCR, nested PCR, bridge PCR,
genome walking, viral detection and quantification standards,
genotyping, sequencing validation, mutation detection, detection of
genetically modified organisms, rare allele detection, and copy
number variation.
[0029] As used herein, droplets may be referred to as sample
portions, sample volumes, or reactions volumes, for example.
[0030] In some embodiments, a flow cytometer, for example, an
acoustic flow cytometer can be used to control the flow of aqueous
droplets dispersed in a substantially immiscible carrier fluid,
through a conduit.
[0031] According to various embodiments, analysis of a sample may
include preparing uniform or variously-sized sample portions. The
sample portions are amplified so that the sample portions contain
the target nucleic acid Amplification may be performed by
polymerase chain reactions (PCR) with target concentration near
terminal dilution. According to various embodiments, amplification
may also be performed by isothermal amplification, thermal
convention, infrared mediated thermal cycling, or helicase
dependent amplification, for example.
[0032] According to various embodiments, detection of a target may
be, but is not limited to, fluorescence detection, detection of
positive or negative ions, pH detection, voltage detection, or
current detection, alone or in combination, for example.
[0033] The volume of the sample portions may be known. If the
sample portions are different sizes, the volume of the sample
portions may need to be determined. The positive and negative
reactions within the plurality of sample portions are counted. More
particularly, the number of sample portions that contain successful
amplification of the target nucleic acid are counted. The sizes and
the positive and negative reactions may be determined by imaging,
for example. The average copy number per reaction is estimated. The
estimation may be made using a Poisson distribution. Then, the
target copy number per unit volume in the starting sample is
estimated. This exemplary method is generally shown in the flow
chart depicted in FIG. 1.
[0034] As shown in FIG. 1, the method may comprise forming a
plurality of discrete sample portions in step 102. Each of the
plurality of a discrete sample portions may include a portion of
the sample, and reaction reagents, such as PCR reagents. The sample
may also be diluted. In some embodiments the plurality of discrete
sample portions may be reactor droplets. The plurality of discrete
sample portions may also be porous beads or magnetic beads, for
example. The discrete sample portions may be a plurality of sizes,
a uniform size, or different predetermined sizes.
[0035] The method may further comprise amplification of the
plurality of discrete sample portions to form a plurality of
processed sample portions in step 104. At least one of the
plurality of discrete processed sample portions contain nucleic
acid amplification reaction products. The amplification may be by
subjecting the reactor droplets to thermal cycling in various
embodiments.
[0036] Then, the method may include detecting fluorescence signals
from the processed sample portions to determine a presence of at
least one target nucleic acid, as in step 106. In other words, the
plurality of discrete processed sample portions may be determined
to be positive or negative for amplification. According to various
embodiments, detection of a target may be, but is not limited to,
fluorescence detection, detection of positive or negative ions, pH
detection, voltage detection, or current detection, alone or in
combination, for example.
[0037] The method further includes determining the respective
volumes of the plurality of discrete processed sample portions in
step 108. The volumes may be determined from imaging the plurality
of discrete processed sample portions. In various embodiments, the
imagining may be done by positioning the plurality of discrete
sample portions in the field of view of an imagining apparatus.
This may be used where the plurality of discrete sample portions
are a plurality of sizes, which may be referred to as a
polydisperse emulsion. In other embodiments, the discrete sample
portions may be of a uniform size and the volume may be known. In
yet other embodiments, the discrete sample portions may be a known
number of different sizes, which may be referred to as an
multi-mono dispersed emulsion. For example, the discrete sample
portions may be two different sizes. In other embodiments, the
discrete sample portions may be three different sizes, for example.
In this way, by determining which size each of the plurality of
discrete sample portions is, the volume may be determined.
[0038] According to various embodiments, a method is provided to
create monodisperse reverse emulsions using T-junctions, as
described for example, in U.S. Patent Application Publication No.
US 2007/0141593 A1 to Lee et al., which is incorporated herein in
its entirety by reference. There would thus be no need to estimate
the size of the discrete sample portions. Some or all of these
workflow steps may be integrated into a single closed system.
[0039] The polydisperse emulsions achieved by the present systems
and methods are relatively easy and cheap to make, and do not
require a consumable. Due to the difference in volume of the
discrete sample portions volumes, they create approximately 3 OM
greater dynamic range of sample input for dPCR without sample
dilution. Acoustic focusing allows massive droplet throughput and
low shear stress to stabilize the plurality of discrete sample
portions in a flow. Also, acoustic focusing has flow speed
flexibility that allows a significant signal to noise increase by
varying flow speeds. This process allows very high numbers of
discrete sample portions to be read, increasing the accuracy,
precision, and dynamic range of dPCR.
[0040] The method may further include estimating the number of
target template copies-per-unit-volume of the at least one target
nucleic acid in the sample based on the number of discrete
processed sample portions determined to contain the at least one
target nucleic acid there, in step 110. As such, the quantity of
the target nucleic acid may be determined.
[0041] In various embodiments, the method may also comprise
averaging the results to improve accuracy and precision and/or
estimating the target copy number per unit volume in the starting
sample. Such methods may increase the dynamic range of dPCR and
provides alternative analysis methods.
[0042] One exemplary method of this type is depicted in the flow
chart shown in FIG. 2. As shown in FIG. 2, the method may first
comprise generating a plurality of discrete sample portions. More
particularly, generating the plurality of discrete sample portions
includes combining reaction components that include sample, assay,
and PCR reagents, with a substantially immiscible continuous phase
liquid, to form a mixture. The mixture is emulsified to form the
plurality of discrete sample portions. In various embodiments, the
plurality of discrete sample portions is an emulsion of reactor
droplets dispersed in the continuous phase liquid.
[0043] Amplification of the plurality of discrete sample portions
may be accomplished by thermal cycling to form a plurality of
discrete processed sample portions. The plurality of discrete
sample portions may be flowed through a conduit before
amplification. In other embodiments, the plurality of discrete
processed sample portions, after amplification, may be flowed the
conduit. The plurality of discrete processed sample portions are
assessed to determine if the discrete sample portions are positive
or negative for amplification. The volumes of the processed sample
portions are estimated. Then, the number of target template
copies-per-unit-volume in the sample may be estimated.
[0044] FIG. 3 is a schematic diagram of an exemplary system and
method according to various embodiments of the present teachings.
In a first step of the method, an emulsion apparatus 20 is provided
where an emulsion 22 is prepared comprising a plurality of discrete
sample portions 24. With reference to FIG. 4, an exemplary dPCR
system 400 is illustrated, including emulsion apparatus 20 for
forming a plurality of discrete sample portions. In some
embodiments, the discrete sample portions 24 may be droplets of an
aqueous sample dispersed in a substantially immiscible carrier 26,
for example, in a fluorinated liquid.
[0045] FIG. 5A illustrates target nucleic acids 500 contained
within discrete sample portions of various sizes. Emulsion 22 is
then amplified. In some embodiments, the amplification is
accomplished by an amplification apparatus 402, referring back to
FIG. 4, thermally cycling such that the discrete sample portions
containing a target nucleic acid are replicated within the discrete
sample portion. The amplification apparatus may be a PCR
instrument. In another example, the amplification apparatus may be
a serpentine thermal cycler. In some embodiments, after amplifying,
emulsion 22 is then moved into and through a conduit 28 in the
direction of flow shown by flow arrow 30, in a flow cytometry
station 32. In conduit 28, the discrete sample portions 24 may be
separated, for example, to form a single-file line of sample
portions. Within, at an exit of, or adjacent an exit of, conduit
28, an excitation detection apparatus 34 can be located where each
discrete sample portion is illuminated by an excitation source 36
and fluorescence that may result from the illumination may be
detected, indicating positive amplification of the target nucleic
acid.
[0046] FIG. 5B illustrates discrete sample portions with positive
amplification 502 as well as discrete sample portions with negative
amplification 504. The plurality of discrete sample portions may
contain a reference dye only, contain no reference dye and no
target nucleic acid, or contain a reference dye and a target
nucleic acid.
[0047] Following excitation and detection by an excitation
detection apparatus 404, with reference again back to FIG. 4, a
plot of the results can be generated as shown graphing the detected
target nucleic acid signal against the detected reference signal.
The excitation and detection apparatus 404 can comprise one or more
of the excitation and detection components described, for example,
in U.S. Patent Application Publication No. US 2007/0141593 A1 to
Lee et al., which is incorporated herein in its entirety by
reference.
[0048] In an exemplary embodiment, a TAQMAN.RTM. fluorescent probes
qPCR reaction with target cDNA is overlayed with a 5-fold higher
volume of mineral oil in a 96-well plate. An inverse emulsion may
be created in an ultrasonic bath. The plurality of discrete sample
portions formed using this method comprises sample portions from
about 1 fL to about 1 pL in volume. The plurality of discrete
sample portions may be thermally cycled in the plate until a
plateau of replicates is produced in the positive discrete sample
portions. With reference back to FIG. 4, an optical imager 404 may
be used to image the plurality of discrete sample portions in order
to determine size and volume of the discrete sample portions. For
example, the discrete sample portions may be analyzed using an
optical camera, or flow cytometry, to estimate the size (volume)
and to determine positivity. Instead of estimating the copy number
per droplet, however, the user determines the droplet volume that
delivered a certain frequency of positivity. For example, one could
ask what discrete sample portion size delivered positive
amplification reactions at a 66% frequency. If 1 pL discrete sample
portions delivered this frequency, then the initial target
concentration would be 1.7.times.10-12 M (1.7 M). If 1 fL discrete
sample portions delivered this frequency, then the initial target
concentration would be 1.7.times.10-9 (1.7 nM). In an alternative
embodiment, the discrete sample portions positivity frequency may
be plotted by discrete sample portions size. The slope of a portion
of the resulting curve may be used to estimate the initial discrete
sample portion concentration.
[0049] Furthermore, dPCR system 400 can include one or more
processors, such as a processor 408. As such, generating any plots
or determining the estimating the number of copies-per-unit-volume
of the at least one target nucleic acid in the sample based on the
number of discrete processed sample portions determined to contain
the at least one target nucleic acid therein, according to
embodiments of the present teachings, may be calculated by
processor 408. Processor 408 can be implemented using a general or
special purpose processing engine such as, for example, a
microprocessor, controller or other control logic. Processor 408
may be included in a separate computing system, or in any one of
optical imager 404, excitation detection apparatus 404,
amplification apparatus 402, or emulsion apparatus 20.
[0050] It will be appreciated that, for clarity purposes, the above
description has described embodiments of the invention with
reference to different functional units and processors. However, it
will be apparent that any suitable distribution of functionality
between different functional units, processors or domains may be
used without detracting from the invention. For example,
functionality illustrated to be performed by separate processors or
controllers may be performed by the same processor or controller.
Hence, references to specific functional units are only to be seen
as references to suitable means for providing the described
functionality, rather than indicative of a strict logical or
physical structure or organization.
Forming Discrete Sample Portions
[0051] According to various embodiments, the present teachings
provide a system including an emulsion apparatus, and a method that
uses the same. The emulsion apparatus generates a plurality of
discrete sample portions. The emulsion apparatus may generate the
plurality of discrete sample portions by various methods, such as
shaking, stirring, sonicating, extruding, or shear electowetting,
for example. In some embodiments, the emulsion apparatus may be a
sonicator, a vortexer, or a plate shaker. Emulsification
parameters, such as emulsification method, strength/power, time,
oil/surfactant chemistry, viscosity, concentration, aqueous phase
composition, and water-to-oil ratio, for example, may be optimized
to produce desired sizes for the discrete sample portions. In some
embodiments, the discrete sample portions have a diameter of
between 10.mu., to 150 .mu.m and a volume of between 1 pL to 1
nL.
[0052] Exemplary systems for methods of preparing and processing
emulsions that may be used according to the present teachings
include those described in U.S. patent application Ser. No.
12/756,547, filed Apr. 8, 2010, to Lau et al. for "System and
method for preparing and using bulk emulsion," which is
incorporated herein in its entirety by reference. Exemplary systems
for methods of processing and thermally cycling emulsions that may
be used according to the present teachings include those described
in U.S. patent application Ser. No. 12/756,783, filed Apr. 8, 2010,
to Liu et al. for "System comprising dual-sided thermal cycler and
emulsion PCR in a pouch," which is also incorporated herein in its
entirety by reference.
[0053] The method may further comprise diluting the sample to form
a diluted sample and forming the plurality of discrete sample
portions from the diluted sample. Dilution may comprise terminally
diluting the sample to achieve an average of less than one of the
at least one target nucleic acid molecules per sample portion. In
some embodiments, the method further comprises: serially diluting
different portions of the sample by different respective dilution
ratios; dividing each serially diluted portion into a plurality of
aliquots; and processing each of the plurality of aliquots of each
of the serially diluted portions.
[0054] According to various embodiments, the components of the
plurality of discrete sample portions may be provided in a
multi-well plate. Forming the plurality of discrete sample portions
may include emulsifying an aqueous sample with a medium that is at
least substantially immiscible with the sample. In some
embodiments, the emulsifying may comprise mixing the aqueous sample
with the medium that is at least substantially immiscible with the
sample in the multi-well plate, sonicating the aqueous sample with
the medium that is at least substantially immiscible with the
sample in the multi-well plate, shaking the aqueous sample in the
medium that is at least substantially immiscible with the sample in
the multi-well plate, or stirring the aqueous sample in the medium
that is at least substantially immiscible with the sample in the
multi-well plate. Emulsification to form a plurality of discrete
sample portions may also take place in the presence of a
surfactant, so that the kinetic stability of the emulsion
increases. Some surfactants which may be used, but are not limited
to, are Span80, STF9, ABIL EM90, and DC BY11-030, for example.
[0055] In various embodiments, polydisperse emulsions may be
generated. Generally, polydisperse emulsions are less difficult to
make, minimally handled, can be formed in batches, and greatly
increase the dynamic range of dPCR. Furthermore, a small reaction
chambers allow analysis without sample dilution that can introduce
error. For example, heat, shaking, sonic energy, ultrasonic baths,
combinations thereof, and the like can be used to produce
emulsions, for example, to process batches of emulsions in 96-well,
384-well plates, or cell culture plates without the need for any
special consumables to physically touch the samples. In other
embodiments, a plate may be used based on the amplification
apparatus. This greatly reduces the chance of cross-contamination.
In addition, polydisperse emulsions typically vary in volume from
about 1 fL to 10 pL, eliminating the need to dilute samples to
achieve terminal dilutions. Since forming an emulsion may result in
very small discrete sample portions, the method may further include
removing very small droplets that may be problematic to detection
or amplification.
[0056] In another embodiment, multi-mono dispersed emulsions may be
formed. A multi-mono dispersed emulsion may include two or more
sizes of discrete sample portions, where the sizes are known or
predetermined. For example, a multi-mono dispersed emulsion may
contain three different sizes of discrete sample portions that are
substantially the same size as three different predetermined sizes.
Substantially means within +/-10% of the predetermined size. By
determining the plurality of discrete sample portions which size of
the different predetermined sizes each discrete sample portion is,
the volume of each discrete sample portion can be determined.
Multi-mono dispersed emulsions may vary in volume from about 1 fL
to 10 pL. In other words, each droplet can be binned into a
predetermined size. In this way, the dynamic range can be increased
and an optical imager may be simplified.
[0057] Any of a variety of substantially or totally immiscible
fluids can be used as the carrier fluid. By immiscible what is
meant is immiscible with respect to the aqueous sample droplets.
The substantially or totally immiscible fluid can comprise, for
example, paraffin oil, mineral oil, silicone oil, a perfluorinated
polyether (PFPE), other fluorinated fluids, fluorinated solvents,
combinations thereof, and the like. Some specific fluids that can
be used as a carrier fluids include GALDEN.RTM. HT170 available
from Solvay Solexis of West Deptford, N.J., other GALDEN.RTM. HT
liquids available from Solvay Solexis, FC-40 available from 3M
Company of St. Paul, Minn., and other FLUORINERT.TM. liquids
available from 3M Company of St. Paul, Minn.
[0058] In some embodiments, forming the plurality of discrete
sample portions may include diluting a sample to near the single
molecule limit, and combining or mixed the sample with all reagents
needed for a PCR. A good input quantity of template may comprise
the dilution limit where each aqueous reactor volume contains 1 or
0 target nucleic acid molecules, for example, such that the Poisson
parameter, .lamda., is close to or equal to 1. If desired, in order
to determine an optimal dilution, an initial quantification using,
for example, a QUBIT.RTM. system (available from the Qubit Systems
Inc., Kingston, Ontario, Canada) may be used. Unlike
lower-replicate dPCR embodiments, the system and method of the
present teachings can accommodate a much larger deviation from an
ideal .lamda.=1 input concentration.
Emulsification and Sampling
[0059] In various embodiments, a method to prevent evaporation of
the aqueous droplets in water-in-fluorocarbon emulsions is
provided. In this way, errors in sampling an emulsion of a
plurality of discrete sample portions are minimized Discrete sample
portions, in this case aqueous droplets, are formed by adding
hydrocarbon oil into a water-fluorocarbon mixture and agitating the
three phases together. As such, after emulsification the
hydrocarbon oil forms a layer covering the aqueous droplets to
prevent evaporation.
[0060] Density of aqueous phase is around 1 g/cc. Density of
fluorocarbon (FC) fluid is about 1.6-1.8 g/cc. Thus, in
water-in-fluorocarbon emulsions, aqueous droplets form a layer on
top of FC fluid due to the density difference. Without a layer to
cover the aqueous droplets, the aqueous droplets may evaporate
during storage. Furthermore, evaporation becomes more severe if the
emulsion is heated, for example, in the thermal cycles of the PCR
process. Droplets may shrink or evaporate completely, inhibiting
biological reactions inside the droplets and making
post-amplification droplet sizing difficult.
[0061] According to various embodiments, hydrocarbon oil may be
layered on top of the layer of aqueous droplets. The density of
hydrocarbon oil is about 0.8 g/cc. Thus, the hydrocarbon oil is
immiscible with both water and fluorocarbon. The hydrocarbon oil
layer reduces evaporation of the aqueous droplets. However, if the
hydrocarbon oil is added after emulsification, this extra step
increases complexity of the process and time. Furthermore, if many
parallel emulsion vessels (e.g. sealed PCR wells) need to be opened
to add hydrocarbon oil, it may increase the chance of cross
contamination among vessels.
[0062] According to various embodiments, hydrocarbon oil is added
into the vessels together with a water-fluorocarbon fluid mixture
before the emulsification or agitation. Thus there are three phases
existing in the vessel. The FC phase contains the fluorocarbon
fluid and a surfactant (fluorosurfactant) to make
water-in-fluorocarbon emulsion. In one example, the fluorocarbon
fluid is HFE7500 (3M), the fluorosurfactant is a block copolymer
containing hydrophilic poly(ethylene glycol) (PEG) block and
fluorophilic poly (perfluoropropylene ether) (PFPE) block, the
hydrocarbon oil is a heavy mineral oil (Sigma-Aldrich 330760), and
the aqueous phase includes sodium chloride (NaCl) solution 100 mM
in distilled water. The aqueous phase contains water and biological
reagents. The hydrocarbon (HC) phase contains only neat hydrocarbon
oil, but no surfactant. The hydrocarbon oil is agitated together
with water-fluorocarbon fluid mixture. After the agitation the
aqueous phase is broken into small aqueous droplets stabilized by
the fluorosurfactant and these aqueous droplets form a layer on top
of the fluorocarbon fluid. The hydrocarbon oil forms a layer on top
of the aqueous droplet layer preventing the evaporation of the
aqueous droplets.
[0063] In one example, the vessel may be a MICROAMP.RTM. Optical
96-Well Reaction Plate (Applied Biosystems, 4316813) with
MICROAMP.RTM. Clear Adhesive Film (Applied Biosystems, 4306311).
Tube strips can also be used, e.g. Molecular BioProducts PCR 8-Tube
Strips.
Example 1
[0064] 50 uL HFE7500 with 2 wt % fluorosurfactant, 10 uL NaCl and
40 uL heavy mineral oil were added in this order into each well on
the plate. The plate was sealed by the adhesive film. When a tube
strip is used, the tubes are sealed with cap strip.
[0065] The plate or the tube strip was then shaken by an
oscillating mixer (RETSCH.RTM. MM301) with appropriate adapters.
The operating parameter used in this example was 15 Hz for 1
min.
[0066] After shaking, the plate was unloaded from the mixer and
kept in the upright position. A layer formed above the HFE7500.
This layer contains the aqueous droplets stabilized by the
fluorosurfactant. A clear mineral oil layer was formed on top the
droplet layer, preventing the evaporation of aqueous droplets. This
three-layered emulsion assembly in a tube-strip is depicted in FIG.
8A.
[0067] The plate or the tube strip with the emulsion sample was
thermocycled between 95.degree. C. and 60.degree. C. for at least
40 cycles and no significant evaporation was observed. This
three-layered assembly in a tube-strip after thermocycles is
depicted in FIG. 8B.
Sampling
[0068] According to other embodiments, a method to sample the
emulsion for subsequent analysis of the individual droplets is
provided.
[0069] As described above, the aqueous droplets form a layer on top
of the fluorocarbon fluid and are covered by the mineral oil. For
subsequent analysis of the individual aqueous droplets, it is
desirable to sample only the aqueous droplets and the fluorocarbon
fluid, and to remove the mineral oil from the sample. An exemplary
method is described below, with reference to FIG. 9.
[0070] In step 1, a capillary is inserted into a tube. The bottom
end of the capillary is in the fluorocarbon phase and close to the
bottom of the tube. In step 2, the fluorocarbon fluid 902 is
withdrawn by applying negative pressure inside the capillary. The
fluorocarbon fluid 902 goes into the capillary, followed by the
aqueous droplets 904, and then the mineral oil 906. Next, the
negative pressure is stopped when only a small amount of mineral
oil 906 enters the capillary.
[0071] In step 3, the capillary is in the upright position. Because
the aqueous droplets 904 have much lower density than the
fluorocarbon fluid 902, they will float up until reaching the top
of the fluorocarbon fluid 902. The mineral oil 906 inside the
capillary tube forms a "plug" under the fluorocarbon fluid, but it
does not float up. Thus the aqueous droplets 904 and the mineral
oil 906 are separated automatically. Additionally, aqueous droplets
904 may be sorted by size because large aqueous droplets have much
higher rising velocity than smaller aqueous droplets. In some
embodiments, a variable amount of backflow can be applied to
prevent small droplets from migrating up the capillary.
[0072] In step 4, sampling, the system can be backflushed to remove
residual droplets and mineral oil.
Example 2
[0073] In this example, the emulsion sample described above in
Example 1 is used to test the sampling procedure. The emulsion held
in an individual well on a 96-well PCR plate. The volume of the
fluorocarbon fluid (HFE 7500) plus that of the aqueous droplet
layer is 60 uL. The volume of the hydrocarbon oil (heavy mineral
oil) is 40 uL. Thus, the total sample volume in the well is 100
uL.
[0074] A GASTIGHT.RTM. syringe (Hamilton #81320) is used together
with a syringe pump (Nexus 3000) to provide negative and positive
pressure. The inner wall of a capillary (I.D. 1 mm, length 10 mm)
is coated with hydrophobic fluoropolymer coating. This is to
prevent the wetting of aqueous droplets on the inner wall. An open
end of the capillary is connected to the syringe needle via
flexible silicone tubing.
[0075] In step 1, the capillary was kept upright and the other open
end is inserted into the well containing the 3-layered emulsion
assembly. The position of this end was inside the fluorocarbon
fluid and close to the bottom of the tube, as shown in the attached
sketch.
[0076] In step 2, the "withdraw" mode of the syringe pump was used
to create negative pressure. The total volume to be withdrawn was
set to be 70 uL and volumetric rate is set to 10 uL/min. At the end
of this step all the fluorocarbon fluid and all the droplets were
collected into the capillary, and only a small fraction of mineral
oil (about 10 uL) was also collected. The pump was then stopped and
no more oil was collected.
[0077] In step 3, the capillary was kept upright for 1 min. It was
seen that majority of the aqueous droplets float to the top of the
fluorocarbon fluid, and the bottom section of the fluorocarbon
fluid became free of droplets. The mineral oil "plug" stayed under
the fluorocarbon fluid.
[0078] In step 4, the "infuse" mode of the syringe pump was used to
apply positive pressure inside the capillary. The volume to be
infused was set to be 15 uL and the volumetric rate was 10
uL/min.
[0079] The mineral oil was pushed out of the capillary, and some
fluorocarbon fluid was also pushed out. At the end of step 4, the
sample inside the capillary contained only aqueous droplets and
fluorocarbon fluid. The mineral oil was separated from the
sample.
[0080] In the embodiments described above, a polydispersed
water-in-fluorocarbon emulsion is created directly inside a common
96-well PCR plate covered by a lighter oil without an additional
step. Aqueous droplets can are sampled, sorted and analyzed by
buoyancy without flow.
[0081] As described, embodiments of these methods reduce the
evaporation of aqueous droplets. Furthermore, the cover oil is
added together with aqueous reagents and fluorocarbon fluid before
emulsification. Eliminating extra step to open the emulsion vessel
to add covering oil after emulsification is avoided. Moreover, the
covering oil can be removed from the emulsion sample and will not
interfere with sample analysis.
[0082] In other embodiments, the plurality of discrete sample
portions may include using non-magnetic beads, magnetic beads, or a
combination thereof may be used in the emulsion mix. In some
embodiments, the plurality of sample portions may comprise
non-magnetic beads, including porous or hollow beads, for example.
The porous or hollow beads may be spherical or cylindrical. In some
embodiments, no beads are used.
Detection of Positive or Negative Amplification
[0083] According to various embodiments, flow cytometry (FC) is
used to count the number of droplets exhibiting specific
fluorescence signals due to, for example, fluorescently-labeled
nucleotide probes or antibody tags. The flow cytometry system can
provide a fluid flow of suspended reactor droplets through an
analysis region in which an excitation and detection system may be
used to serially count the number of droplets with one or more
specific fluorescent emission characteristics. Multi-color FC
systems may be used for labeling or tagging droplets and/or target
molecules with different markers.
[0084] TAQMAN.RTM. fluorescent probes, SYB.RTM., LUX.TM., or other
real-time fluorescence detection methods may be used. If beads and
SYBR.RTM. are used, and the method further comprises a flow
cytometry step, SYBR.RTM. may subsequently be included in the
running buffer of a flow cytometer to bind and label beads
containing dsDNA. If beads and TAQMAN.RTM. are used, probes may be
attached to the beads following cleavage. In some embodiments,
labeled primers, such as LUX.TM. primers, may be used to label
beads, and the method may allow multiplexing without a need for
using a dye-loaded buffer in a flow cytometry step.
[0085] According to various embodiments, a passive reference or
spectator reference can be used in the method, for example,
included in the aqueous amplification reagent mix. For bead-based
approaches, the passive reference may be made to attach to the bead
during or after the amplification, for example, prior to breaking
the emulsion. In an exemplary embodiment, a biotinylated
fluorescent ROX.TM. dye is used with a streptavidin-terminated
bead. For non-bead-based emulsions, a water-soluble dye, such as
ROX.TM. dye, may be used.
[0086] The present teachings expand the capabilities of dPCR by
greatly increasing the number of replicates that may be processed
in a given time period when compared to current dPCR methods. The
improvements may be use available instrumentation and overcome the
limitations of current m.times.n dPCR configurations. In some
embodiments, the present teachings provide serial processing using
conventional flow cytometry systems applied to dPCR. In some
embodiments, the system and method processes micelles containing
PCR reactants and dispersed in a substantially immiscible carrier
fluid. In some embodiments, magnetic focusing is used in
conjunction with flow cytometry and post-read, downstream
collecting and processing of single droplets is enabled.
[0087] According to various embodiments, emulsion PCR (emPCR) is
used and may involve a massively parallel PCR amplification of
nucleic acid samples in aqueous reactors immersed in a
substantially immiscible continuous phase, for example, in a
non-soluble oil medium. The method may be used, for example, to
prepare poly-disperse collections of reactor droplets or libraries
for analysis.
[0088] According to various embodiments, fluorescent-activated cell
sorting is used such that, following analysis, various fluid
manipulation methods divert droplets with specific fluorescence
signatures to different locations or chambers for enrichment or
other further processing.
[0089] According to the present teachings, a system and method are
provided for using emulsion PCR with a flow cytometry read-out to
provide a digital PCR answer. Unlike conventional flow cytometry
systems and methods that process cells, the "cells" in the present
flow cytometer system are instead individual PCR
reactant-containing droplet reactors, some of which may comprise
replicates of PCR reactions, also referred to as positive
results.
[0090] In various embodiments, following the formation of the
discrete sample portions, the resulting amplification products can
be introduced into a flow cytometer. This can be accomplished in
any suitable way. For example, when beads are used, the emulsion
can be broken with detergent, rapid aqueous dilution, or another
standard emulsion breaking method. An aqueous suspension of the
beads can then be introduced into a flow cytometer in a manner
similar to how cells are typically loaded in a conventional cell
manipulating flow cytometer. In some embodiments, for bead and
bead-less emulsions, lipids may be included in the emulsion
followed by aqueous dilution to result in micelle formation,
wherein the PCR product may be contained, for example, within a
lipid bilayer. Such a droplet can be referred to as a "PCR cell"
that may then be introduced and analyzed in a manner used to
introduce and analyze a conventional biological cell in a flow
cytometer.
[0091] Plates of emulsified samples can be bulk thermal cycled
using a standard thermal cycler, for example, using any of the many
thermal cyclers available from Applied Biosystems, LLC of Foster
City, Calif., for example. Furthermore, as used herein, thermal
cycling may include using a thermal cycler, isothermal
amplification, thermal convention, infrared mediated thermal
cycling, or helicase dependent amplification, for example. In some
embodiments, the chip may be integrated with a built-in heating
element.
[0092] Amplification may be run to completion to create strong
signals for the reporters present in droplets containing a target
molecule. In some embodiments, a microfluidic meandering PCR method
may be used or isothermal amplification techniques may be used to
keep emulsions intact.
[0093] According to various embodiments, the method may comprise
subjecting the plurality of discrete sample portions to nucleic
acid amplification conditions for carrying out a polymerase chain
reaction, for example, thermal cycling conditions.
[0094] In some embodiments, the flow cytometer can be set to detect
at least two colors, for example, the passive reference and the
indicator dye. In an example, the flow cytometer may be set up to
detect fluorescence from a SYBRO dye and from a FAM.TM. dye. Using
TAQMANO fluorescent probes available from Applied Biosystems, LLC
of Foster City, Calif., multiplexing can also be applied generating
more than one probe signal, for example, to generate signals form
FAM.TM., VIC.RTM., and other dyes. For simplex reactions, 3 types
of "cells" should be distinguishable: unlabeled cells from
beads/micelles which were not in contact with the sample-PCR mix,
cells labeled with the spectator dye only from beads/micelles which
included PCR mix but no template, and cells labeled with both dyes,
where the bead/micelle was in contact with both the PCR mix and a
target template.
[0095] A digital read-out may be calculated as follows. Two-dye
"cells" are 1 (indicating mix+ template present), passive dye-only
"cells" are 0 (indicating mix but no template), and un-dyed "cells"
are not used in the calculation. The number of 0 and 1 "cells" is
used to fit the Poisson equation, and estimate the number of
template molecules present in the original diluted sample. From
that, the number of template molecules present in the original
undiluted sample can also be estimated.
[0096] According to various embodiments, magnetic beads may be used
in the assay. Magnetic forces may be used to break the emulsion.
Cylindrical solenoid fields may be used during flow cytometry to
focus beads into the center of the flow stream, increasing the
sensitivity and throughput of the flow system. In some embodiments,
focusing may be enabled by an acoustic focusing system as described
below. Following analysis, variable magnetic fields can be used to
direct particular "cells" to an appropriate reservoir.
[0097] In some embodiments, if non-magnetic beads are used in the
emulsion, a magnetic passive reference may be included in the PCR
mix. For example, iron nanoparticles may be used which are
functionalized for aqueous solubility and covalent attachment to
polystyrene beads, silica beads, or other beads. In this way, a
magnet can be used to retain beads which are exposed to the
reaction mix (true PCR positives and negatives) while beads which
were not exposed to the mix during formation may be easily washed
away. Such an approach may also be used to facilitate clean-up for
SOLiD.TM. library preparation.
[0098] By using an acoustic focused flow system, as in some
embodiments, a method is provided that has very high speed and
flexibility. Also very low sheer can help keep the emulsion intact
during the reading process, as opposed to higher sheer that might
disrupt the emulsion and cause inaccurate results. The present
systems and methods can measure up to 1,000,000 discrete sample
portions per sample, for example, from about 1,000 to about
1,000,000 discrete sample portions per sample, from about 10,000 to
about 1,000,000 discrete sample portions per sample, or from about
100,000 to about 1,000,000 discrete sample portions per sample.
[0099] Exemplary acoustic flow systems and methods and components
thereof that can be used in the present systems and methods include
those described, for example, in U.S. Published Patent Applications
Nos. US 2009/0050573 A1 to Ward et al. and US 2009/0178716 A1 to
Kaduchak et al., both of which are incorporated herein in their
entireties by reference. Other flow cytometry systems and methods
having components that can be used in the present teachings include
those described, for example, in U.S. Published Patent Application
No. US 2009/013066 A1 to Oldham and in U.S. Pat. No. 7,280,207 B2
to Oldham et al., both of which are also incorporated herein in
their entireties by reference.
Fluorescent-Activated Cell Sorting (FACS)
[0100] Determining a presence or an absence of the at least one
target nucleic acid in each of the plurality of processed sample
portions may comprise measuring a fluorescence signal. After
determining the presence or absence of the at least one target
nucleic acid in each of the plurality of processed sample portions,
fluorescent-activated cell sorting (FACS) may be used to sort the
plurality of processed sample portions according to the fluorescent
signal measured. In some embodiments, the volume of one or more of
the processed sample portions may be estimated by measuring a
fluorescence signal. The determining a presence or an absence may
comprise introducing each of the plurality of processed sample
portions individually into a flow cytometer.
[0101] If a FACS platform is used, after flow through the analysis
region, a bead/micelle can be segregated according to the
fluorescent signals detected. For example, all positive beads can
be shunted, directed, or diverted to a separate reservoir for
collection and subsequent downstream analysis with other methods,
for example, for a sequencing reaction or a sequence detection
reaction.
[0102] The system and method provide the ability to estimate the
droplet volume, as well as the presence of a reporter for PCR
amplification. For this purpose, a flow cytometer such as a
modified acoustic focusing flow cytometer may be used with an
autosampler. The flow system may autosample from 96- or 384-well
plates. Acoustic focusing may be sued to enable excellent focus
control and can achieve read speeds of 20,000 events per second.
The size of a droplet can be measured using any of a variety of
properties of the droplet, for example, by measuring laser forward
or side scatter properties. In some embodiments, a reference dye
may optionally be added to the aqueous phase, and the signal from
the reference dye may be used to estimate droplet volume. Positive
and negative counts are available for each droplet that passes the
laser interrogation zone in the center of the acoustically focused
flow. High throughput using parallel acoustic focusing streams may
be achieved. Sorting positive droplets may be employed as a
preparatory method for DNA sequencing.
Non-Flow Cytometry Read-Out Alternatives
[0103] In some embodiments, read-out methods after emPCR are used
that are alternatives to flow cytometry. For example, emulsion
products with beads can be dispersed on a glass slide, for example,
containing a thin gel matrix for immobilization. The dispersed,
fluorescent beads can be read using a SOLiD.TM. platform (available
from Applied Biosystems, Foster City, Calif.), a conventional
fluorescence microscope, or a COUNTESS.TM. cell counting system
available from Invitrogen, Carlsbad, Calif. The presence of passive
reference-only beads indicates the stochastic limit has been
reached, and these beads do not need to be quantified. Counting the
number of positive beads then provides quantitation of the template
molecules present in the initial mix. Micelles and/or beads may
also be read serially using Capillary Electrophoresis systems with
polymer (or no polymer), for example, using a polymer specially
optimized for such reactors.
[0104] According to various embodiments, a system that uses a
manual PCR setup is provided that may use SYBR.RTM. or TAQMAN.RTM.
assays and standard qPCR SuperMixes. The reactions may be set up in
a standard 96-well plate using typical PCR volumes, for example,
from about 5 .mu.L to about 15 .mu.L, covered with approximately 50
.mu.L of oil or fluorinated fluid for emulsion PCR. The plate may
be sealed using an adhesive cover. More complicated microfluidic
reaction assemblies may also be used.
Dilution and Fluorescent-Activated Cell Sorting (FACS)
[0105] The present teachings can achieved a throughput of thousands
of sample droplets per minute. As a result, the dynamic range of an
assay may be expanded and the method can be extended to
applications typically reserved for qPCR, such as gene expression,
genotyping, and miRNA analysis. The present systems and methods are
well suited for small sample inputs, for example, single cell
samples. In some embodiments, a fluorescent-activated cell sorting
(FACS) system is used and the method may comprise collecting and
purifying selected post-PCR samples from other samples, obviating
the need for user accessibility to and manipulation of samples in
plate or array formats. For example, all mutant amplicons
identified by dPCR may be sorted from wild-type amplicons, for
subsequent sequencing.
[0106] In some embodiments, the method involves using FACS before
and after emPCR. Prior to emPCR, the FACS can be used to prepare
and isolate single cells (or types of cells) for analysis. The
single cells can be lysed and introduced into the emPCR step for
subsequent FC analysis. As an example, circulating tumor cells, as
detected by a fluorescent antibody, may be counted and enriched by
FACS. The resulting cancer cell product can then be analyzed using
the dPCR approach described above, that is, emPCR followed by
FC/FACS, which provides an extremely low limit of detection for
quantifying expression levels in these purified cells.
[0107] The fraction of beads/micelles containing neither passive
reference nor template may be large if the emulsion PCR process is
not optimized. With a throughput of 10,000 cells per minute, if 10%
of beads/micelles are in contact with the PCR mix, 60,000 usable
digital replicate results may be obtained in one hour of flow
cytometer time. Moreover, the emPCR+FC of the present teachings can
produce answers beyond typical dPCR results. For example, the
method may be used to simply genotype a sample by measuring the
ratio of the FAM/ROX cells and the VIC.RTM./ROX.TM. cells. In some
embodiments, with such a high throughput, a 3-5 log dynamic range
may be attainable for gene expression or miRNA analysis. Using this
approach, with 60,000 observations per hour, roughly 1,000-fold
differences in expression of two targets may be measured. For
example, 21,045 cells of target A vs. 24 cells of target B may be
quantified, a 1000-fold difference in expression levels measured
which is absolute in quantity and independent of PCR efficiency and
other differences between the two targets.
Volume Estimation of Discrete Sample Portions
[0108] In yet other embodiments of the present teachings, an
alternative data analysis method for absolute target molecule
quantification from heterogeneously sized dPCR reactions, is
provided. The method may comprise determining absolute target
concentrations from reactions of various volumes. The reactions may
be created with random sizes, randomly or deliberately. The method
is useful in analyzing dPCR data from polydisperse emulsions, such
as emulsions created with mechanical or sonic energy.
[0109] The method may further comprise comparing the plurality of
processed portions to a plurality of standards of known respective
volumes, for example, a plurality of standards of known respective
volume that uniformly sized or that are of different known volumes.
The method may further comprise subjecting a plurality of portions
of a standard to the same nucleic acid amplification conditions to
form a plurality of processed standards, wherein each of the
processed standards are of a known respective volume, and then
comparing the plurality of processed standards to the plurality of
processed sample portions. In some embodiments, the plurality of
sample portions have an average of from about 0.1 to about 0.8 copy
of the target nucleic acid per discrete loaded mixture. The
plurality of sample portions may have an average diameter of from
about 0.3 micrometer (.mu.m) to about 600 .mu.m, or an average
diameter of from about 1.0 .mu.m to about 100 .mu.m, or an average
volume of from about 0.5 femtoliter (fL) to about 1 microliter
(.mu.L), or an average volume of from about 10.0 fL to about 100
nanoliters (nL).
[0110] According to various embodiments, the volume of the discrete
sample portions is estimated. The present systems and methods may
use an optical imager for using optical scatter properties, a
passive reference dye, optical refraction properties, optical
imaging for measurement, optical reflection properties, optical
absorbance properties, optical transmission properties, a ladder of
standard droplet sizes using different reference dyes, or a
combination thereof to estimate discrete sample portion volumes. An
exemplary optical imager 600 is depicted in FIG. 6 to detect
samples in flow cell 602. An optical imager may also be camera
configured to take images of the discrete sample portions, for
example.
[0111] In an example, discrete sample portions of first, second,
and third volumes of different known respective standard sizes may
contain first, second, and third respective detectably unique dyes
and may be identified and used to scale the size of the discrete
sample portions having unknown volume sizes. The systems and
methods of the present teachings may produce discrete sample
portion sizes of from about 0.3 .mu.m in diameter up to about 1000
.mu.m in diameter, for example, from about 0.4 .mu.m in diameter up
to about 300 .mu.m in diameter, from about 0.5 .mu.m in diameter up
to about 200 .mu.m in diameter, or from about 1.0 .mu.m in diameter
up to about 100 .mu.m in diameter. Discrete sample portion volumes
of up to about 1.0 uL in size may be produced and processed
according to various embodiments. Discrete sample portion volumes
based on spherical diameters can be estimated, for example, using a
conversion chart such as this one:
TABLE-US-00001 Radius diameter volume 0.6 uM 1.2 uM 1 fL e. coli
1.4 uM 2.8 uM 10 fL 3 uM 6 uM 100 fL 6 uM 12 uM 1 pL 14 uM 28 uM 10
pL human cell 30 uM 60 uM 100 pL 60 uM 120 uM 1 nL 140 uM 280 uM 10
nL 300 uM 600 uM 100 nL 600 uM 1200 uM 1 uL
[0112] Measuring the size of each of the plurality of processed
sample portions may comprise analyzing each of the plurality of
processed sample portions, and the analyzing may comprise one or
more of measuring or analyzing an index of refraction, a light
scattering property, a forward light scattering property, a side
light scattering property, an optical absorption property, an
optical transmission property, a peak height of an optical signal,
a peak width of an optical signal, a fluorescent property, a
time-of-flight fluorescent property, or a combination thereof. The
method may further comprise estimating what size of processed
sample portion provides a specific percentage of processed sample
portions of that size that test positive for the presence of one or
more of the at least one target nucleic acid, or estimating what
size processed sample portion of the differently-sized processed
sample portions provides a 50% positivity rate with regard to
determining the presence of one or more of the at least one target
nucleic acid.
Estimating Number of Copies-Per-Unit-Volume of Sample
[0113] According to yet other embodiments of the present teachings,
a method for estimating the number of copies-per-unit-volume of at
least one target nucleic acid in a sample is provided. The method
may comprise: forming a plurality of discrete sample portions each
comprising a portion of a sample, and a reaction mixture;
subjecting each of the plurality of discrete sample portions to
nucleic acid amplification conditions to form a plurality of
discrete processed sample portions including at least one discrete
processed sample portion containing nucleic acid amplification
reaction products; generating and measuring fluorescence signals
from at least some of the plurality of discrete processed sample
portions; analyzing each of the plurality of discrete processed
sample portions individually to determine a presence or an absence
of one or more of the at least one target nucleic acid in each of
the plurality of discrete processed sample portions based on the
fluorescence signals, and to determine the respective volumes of
the plurality of discrete reacted mixtures; and estimating the
number of copies-per-unit-volume of the at least one target nucleic
acid in the sample based on (1) the number of discrete processed
sample portions determined to contain one or more of the at least
one target nucleic acid present therein, and (2) the determined
respective volumes. Each of the plurality of discrete sample
portions may be provided in a multi-well plate and the method may
further comprise emulsifying to form the plurality of discrete
sample portions.
[0114] In some embodiments, the method further comprises estimating
what size of processed sample portion provides a specific average
number of copies-per-unit-volume, or estimating what size processed
sample portion of the differently-sized processed sample portions
provides an average of from 0.25 to 0.75 copy-per-processed sample
portion.
Bayesian Inference to Concentration Estimation
[0115] According to various embodiments, an algorithm may be used
for estimating target concentration within a dPCR system with
multiple discrete sample portions sizes and/or multiple sample
dilutions and/or low precision in resolving positive and negative
amplification reactions. Typically, data analysis methods for
digital PCR systems are based on a strict set of assumptions that
may not be possible to meet in practice, or may even be
undesirable. For example, typical assumptions may be: all dPCR
discrete sample portions have identical volume, all dPCR discrete
sample portions have identical sample dilution, and the distinction
between positive and negative discrete sample portions can always
be made with very high precision
[0116] If any of the above assumptions are violated in a practical
system, by design and/or due to system imperfections, for example,
following standard data analysis methods may lead to suboptimal
results.
[0117] According to various embodiments, methods of data analysis
may be utilized that explicitly takes into account that discrete
sample portions have varying/uncertain volumes and concentrations,
and account for measurements of varying quality. This may be
achieved by estimating the target concentration through Bayesian
inference, although other statistical estimation techniques are
also appropriate.
[0118] Some of the following assumptions may need to be
incorporated into the model statistically linking target
concentration to measurements: treating discrete sample portion
volumes as constants, specified separately for each discrete sample
portion, or treating discrete sample portions volumes as random
variables with known distributions (obtained by characterizing or
calibrating the system), or treating discrete sample portion
volumes as random variables with known conditional distributions,
dependent on volume dependent measurements obtained at the run
time, such as discrete sample portion diameter estimate, discrete
sample portion area estimate, passive reference intensity, etc.
[0119] One assumption may be treating discrete sample portion
concentrations as constants, specified separately for each discrete
sample portion, or treating discrete sample portion concentrations
as random variables with known distributions, obtained by
characterizing the system and dilution preparation protocol.
[0120] Another assumption that may be made is treating end-point
intensity reads as random variables with known "positive" and
"negative" distributions, obtained by characterizing the
measurement system.
[0121] A goal of Bayesian inference is to produce posterior
probability distribution of target concentration, conditional on
all available information and measurements. Such posterior
distribution may then be used to derive maximum likelihood
estimate, unbiased estimate, and/or confidence interval for target
concentration.
[0122] Central to these methods, according to various embodiments,
is a score function that depends on both the observed results and a
specific value of the target concentration. The score function
conveys information on how good the agreement between a
hypothetical value of the concentration and the actually observed
measurements. In other words, a value of the score function
obtained for certain concentration and certain measurements is a
measure of the likelihood that these measurements could arise if
the sample under consideration indeed had this concentration.
Maximum Likelihood Estimation
[0123] In one embodiment, the process of estimating the target
concentration involves finding the value of concentration for
which, given a set of measurements, the score function attains the
maximum value (or appropriately high value; or value appropriately
close to maximum). This search for maximum value may be performed
by evaluating the score function on a predefined set of candidate
solutions and choosing the maximum, a successive approximation
method, evaluating the analytical solution to the maximization
problem, or any number of other maximization method or combination
of methods.
Unbiased Estimation
[0124] In another embodiment, the process of estimating the target
concentration involves finding the weighted average of candidate
concentration values (the candidate values coming from a predefined
or dynamically established set of discrete of continuous
concentration values), where the values of the score function
(obtained for the candidate concentrations) serve as the weights in
the averaging process. This process can be achieved by evaluating
the score function for all or subset of candidate values and
directly calculating the weighted average; or by evaluating an
analytical solution; or by any other appropriate method.
Confidence Interval
[0125] In addition to producing the estimate of the target
concentration, a confidence interval may be generated. The
confidence interval is a range of target concentration values,
delimited by the upper value and lower value, calculated from the
measurements, such that the likelihood that the true target
concentration value is outside of this range is small. For example,
a 95%-confidence interval is where the model-based probability of
true target concentration being outside of it is 5%. The confidence
interval can be established as the range of target concentration
values for which the score function is above certain threshold. The
threshold may be dynamically calculated. For example, the threshold
may be selected such that the sum (or integral) of score function
values exceeding the threshold is greater by a predefined factor
from the sum (or integral) of score function values below the
threshold. (for example, if the score function is in fact a
model-based likelihood function and 95%-confidence interval is
sought, the threshold is selected so that the integral of
likelihoods above the threshold is 95/5 times greater that the
integral of likelihoods below the threshold).
Score Function
[0126] In some embodiments, the score function can be: a measure of
probability; a measure of conditional probability; a measure of
likelihood; a logarithm of probability. The score function may use
the following probabilistic model that expresses the conditional
probability of the positive/negative measurements for individual
PCR reactions conditioned on the target concentration and volumes
of the PCR reactions. Alternatively, the score function may be a
model-based conditional probability of fluorescence measurements
for individual PCR reactions conditioned on the target
concentration and on volume measurements for the PCR reactions.
[0127] Under the constraint on the total number of droplets,
digital PCR systems with multiple droplet sizes and/or multiple
sample dilutions can carry significant dynamic range advantage over
system with equally sized and diluted droplets. However, such a
system may need much different data analysis to determine target
concentration in the original sample. For example, the system may
need to take into account discrete sample portion volumes and
dilutions, dilution information may need to be provided by the user
or is system-specific, or discrete sample portion volume
information may be either provided or estimated at run time based
on a size measurement, passive reference intensity, or features of
real-time amplification curve, for example.
.lamda.--true target concentration in undiluted sample N--number of
droplets V.sub.i--true volume of droplet i C.sub.i--true dilution
coefficient of droplet i (what fraction of V.sub.i is taken up by
the undiluted sample) W.sub.i--set of measurements or prior
information about volume of droplet i D.sub.i--prior information
about dilution coefficient of droplet i X.sub.i--true number of
target molecules in droplet i Y.sub.i--plus/minus measurement for
droplet i
P ( V i | W i ) ##EQU00001## P ( C i | D i ) ##EQU00001.2## P ( X i
| V i , C i , .lamda. ) ##EQU00001.3## P ( Y i | X i )
##EQU00001.4## P ( Y i = 0 | X i = 0 ) = 1 - f p ##EQU00001.5## P (
X i | V i , C i , .lamda. ) ##EQU00001.6## P ( Y i | D i , W i ,
.lamda. ) = .intg. 0 .infin. .intg. 0 .infin. X i = 0 .infin. P ( Y
i | X i ) P ( X i | V i , C i , .lamda. ) P ( C i | D i ) P ( V i |
W i ) dC i dV i ##EQU00001.7## P ( Y | D , W , .lamda. ) = i = 1 N
P ( Y i | D i , W i , .lamda. ) ##EQU00001.8## .lamda. ^ ML =
argmax .lamda. i = 1 N P ( Y i | D i , W i , .lamda. )
##EQU00001.9##
Multi-Level Digital PCR
[0128] In another embodiment, the number of copies of the target
nucleic acid may be estimated based on real-time measurements and
C.sub.q values to discriminate between the number of starting
template target nucleic acid copies. C.sub.q values are also
referred to as C.sub.T values in some examples. In this way,
accuracy and dynamic range of dPCR can be enhanced.
[0129] As described in various embodiments above, dPCR is based at
least in part on partitioning the sample into a plurality of
discrete sample portion, which may be viewed as a large number of
separate PCR reactors. A positive/negative PCR test is performed on
each PCR reactor, whereby each individual plus/minus test
discriminates between zero starting copies and nonzero starting
copies.
[0130] According to this embodiment, a method using C.sub.q values
from each reactor is provided to achieve higher dynamic range and
accuracy than would be possible from simple plus/minus calls.
[0131] As described above, a positive/negative data analysis is
generally described as determining the number of positive and
negative reactions. Then, making the assumption that the number of
starting template copies per well follows Poisson distribution, it
can be inferred the most likely total number of copies from the
number of positive and negative wells.
[0132] According to this embodiment, a C.sub.q-based dPCR data
analysis method includes determining a number of negative
reactions. For reactions that showed amplification, the most likely
number of starting template copies from C.sub.q is determined. The
relationship between C.sub.q and copy number can be determined by
finding peaks in the C.sub.q histogram, with rightmost peak
corresponding to 1 copy, as illustrated in FIGS. 10-15. The method
further includes determining the most likely total number of
template copies by summing up template copy numbers from individual
reactors.
[0133] Furthermore, for each well the likelihoods of starting copy
number 0, 1, 2, etc, may be determined instead of making a copy
number call. This can be used to determine a total number of copies
more precisely with tighter confidence interval. Moreover, the
process of associating C.sub.q with copy number may also assume
Poisson copy number distribution and logarithmic spacing between
C.sub.qs.
[0134] As the result of using Cq-based dPCR with the above analysis
according to embodiments described herein, better accuracy and
dynamic range with the same number of reactors, and the same
accuracy and dynamic range with lower number of reactors compared
to plus/minus-based dPCR may be achieved.
Initial PCR Efficiency Determination from dPCT C.sub.q Spectrum
[0135] According to another embodiment, a method of measuring PCR
efficiency in the first PCR cycle from the Cq spectrum of a dPCR
experiment is provided. The method includes measuring the fraction
of replicate PCR reactions with one copy of starting template that
did not amplify in the first PCR cycle.
[0136] PCR efficiency can be described as the percentage of DNA
template that produces a copy during a single PCR cycle. PCR
efficiency is an important unknown parameter in downstream analysis
of real-time amplification curves. PCR efficiency can differ
between cycles of PCR, generally decreasing with each cycle. During
real-time PCR, once the amount of PCR template exceeds the
detection level, the fluorescence intensity can be used to monitor
the rate of change of the template amplicon and thus to infer the
efficiency.
[0137] However, this approach cannot be used to directly measure
the efficiency at the initial cycles when the fluorescence is below
detection level. It is common to make various assumptions about
this initial efficiency, such as assuming 100% initial efficiency,
assuming initial efficiency is the same as later, directly observed
efficiency, or to use various models that extrapolate the initial
efficiency backward from the observed efficiency.
[0138] According to this embodiment, the method includes using a
target nucleic acid template dilution that targets the average
number of starting template copies per reaction close to 1. This
yields high fraction of reactions to have precisely one starting
copy (assuming a Poisson-distribution across reactions). An
amplification curve is collected for all reactions and C.sub.q
values are obtained.
[0139] The method may establish the number of reactions A that had
one starting target nucleic acid template copy and that copy was
amplified in the first PCR cycle, and the number of reactions B
that had one starting target nucleic acid template copy and that
copy was not amplified in the first PCR cycle, but did amplify in
subsequent PCR cycles.
[0140] The cycle-1 efficiency can be then calculated as A/(A+B),
where A and B are determined from the C.sub.q histogram. This is
based on identification of the histogram peak corresponding to the
groups A and B, which is illustrated in FIG. 33.
[0141] Furthermore, knowledge of the efficiency at the initial PCR
cycle can be used as a reagent research and QC tool. Additionally,
this method may also enhance our qPCR models possibly leading to
improved accuracy of qPCR data analysis.
[0142] Also, as mentioned above, in various embodiments, the
methods and systems described herein may be used to detect other
biological components of interest. These biological components of
interest may include, but are not limited to, cells and circulating
tumor cells, for example. Furthermore, in addition to dPCR, the
methods and systems in various embodiments may be used in
applications, such as fetal diagnostics, multiplex DPCR, viral
detection, genotyping, and rare allele detection copy number
variation.
[0143] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
[0144] While embodiments of the present disclosure have been shown
and described herein, it will be obvious to those skilled in the
art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions will now occur to
those skilled in the art without departing from the disclosure. It
should be understood that various alternatives to the embodiments
of the disclosure described herein may be employed in practicing
the disclosure. It is intended that the following claims define the
scope of the disclosure and that methods and structures within the
scope of these claims and their equivalents be covered thereby.
* * * * *