U.S. patent application number 17/502251 was filed with the patent office on 2022-02-03 for method for detecting and quantifying latent retroviral rna species.
The applicant listed for this patent is Bio-Rad Laboratories, Inc.. Invention is credited to Sirshendu Roopom Banerjee, Darren R. Link, Michael L. Samuels.
Application Number | 20220033919 17/502251 |
Document ID | / |
Family ID | 53481066 |
Filed Date | 2022-02-03 |
United States Patent
Application |
20220033919 |
Kind Code |
A1 |
Samuels; Michael L. ; et
al. |
February 3, 2022 |
METHOD FOR DETECTING AND QUANTIFYING LATENT RETROVIRAL RNA
SPECIES
Abstract
The invention includes methods for determining the presence of a
latent viral population by analyzing an RNA population from the
virus with digital techniques, such as digital PCR or by sequencing
cDNA produced from the RNA. The invention additional includes
methods for determining the presence of latent viral populations by
detecting and/or quantifying enzymes that are uniquely associated
with the virus, e.g., reverse transcriptases.
Inventors: |
Samuels; Michael L.;
(Windham, NH) ; Link; Darren R.; (Lexington,
MA) ; Banerjee; Sirshendu Roopom; (Lexington,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bio-Rad Laboratories, Inc. |
Hercules |
CA |
US |
|
|
Family ID: |
53481066 |
Appl. No.: |
17/502251 |
Filed: |
October 15, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14587365 |
Dec 31, 2014 |
11193176 |
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17502251 |
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62036419 |
Aug 12, 2014 |
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62001963 |
May 22, 2014 |
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61922307 |
Dec 31, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/70 20130101; C12Q
1/702 20130101; C12Q 1/70 20130101; C12Q 2563/159 20130101 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70 |
Claims
1. A method for detection of latent retrovirus, comprising the
steps of: isolating a virion particle in an aqueous droplet
surrounded by an immiscible fluid; lysing the virion particle in
the aqueous droplet to release an RNA molecule; exposing the RNA
molecule to at least one primer capable of hybridizing to at least
a portion of the RNA molecule; synthesizing a cDNA product from the
RNA molecule; and detecting the cDNA product.
2. The method of claim 1, wherein detecting comprises amplifying
the cDNA product to produce a plurality of substantially identical
copies of the cDNA product.
3. The method of claim 2, further comprising sequencing the
substantially identical copies of the cDNA product to produce
sequence reads.
4. The method of claim 2, wherein the copies of cDNA product are
detected with fluorescence detection.
5. The method of claim 1, wherein said detecting step comprises
quantifying said cDNA.
6. The method of claim 1, further comprising introducing an amount
of exogenous synthetic RNA, DNA, or mRNA.
7. The method of claim 6, further comprising normalizing the
detected cDNA against the exogenous synthetic RNA, DNA, or
mRNA.
8. The method of claim 1, further comprising collecting the virion
particle from an isolated biological sample.
9. The method of claim 8, wherein the isolated biological sample is
a T-cell.
10. The method of claim 9, wherein the T-cell is a CD4+ T cell.
11. The method of claim 1, wherein the latent retrovirus a human
immunodefficiency virus (HIV).
12. A method for detecting a latent retrovirus species, the method
comprising the steps of: isolating virion RNA in an aqueous droplet
surrounded by an immiscible fluid, exposing the virion RNA in the
droplet to at least one primer capable of hybridizing to at least a
portion of the virion RNA; synthesizing a cDNA product from the
virion RNA; and detecting the cDNA product.
13. The method of claim 12, wherein detecting comprises amplifying
the cDNA product to produce a plurality of substantially identical
copies of the cDNA product.
14. The method of claim 13, further comprising sequencing the
substantially identical copies of the cDNA product to produce
sequence reads.
15. The method of claim 13, wherein the copies of cDNA product are
detected with fluorescence detection.
16. The method of claim 12, wherein said detecting step comprises
quantifying said cDNA.
17. The method of claim 12, further comprising introducing an
amount of exogenous synthetic RNA, DNA, or mRNA.
18. The method of claim 17, further comprising normalizing the
detected cDNA against the exogenous synthetic RNA, DNA, or
mRNA.
19. The method of claim 12, further comprising collecting the
virion particle from an isolated biological sample.
20. The method of claim 19, wherein the isolated biological sample
is a T-cell.
Description
FIELD OF THE INVENTION
[0001] The invention generally relates to droplet based
amplification and methods for analyzing, detecting, and quantifying
RNA including, but not limited to, miRNA and viral RNA.
BACKGROUND
[0002] Classical methods for detecting and quantifying RNA have
proven challenging in many applications due to the characteristics
of particular RNA species and/or the particular environment in
which the RNA species exists. The methods for detection of small
RNA species have included northern hybridization analysis and
microarray analysis. However, it is generally appreciated that the
sensitivity and selectivity of those methods is poor for detection
of low-abundance small RNA species, and the workflow can be
challenging and expensive. (What is generally referred to as "small
RNA" comprises a genus of RNA molecules that include species of
what are generally referred to as non-coding RNA. Examples of small
RNA species include miRNA (micro RNA), piRNA (P-element induced
wimpy testis interacting RNA), siRNA (small interfering RNA), ceRNA
(competing endogenous RNA), saRNA (small activating RNA), etc.) For
example, traditional approaches used for miRNA analysis include a
step of cDNA synthesis using a reverse transcriptase enzyme
(RNA-dependent DNA polymerase isolated from a retrovirus) and
subsequent amplification from the cDNA, where the process consists
of at least two sample processing steps requiring user
intervention, carried out separately (e.g. what may be referred to
as "2-step RT-PCR"). For example, in the first step, purified RNA
molecules are converted to cDNA using target-specific primers
(sometimes referred to as "RT primers") or random hexamers, and
reverse transcriptase enzyme. The cDNA products are then amplified
and detected in a second qPCR or digital PCR step. Numerous
attempts to convert the two-step method into a one-step method for
measuring miRNAs have failed, primarily because they typically
yield significant background noise that in many instances makes
discrimination of true signals impossible. This is especially true
when low amounts of the miRNA targets of interest are present in
the sample, when multiplexing multiple miRNAs, or when multiplexing
miRNA in combination with other targets.
[0003] As described above, one example of a small RNA species
includes what is referred to as microRNA (miRNA), which is a short
non-coding RNA that plays important roles in various physiological
processes through the post-transcriptional regulation of gene
expression. Species of miRNA generally function via base-pairing
with complementary sequences within mRNA molecules causing
translational repression or target degradation thus silencing the
expression of the target gene. Typical mature miRNA species are
about 22 nucleotides in length, and are differentially expressed in
various cell and/or tissue types where an analysis of quantitative
expression of miRNA species can be used to accurately identify a
cell and/or tissue type. Species of miRNA are also found as stable
molecules in peripheral body fluids accessible using non or
minimally invasive methods (e.g. cerebral spinal fluid, blood,
urine, etc.), where similar quantitative analysis can indicate the
presence of cell and/or tissue types elsewhere in the system. For
example miRNA may be found as cell free RNA or packaged into
exosomal and microvesicluar compartments that can be analyzed from
bodily fluids such as blood and/or serum.
[0004] Species of miRNA have been implicated in a number of
diseases, such as cancer, diabetes, immune system diseases, muscle
disorders, and neurological development and degeneration. For
example, miRNA-21 is involved in several cancer types such as
glioblastoma and astrocytoma, and miRNA deregulation has been found
to be associated with some types of cancer. By measuring quantity
of specific miRNA types, diseases can be identified and
distinguished to aid in the determination of a treatment
course.
[0005] Also, available methods for quantifying "latent" reservoirs
of retrovirus in patients have been shown to be suboptimal where
such quantification is important for effective evaluation of
available therapeutic strategies to fully cure retroviral
infections. Typically, retrovirus is a single stranded RNA virus
capable of producing a DNA copy of itself that integrates into and
is treated as part of the genome of the host cell (sometimes
referred to as a "provirus"). This DNA copy may then remain dormant
or "latent" for long periods of time until cues are received by the
host cell and the provirus initiates the process of
transcription-translation to produce active virus and its
associated proteins (sometimes referred to as
"replication-competent provirus").
[0006] One particularly important example of retrovirus is human
immunodeficiency virus (HIV). While antiretroviral therapies have
greatly improved the lives of many infected with HIV, it is
generally understood that HIV establishes latent infections in
long-lived cells that form a reservoir of the virus even after
years of treatment with highly active antiretroviral therapy
(HAART). Actually curing HIV infection, thus, requires eliminating
these long-lived reservoir cells. A typical reservoir of latent HIV
resides in resting CD4+ memory T cells.
[0007] Because the latent population of cells represents an
important metric in HIV health, researchers have long sought a
method to quickly and precisely quantify the number of cells
harboring latent provirus. However, the low frequency of these
cells makes such an assay technical challenging. For example, for
HIV-positive individuals on HAART, only about one in a million
resting CD4 T cells contain latent proviruses capable of producing
replication-competent virus. Accordingly, the low counts are often
lost in the "noise" associated with biological assays. See, e.g.,
Eriksson et al., "Comparative Analysis of Measures of Viral
Reservoirs in HIV-1 Eradication Studies," PLOS, Pathogens, vol. 9
(2), e1003174, p. 1-17 (2013), incorporated herein by reference in
its entirety.
[0008] The current gold-standard method of detecting latent HIV
infection involves a limiting dilution viral outgrowth assay (also
referred to as "VOA") that is slow, resource-intensive, and
relatively imprecise. The VOA assay also requires 120-160 ml of
blood. Complicating matters further is the fact that defective HIV
DNA is present at approximately 100- to 1000-fold excess over
functional proviral HIV DNA resulting in overestimation of
replication competent provirus, and underestimation can also occur
due to replication competent provirus not being scored in the
VOA.
[0009] Rapid, simple, accurate, and cost effective quantification
methods for RNA species including small RNA and viral species are
needed both to accelerate understanding of the role and biological
function of these RNA species in normal and disease states, as well
as for use in clinical diagnostics.
SUMMARY
[0010] Embodiments of the invention provide improved detection of
RNA species that include small RNA, mRNA, and viral populations,
such as latent retroviral populations, by isolating and quantifying
RNA such as that associated with the latent virus or enzymes (e.g.,
reverse transcriptase) associated with the latent virus. Because
the methods of the invention individually isolate each RNA (and/or
enzyme) molecule in a fluidic droplet, it is possible to quickly
quantify the amount of replication competent virus in a sample by
simply counting the number of droplets meeting a criterion, or by
determining the number of RNA or enzyme samples that have a viable
sequence. Furthermore, because the isolated microenvironment
facilitates superefficient reverse transcription, strand switching
in the nucleic acid is minimized, resulting in sequence reads with
higher fidelity, and greater confidence in the final results. For
example, the methods are broadly applicable to determine RNA viral
load, and the presence of enzymes associated with retroviruses. In
some embodiments, the retrovirus is HIV.
[0011] The RNA detection process involves exposing the RNA to a
primer and then synthesizing cDNA. The cDNA may optionally be
amplified and quantified, or the cDNA may be directly sequenced.
For example, the method may involve a one-step reverse
transcription/amplification process. In some embodiments, the
resulting cDNA will be labeled with a unique or semi-unique barcode
that will facilitate identification of individual RNA molecules
after sequencing. In some embodiments, a virion particle containing
the RNA is isolated in an aqueous droplet, and the virion particle
subsequently lysed to release the RNA. In other embodiments the
virion particles may be lysed and then the RNA isolated into
aqueous droplets. In some embodiments, the RNA sample is collected
from highly purified resting CD4+ T cells.
[0012] In other embodiments, the detection process involves
exposing the enzyme to a labeled probe with a specific binding for
the enzyme. In some embodiments, the probe may include an antibody,
i.e., a fluorescently-labeled antibody, or an ELISA-type probe. In
other embodiments, enzymatic activity can be evaluated by
encapsulating each enzyme with a known substrate (e.g. exogenous
RNA) and the detecting the enzymatic products (e.g. qPCR type assay
for the exogenous RNA that may include use of hydrolysis probes, or
the use of a fluorogenic RNA substrate). In embodiments where the
enzymes are quantified, the methods may involve collecting enzymes
from a lysate of a cell population containing the latent virus.
[0013] Using the methods of the invention, it is straightforward to
quickly and accurately quantify the latent viral load in a sample,
e.g., a biological sample from a subject diagnosed as suffering
from the virus. Accordingly, the methods allow for faster clinical
evaluation of treatments for the virus. The methods also allow
health care providers to evaluate the progress of an individual's
latent viral load and response to treatment. Thus, some patients
may be able to scale back on the amount or type of antiretroviral
agents that are administered. This will reduce overall healthcare
costs for the population and potentially increase the effectiveness
of the antiretroviral agents because the virus will not develop a
resistance to the antiretroviral agents as quickly.
[0014] Because measurements can be performed on individual
droplets, quantification typically only involves counting droplets
with and without the targeted properties. For example, the amount
of RNA in a sample that is replication competent is quantified. In
another embodiment, enzyme molecules are quantified based on their
activity inside individual droplets. For example, droplets may be
identified as "negative" and/or "positive" droplets for the RNA or
the enzyme, and the number of RNA molecules or enzyme molecules
within positive droplets may be determined. In other embodiments,
the results may not be "digital," but exhibit a variety of
characteristics, e.g., a variety of closely-related sequences and a
variety of enzymatic activity. In such instances, it may be
necessary to invoke statistical models to deconvolve the
results.
[0015] The above embodiments and implementations are not
necessarily inclusive or exclusive of each other and may be
combined in any manner that is non-conflicting and otherwise
possible, whether they are presented in association with a same, or
a different, embodiment or implementation. The description of one
embodiment or implementation is not intended to be limiting with
respect to other embodiments and/or implementations. Also, any one
or more function, step, operation, or technique described elsewhere
in this specification may, in alternative implementations, be
combined with any one or more function, step, operation, or
technique described in the summary. Thus, the above embodiment and
implementations are illustrative rather than limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The above and further features will be more clearly
appreciated from the following detailed description when taken in
conjunction with the accompanying drawings. In the drawings, like
reference numerals indicate like structures, elements, or method
steps and the leftmost digit of a reference numeral indicates the
number of the figure in which the references element first appears
(for example, element 120 appears first in FIG. 1). All of these
conventions, however, are intended to be typical or illustrative,
rather than limiting.
[0017] FIG. 1 is a functional block diagram of one embodiment of a
system for droplet generation and detection;
[0018] FIG. 2 is a simplified graphical representation of one
embodiment of a droplet generation device of the system of FIG.
1;
[0019] FIG. 3 is a simplified graphical representation of one
embodiment of a miRNA qPCR detection processes;
[0020] FIG. 4 is a simplified graphical representation of one
embodiment of one-step RT-PCR results from miR-16 and Xeno assays
of Samples A-H;
[0021] FIG. 5 is a simplified graphical representation of one
embodiment of data comprising droplet counts of the results of FIG.
4;
[0022] FIG. 6 is a simplified graphical representation of one
embodiment of one-step RT-PCR results from miR-16, miR-21, and
miR-92a assays with Xeno spike in for Samples A-F;
[0023] FIG. 7 is a simplified graphical representation of one
embodiment of one-step RT-PCR results from miR-21 and miR-15, for
Samples A-C;
[0024] FIG. 8 is a simplified graphical representation of one
embodiment of one-step RT-PCR results from endogenous mRNA and
miR-21, for Samples A and B;
[0025] FIG. 9 is a schematic showing sandwich formation for digital
droplet ELISA;
[0026] FIGS. 10A-10D illustrate different digital droplet ELISA
readout counting modes; and
[0027] FIG. 11 is a simplified graphical representation of one
embodiment of a workflow for encapsulating HIV virions in droplets
and incorporating barcode for whole genome sequencing of individual
virus.
DETAILED DESCRIPTION
[0028] As will be described in greater detail below, embodiments of
the described invention include systems and methods for
super-efficient reverse transcription and detection of RNA species
in a single molecule format. More specifically, embodiments of a
unique one step approach are described in detail below that enable
highly accurate and quantifiable detection of RNA species such as,
but not limited to, small RNA species and viral RNA species.
Further, in the described embodiments the RNA species are
compartmentalized in droplets prior to amplification which enables
massive parallelization of single molecule targets that provide
advantages in statistical significance and throughput.
[0029] One embodiment of the invention includes super-efficient
reverse transcription of virion RNA into a cDNA product and
sequencing the cDNA product. Embodiments of the invention
additionally include methods for estimation of latent viral loads
by detecting an amount of enzyme that is attributable only to the
presence of the viral load. For example, certain families of
reverse transcriptase are only attributable to the presence of HIV,
a retrovirus. Each HIV virion contains 50-100 molecules having
enzymatic activity (either RT or RNAseH or both), and these species
are also present in the host cell during various stages of the HIV
life cycle (i.e. infection or escape from latency). This invention
relates to absolute quantification of each individual enzyme
molecule confined into separate compartments for a digital counting
assay.
[0030] For the enzyme quantification methods described, test
materials (e.g. cell lysate, culture supernate, etc.) are
recovered, diluted, and partitioned to create a single molecule
(`digital`) environment together with additional assay components,
enzymatic reaction to a fluorescent endpoint. Once the reactions
are complete the latent viral load and be quantified by simply
counting `positive` compartments. In some embodiments, the total
number of positive compartments will be identical to the total
number of starting enzyme molecules present in the measured test
fluid, and can be correlated to the number of viral species
present. Furthermore, measurement of RT activity can be performed
by co-encapsulation with a known substrate (e.g. exogenous RNA) and
detection assay (e.g. qPCR assay for the exogenous RNA, or the use
of a fluorogenic RNA substrate). Similarly, RNAseH detection would
utilize co-encapsulation of a substrate (e.g. synthetic RNA/DNA
hybrid) together with a detection assay (e.g. qPCR assay for the
exogenousDNA that is released from the RNA/DNA hybrid after RNAseH
activity, or the use of a fluorogenic RNA/DNA substrate).
[0031] In some embodiments, exogenous or endogenous controls are
utilized to improve the accuracy of quantification. For example, a
"spike-in" (e.g. an addition from user 101, FIG. 1) nucleic acid
(e.g. synthetic control RNA, cDNA, or DNA species) may be added at
known concentrations to the sample with target small RNA molecules.
In the present example, a microfluidic device can be used to
encapsulate the target small RNA and control nucleic acid in
droplets that are subjected to a first strand cDNA synthesis step,
and in some embodiments an amplification reaction, the cDNA and/or
amplification products are then detected in the droplets (e.g. via
digital PCR) or released from the droplets and detected (e.g. via
sequencing). In the present example, the detected numbers for the
spike-in nucleic acid can be compared to the known numbers from the
concentration and, if necessary, the detected numbers for the small
RNA numbers may be normalized to increase accuracy. Also in the
present or different example, an endogenous mRNA target may be
utilized together or separately with the exogenous spike-in nucleic
acid for additional normalization of comparison.
[0032] Some exemplary embodiments of systems and methods associated
with sample preparation and processing, generation of data, and
analysis of data are generally described below, some or all of
which are amenable for use with embodiments of the presently
described invention. In particular, the exemplary embodiments of
systems and methods for preparation of nucleic acid template
molecules, amplification of template molecules, detection of
template molecules and/or substantially identical copies thereof.
Embodiments that execute methods of detection such as digital PCR
and/or sequencing methods utilizing exemplary instrumentation and
computer systems are described.
[0033] Typical embodiments of "emulsions" include creating a stable
emulsion of two immiscible substances, and in the embodiments
described herein generally refer to an emulsion of aqueous droplets
in a continuous oil phase within which reactions may occur. In
particular, the aqueous droplets of an emulsion amenable for use in
methods for conducting reactions with biological samples and
detecting products may include a first fluid, such as a water based
fluid (typically referred to as "aqueous" fluid) suspended or
dispersed as droplets (also referred to as a discontinuous phase)
within another fluid, such as a hydrophobic fluid (also referred to
as a continuous phase) that typically includes some type of oil.
Examples of oil that may be employed include, but are not limited
to, mineral oils, silicone based oils, fluorinated oils, partially
fluorinated oils, or perfluorinated oils.
[0034] One example of an aqueous fluid compatible with embodiments
of the invention may include an aqueous buffer solution, such as
ultrapure water (e.g., 18 mega-ohm resistivity, obtained, for
instance by column chromatography), 10 mM Tris HCl and 1 mM EDTA
(TE) buffer, phosphate buffer saline (PBS) or acetate buffer. In
the presently described example, any liquid or buffer that is
physiologically compatible with nucleic acid molecules or
encapsulated biological entity can be used. Also, in the same or
alternative example a carrier fluid compatible with embodiments of
the invention includes a non-polar solvent, decane (e g.,
tetradecane or hexadecane), fluorocarbon oil, silicone oil or
another oil (for example, mineral oil). In certain embodiments, the
carrier fluid may contain one or more additives, such as agents
which increase, reduce, or otherwise create non-Newtonian surface
tensions (surfactants) and/or stabilize droplets against
spontaneous coalescence on contact.
[0035] Embodiments of surfactants that act to stabilize emulsions,
which may be particularly useful for embodiments that include
conducting reactions with biological samples such as PCR may
include one or more of a silicone or fluorinated surfactant. For
example, in microfluidic embodiments the addition of one or more
surfactants can aid in controlling or optimizing droplet size, flow
and uniformity, for example by reducing the shear force needed to
extrude or inject droplets into an intersecting channel. This can
affect droplet volume and periodicity, or the rate or frequency at
which droplets break off into an intersecting channel. Furthermore,
the surfactant can serve to stabilize aqueous emulsions in
fluorinated oils and substantially reduce the likelihood of droplet
coalescence.
[0036] In some embodiments, the aqueous droplets may be coated with
a surfactant or a mixture of surfactants, where those of skill in
the art understand that surfactant molecules typically reside at
the interface between immiscible fluids, and in some cases form
micelles in the continuous phase when the concentration of
surfactant(s) is greater than what is referred to as the critical
micelle concentration (also sometimes referred to as CMC). Examples
of surfactants that may be added to the carrier fluid include, but
are not limited to, surfactants such as sorbitan-based carboxylic
acid esters (e.g., the "Span" surfactants, Fluka Chemika),
including sorbitan monolaurate (Span 20), sorbitan monopalmitate
(Span 40), sorbitan monostearate (Span 60) and sorbitan monooleate
(Span 80), and perfluorinated polyethers (e.g., DuPont Krytox 157
FSL, FSM, and/or FSH). Other non-limiting examples of non-ionic
surfactants which may be used include polyoxyethylenated
alkylphenols (for example, nonyl-, p-dodecyl-, and dinonylphenols),
polyoxyethylenated straight chain alcohols, polyoxyethylenated
polyoxypropylene glycols, polyoxyethylenated mercaptans, long chain
carboxylic acid esters (for example, glyceryl and polyglycerl
esters of natural fatty acids, propylene glycol, sorbitol,
polyoxyethylenated sorbitol esters, polyoxyethylene glycol esters,
etc.) and alkanolamines (e.g., diethanolamine-fatty acid
condensates and isopropanolamine-fatty acid condensates).
[0037] In one embodiment, a fluorosurfactant can be prepared by
reacting the perflourinated polyether DuPont Krytox 157 FSL, FSM,
or FSH with aqueous ammonium hydroxide in a volatile fluorinated
solvent. The solvent and residual water and ammonia can be removed
with a rotary evaporator. The surfactant can then be dissolved
(e.g., 2.5 wt %) in a fluorinated oil (e.g., Flourinert (3M)),
which then serves as the carrier fluid (e.g. continuous phase). In
the presently described embodiment, the surfactant produced is an
ionic salt, and it will be appreciated that other embodiments of
non-ionic surfactant compositions may also be used. For example,
non-ionic surfactant composition may include what are referred to
as block copolymers (e.g. di-block, or tri-block copolymers)
typically comprising a head group and one or more tail groups. A
more specific example of a fluorinated block copolymer includes a
polyethylene glycol (PEG) head group and one or more
perfluoropolyether (PFPE) tail groups.
[0038] Further, in some embodiments other reagents that act as
droplet stabilizers (also referred to as passivating agents) may be
included. Useful droplet stabilizing reagents may include, but are
not limited to, polymers, proteins, BSA, spermine, or PEG.
[0039] In some embodiments, desirable characteristics may be
achieved by adding a second surfactant, or other agent, such as a
polymer or other additive, to the aqueous fluid. Further, in
certain embodiments utilizing microfluidic technology the carrier
fluid may be caused to flow through the outlet channel so that the
surfactant in the carrier fluid coats the channel walls.
[0040] In the embodiments described herein, droplets of an emulsion
may be referred to as compartments, microcapsules, microreactors,
microenvironments, or other name commonly used in the related art.
The aqueous droplets may range in size depending on the composition
of the emulsion components or composition, contents contained
therein, and formation technique employed. The described emulsions
are microenvironments within which chemical reactions that may
include binding reactions, such as Reverse Transcription, PCR, or
other process may be performed. For example, template nucleic acids
and all reagents necessary to perform a desired PCR reaction may be
encapsulated and chemically isolated in the droplets of an
emulsion. Additional surfactants or other stabilizing agent may be
employed in some embodiments to promote additional stability of the
droplets as described above. Thermocycling operations typical of
PCR methods may be executed using the droplets to amplify an
encapsulated nucleic acid template resulting in the generation of a
population comprising many substantially identical copies of the
template nucleic acid. In some embodiments, the population within
the droplet may be referred to as a "clonally isolated",
"compartmentalized", "sequestered", "encapsulated", or "localized"
population. Also in the present example, some or all of the
described droplets may further encapsulate a solid substrate such
as a bead. In some embodiments, beads may be employed for
attachment of template and amplified copies of the template,
amplified copies complementary to the template, or combination
thereof. Further, the solid substrate may be enabled for attachment
of other type of nucleic acids, reagents, labels, or other
molecules of interest. It will also be appreciated that the
embodiments described herein are not limited to encapsulating
nucleic acids in droplets, but rather the droplets may be
configured to encapsulate a variety of entities that include, but
are limited to, cells, antibodies, enzymes, proteins, or
combinations thereof. As with nucleic acids, the droplets may
further be amenable to performing various reactions on the entities
encapsulated therein and/or detection methods such as, for
instance, ELISA assays.
[0041] Various methods of forming emulsions may be employed with
the described embodiments. In the some embodiments methods involve
forming aqueous droplets where some droplets contain zero target
nucleic acid molecules, some droplets contain one target nucleic
acid molecule, and some droplets may contain multiple target
nucleic acid molecules. It will be appreciated by those of skill in
the art that in some embodiments it may be desirable for individual
droplets to contain multiple nucleic acid molecules from a sample,
however in certain assays there may be a discrete number of targets
of interest where droplets are generated based on the likelihood
that there is at most a single target of interest in each droplet
in the presence of other nucleic acid molecules that are not
targets of interest.
[0042] In some embodiments the number of target nucleic acid
molecules in the droplets is controlled via a limiting dilution of
the target nucleic acid molecules in the aqueous solution.
Alternatively, in some embodiments the number of target nucleic
acid molecules in the droplets is controlled via a method of
partitioning very small volumes of the aqueous fluid (e.g.
picoliter-nanoliter volumes such as a volume of about 5 picoliters)
into the droplet where the statistical likelihood of distributing
multiple target nucleic acid molecules in the same droplet is very
small. In some or all of the described embodiments, the
distribution of molecules within droplets can be described by
Poisson distribution. However, it will be appreciated that methods
for non-Poisson loading of droplets may be employed in some
embodiments and include, but are not limited to, active sorting of
droplets such as by laser-induced fluorescence, or by passive
one-to-one loading.
[0043] Systems and methods for generation of emulsions include what
are referred to as "bulk" emulsion generation methods that
generally include an application of energy to a mixture of aqueous
and carrier fluids. In the example of bulk generation methods
energy may be applied by agitation via vortexing, shaking, spinning
a paddle (to create shear forces) in the combined mixture or in
some embodiments the agitation of the aqueous solution may applied
when separate from the immiscible fluid where the agitation results
in droplets being added to the immiscible fluid as for example when
piezo-electric agitation is employed. Alternatively, some bulk
generation methods include adding the aqueous fluid drop-wise to a
spinning carrier fluid. Bulk emulsion generation methods typically
produce emulsions very quickly and do not require complicated or
specialized instrumentation. The droplets of the emulsions
generated using bulk generation techniques typically have low
uniformity with respect to dimension and volume of the droplets in
the emulsion.
[0044] Other embodiments of emulsion formation methods include
"microfluidic" based formation methods that may employ a junction
of channels carrying aqueous and carrier fluids that result in an
output of droplets in a stream of flow. Some embodiments of
microfluidic based droplet generation approaches may utilize one or
more electric fields to overcome surface tension. Alternatively,
some embodiments do not require the addition of an electric field.
For example, a water stream can be infused from one channel through
a narrow constriction; counter propagating oil streams (preferably
fluorinated oil) hydrodynamically focus the water stream and
stabilize its breakup into droplets as it passes through the
constriction. In order to form droplets, the viscous forces applied
by the oil to the water stream must overcome the water surface
tension. The generation rate, spacing and size of the water
droplets is controlled by the relative flow rates of the oil and
the water streams and nozzle geometry. While this emulsification
technology is extremely robust, droplet size and rate are tightly
coupled to the fluid flow rates and channel dimensions.
[0045] Continuing with the present example, some embodiments of
microfluidic devices can incorporate integrated electric fields,
thereby creating an electrically addressable emulsification system.
For instance, this can be achieved by applying high voltage to the
aqueous stream and charge the oil water interface. The water stream
behaves as a conductor while the oil is an insulator;
electrochemical reactions charge the fluid interface like a
capacitor. At snap-off, charge on the interface remains on the
droplet. The droplet size decreases with increasing field strength.
At low applied voltages the electric field has a negligible effect,
and droplet formation is driven exclusively by the competition
between surface tension and viscous flow
[0046] Additional examples of systems and methods for forming
aqueous droplets surrounded by an immiscible carrier fluid in
microfluidic structures are described U.S. Pat. Nos. 7,708,949; and
7,041,481 (reissued as RE 41,780) and U.S. Published Patent
Application numbers 2006/0163385 A1; 2008/0014589; 2008/0003142;
and 2010/0137163; and 2010/0172803 each of which is hereby
incorporated by reference herein in its entirety for all
purposes.
[0047] In some embodiments, emulsion formation methods also include
merging already formed emulsion droplets with other droplets or
streams of fluid to produce combined droplets. The merging of
droplets can be accomplished using, for example, one or more
droplet merging techniques described for example in Link et al.
(U.S. patent application numbers 2008/0014589; 2008/0003142; and
2010/0137163) and European publication number EP2047910 to
RainDance Technologies Inc.
[0048] In certain embodiments, a reverse transcriptase reaction
(referred to as an "RT" reaction) may be used to convert from RNA
starting material to a nucleic acid such as cDNA or other synthetic
nucleic acid derivative. Reverse transcriptase reaction refers to
methods known in the art, for example by methods described by
Yih-Horng Shiao, (BMC Biotechnology 2003, 3:22;
doi:10.1186/1472-6750-3-22). See also J Biomol Tech. 2003 March;
14(1): 33-43, which includes a discussion of RT reaction methods,
each of which is incorporated by reference. For example, the
process includes a first step of introducing a reverse
transcriptase enzyme used to generate single stranded complementary
DNA (cDNA) from an RNA template using target-specific primers
(sometimes referred to as "RT primers") or random hexamers. For
embodiments of conversion of small RNA to cDNA a target-specific
stem loop primer may be used to add length and optimize
characteristics such as melting temperature and specificity. In
some embodiments, the single stranded cDNA is then used as a
template for conversion of a second strand complementary to the
single stranded cDNA. The single or double stranded cDNA may then
be used as a template for amplification, such as by PCR. The
process for amplifying the target sequence can include introducing
an excess of oligonucleotide primers to a DNA or cDNA mixture
containing a desired target sequence, followed by a precise
sequence of thermal cycling in the presence of a DNA polymerase.
The primers are complementary to their respective strands of the
double stranded target sequence.
[0049] As described elsewhere in this description, the described
embodiments include conducting reactions with biological entities
within the emulsion droplets. An example of a very useful class of
reactions includes nucleic acid amplification methods. The term
"amplification" as used herein generally refers to the production
of substantially identical copies of a nucleic acid sequence
(typically referred to as "amplicons"). One of the most well-known
amplification strategies is the polymerase chain reaction (e.g.,
Dieffenbach and Dveksler, PCR Primer, a Laboratory Manual, Cold
Spring Harbor Press, Plainview, N.Y. [1995]). The amplification
reaction may include any amplification reaction known in the art
that amplifies nucleic acid molecules, such as Loop-mediated
Isothermal Amplification (also referred to as LAMP), Recombinase
Polymerase Amplification (RPA), Helicase-dependent amplification
(HDA), Nicking enzyme amplification reaction (NEAR), polymerase
chain reaction, nested polymerase chain reaction, ligase chain
reaction (Barany F. (1991) PNAS 88:189-193; Barany F. (1991) PCR
Methods and Applications 1:5-16), ligase detection reaction (Barany
F. (1991) PNAS 88:189-193), strand displacement amplification
(SDA), transcription based amplification system, nucleic acid
sequence-based amplification, rolling circle amplification, and
hyper-branched rolling circle amplification.
[0050] In some embodiments, generally referred to as
"multiplexing", emulsion droplets comprise a plurality of species
of primer pairs each specific to amplify a different region of
nucleic acid sequence. Optimization of traditional multiplexing of
standard PCR primers in tubes or wells is known to be difficult.
Multiple PCR amplicons being generated in the same reaction can
lead to competition between amplicons that have differing
efficiencies due to differences in sequence or length or access to
limiting reagents. This results in varying yields between competing
amplicons which can result in non-uniform amplicon yields. However,
because droplet based digital amplification utilizes only one
template molecule per droplet, even if there are multiple PCR
primer pairs present in the droplet, only one primer pair will be
active. Since only one amplicon is being generated per droplet,
there is no competition between amplicons or reagents, resulting in
a more uniform amplicon yield between different amplicons.
[0051] In some embodiments, even though the number of PCR primer
pairs per droplet is greater than one, there is still at most only
one template molecule per droplet and thus there is only one primer
pair per droplet that is being utilized at one time. This means
that the advantages of droplet amplification for eliminating bias
from either allele specific PCR or competition between different
amplicons is maintained.
[0052] Additional examples describing systems and methods for
performing amplification in droplets are shown for example in Link
et al. (U.S. patent application numbers 2008/0014589, 2008/0003142,
and 2010/0137163), Anderson et al. (U.S. Pat. No. 7,041,481 and
which reissued as RE 41,780) and European publication number
EP2047910 to RainDance Technologies Inc. The content of each of
which is incorporated by reference herein in its entirety.
[0053] In certain cases it is desirable to release the contents of
the droplets to use in further processing and/or detection
processes. In some embodiments, the contents of many droplets are
released and pooled together, however it will be appreciated that
in some embodiments the contents of droplets are released
individually and maintained separately. Various methods for
releasing the contents of droplets may be employed, typically
depending on the composition of the droplets. For example, in cases
where aqueous droplets are in a silicone based oil, an organic
solvent may be used to "break" the integrity of the interface
between the aqueous fluid and silicone oil combining into a single
solution that may be separated using various techniques.
Alternatively, in cases where aqueous droplets are in a fluorinated
oil, a perfluorinated alcohol reagent may be used. In the present
example, the perfluorinated alcohol provides advantages for use as
a releasing agent in that it is not immiscible with aqueous fluid
(e.g. will not be present in aqueous phase post release) and works
very well to disrupt surfactants typically used with fluorinated
oils. One specific example of perfluorinated alcohol useful for
release applications includes perfluoro decanol.
[0054] In some embodiments, often referred to as digital PCR, after
amplification the emulsion droplets are introduced into an
instrument for optical detection of amplification products. In some
embodiments the generation and amplification of the nucleic acid
molecules occurs in a single fluidic chip that is also used for
detection, alternatively the emulsion droplets may be removed or
dispensed from a fluidic chip used for droplet generation in order
to conduct the amplification "off-chip". For embodiments of the
off-chip application, post amplification the droplets may be
introduced into either a second fluidic chip used for detection of
the amplicons or into the original fluidic chip used for droplet
generation. Further, in embodiments where the emulsion droplets are
generated using bulk methods, after amplification the droplets may
be introduced into a fluidic chip used for detection of amplicon
products. In the same or alternative embodiments detection of
reaction products produced from PCR thermocycling may be performed
during or after each amplification cycle (e.g. sometimes referred
to as "real time" PCR). The detected signals from the reaction
products may be used to generate what are referred to as "melt
curves" sometimes used with known concentrations as standards for
calibration. Melt curves may also be based on the melting
temperature of probes in the reaction where combinations of probes
are associated with specific sequence composition of a target (e.g.
as an identifier or type of molecular barcode) where the presence
of the target can be identified from the melt curve signature.
[0055] In some embodiments, when droplets are introduced into a
fluidic chip used for detection it may be highly desirable to add
additional carrier fluid to increase the spacing between successive
droplets. Examples of increasing the spacing between droplets is
described in U.S. patent application Ser. No. 12/087,713 which is
hereby incorporated by reference herein in its entirety for all
purposes.
[0056] The emulsion droplets may be individually analyzed and
detected using any methods known in the art, such as detecting the
presence and/or amount of signal from a reporter. Generally, the
instrument for detection comprises one or more detection elements.
The detection elements can be optical, magnetic, electromagnetic,
or electrical detectors, other detectors known in the art, or
combinations thereof. Examples of suitable detection elements
include optical waveguides, microscopes, diodes, light stimulating
devices, (e.g., lasers), photo multiplier tubes, charge-coupled
devices (CCD), and processors (e.g., computers and software), and
combinations thereof, which cooperate to detect a signal
representative of a characteristic, marker, or reporter. Further
description of detection instruments and methods of detecting
amplification products in droplets are shown in Link et al. (U.S.
patent application numbers 2008/0014589, 2008/0003142, and
2010/0137163) and European publication number EP2047910 to
RainDance Technologies Inc., each of which is hereby incorporated
by reference herein in its entirety for all purposes.
[0057] In certain embodiments, amplified target nucleic acid
molecules are detected using detectably labeled probes, such as
hybridization probes. In some or all of the described embodiments a
probe type may comprise a plurality of probes that recognize a
specific nucleic acid sequence composition. For example, a probe
type may comprise a group of probes that recognize the same nucleic
acid sequence composition where the members of the group have one
or more detectable labels specific for that probe type and/or
members that do not include a detectable label (that may be
included to modulate intensity of reporter signal). Further the
probe members may be present at different concentrations relative
to each other within the droplets. Thus, the combination of
detectable labels and relative intensities detected from the
concentrations of probes are specific to and enable identification
of the probe type. Those of ordinary skill in the related art
appreciate that the embodiments described herein are compatible
with any type of fluorogenic DNA hybridization probes or hydrolysis
probes, such as TaqMan probes, molecular beacons, Solaris probes,
scorpion probes, and any other probes that function by sequence
specific recognition of target DNA by hybridization and result in
increased fluorescence on amplification of the target sequence.
Further in the embodiments described herein, probe types may also
be multiplexed in emulsion droplets in the same way as described
elsewhere with respect to multiplexing primer species.
[0058] As described elsewhere, the droplets may contain a plurality
of detectable probes that hybridize to amplicons produced in the
droplets. Members of the plurality of probes can each include the
same detectable label, or a different detectable label. The
plurality of probes can also include one or more groups of probes
at varying concentration. The groups of probes at varying
concentrations can include the same detectable label which varies
in intensity, due to varying probe concentrations. In the
embodiments described herein, the fluorescence emission from each
fused droplet may be determined and plotted on a scattered plot
based on its wavelength and intensity. Examples of probe detection
and analysis using wavelength and intensity is described in US
Patent Application Serial No 2011/0250597, which is hereby
incorporated by reference herein in its entirety for all
purposes.
[0059] Types of detectable labels suitable for use with probes
specific to bridge regions of a primer and other probes for use in
methods of the invention are described hereinafter. In some
embodiments, the detectably labeled probes are optically labeled
probes, such as fluorescently labeled probes. Examples of
fluorescent labels include, but are not limited to, Atto dyes,
4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine
and derivatives: acridine, acridine isothiocyanate;
5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);
4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;
N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY;
Brilliant Yellow; coumarin and derivatives; coumarin,
7-amino-4-methylcoumarin (AMC, Coumarin 120),
7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes;
cyanosine; 4',6-diaminidino-2-phenylindole (DAPI);
5'5''-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;
diethylenetriamine pentaacetate;
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid;
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS,
dansylchloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate
(DABITC); eosin and derivatives; eosin, eosin isothiocyanate,
erythrosin and derivatives; erythrosin B, erythrosin,
isothiocyanate; ethidium; fluorescein and derivatives;
5-carboxyfluorescein (FAM),
5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),
2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein,
fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;
IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho
cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;
B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives:
pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum
dots; Reactive Red 4 (Cibacron.TM. Brilliant Red 3B-A) rhodamine
and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine
(R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod),
rhodamine B, rhodamine 123, rhodamine X isothiocyanate,
sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative
of sulforhodamine 101 (Texas Red);
N,N,N',N'tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl
rhodamine; tetramethyl rhodamine isothiocyanate (TRITC);
riboflavin; rosolic acid; terbium chelate derivatives; Cy3; Cy5;
Cy5.5; Cy7; IRD 700; IRD 800; La Jolta Blue; phthalo cyanine; and
naphthalo cyanine. Preferred fluorescent labels for certain
embodiments include FAM and VIC, and in the same or alternative
embodiments may also include TET, Yakima yellow, Calcein orange,
ABY and JUN dyes (from Thermo Fisher Scientific). Labels other than
fluorescent labels are contemplated by the invention, including
other optically-detectable labels.
[0060] Additional examples of digital amplification and detection
of reporters are described in U.S. Pat. No. 8,535,889, which is
hereby incorporated by reference herein in its entirety for all
purposes.
[0061] In embodiments of digital PCR, data analysis typically
involves a scatter plot type of representation for identifying and
characterizing populations of statistically similar droplets that
arise from unique probe signatures (wavelength and intensity), and
for discriminating one population of droplets from the others. In
some embodiments, a user and/or computer application may select
data points associated with specific droplets or groups of droplets
within histograms, either for counting, or for assay selection as
in the use of optical labels, or for any other purpose. Some
methods of selection may include the application of boundaries
surrounding one or more selections, either closed or unclosed, of
any possible shape and dimension.
[0062] The embodiments described herein are not limited to the use
of a specific number of probe species. In some embodiments a
plurality of probe species are used to give additional information
about the properties of nucleic acids in a sample. For example,
three probe species could be used wherein a first probe species
comprises a fluorophore that has a particular excitation and
emission spectra (e.g., VIC), and a second probe species comprises
a fluorophore that has a particular excitation and emission spectra
(e.g., FAM) where the excitation spectra for the first and second
probe species may overlap but have clearly distinct emission
spectra from each other. Detected differences in intensity can be
used to discriminate between different probe species that employ
the same fluorophore, where the intensity may be tunable of emitted
light.
[0063] In some of the described embodiments, a further step of
releasing converted or amplified target molecules from the emulsion
droplets for further analysis. The released converted or amplified
material can also be subjected to further processing and/or
amplification. Additional examples of systems and methods of
releasing amplified target molecules from the droplets are
described in Link et al. (U.S. patent application numbers
2008/0014589, 2008/0003142, and 2010/0137163) and European
publication number EP2047910 to RainDance Technologies Inc.
[0064] In certain embodiments, the amplified target molecules are
sequenced using any suitable sequencing technique known in the art.
In one example, the sequencing is single-molecule
sequencing-by-synthesis. Single-molecule sequencing is shown for
example in Lapidus et al. (U.S. Pat. No. 7,169,560), Quake et al.
(U.S. Pat. No. 6,818,395), Harris (U.S. Pat. No. 7,282,337), Quake
et al. (U.S. patent application number 2002/0164629), and
Braslaysky, et al., PNAS (USA), 100: 3960-3964 (2003), the contents
of each of these references is incorporated by reference herein in
its entirety. Other examples of sequencing nucleic acids may
include Maxam-Gilbert techniques, Sanger type techniques,
Sequencing by Synthesis methods (SBS), Sequencing by Hybridization
(SBH), Sequencing by Ligation (SBL), Sequencing by Incorporation
(SBI) techniques, massively parallel signature sequencing (MPSS),
polony sequencing techniques, nanopore, waveguide and other single
molecule detection techniques, reversible terminator techniques, or
other sequencing technique now know or may be developed in the
future.
[0065] In one embodiment, the sequencing is Illumina sequencing.
Illumina sequencing is based on the amplification of DNA on a solid
surface using fold-back PCR and anchored primers. Genomic DNA is
fragmented, and adapters are added to the 5' and 3' ends of the
fragments. DNA fragments that are attached to the surface of flow
cell channels are extended and bridge amplified. The fragments
become double stranded, and the double stranded molecules are
denatured. Multiple cycles of the solid-phase amplification
followed by denaturation can create several million clusters of
approximately 1,000 copies of single-stranded DNA molecules of the
same template in each channel of the flow cell. Primers, DNA
polymerase and four fluorophore-labeled, reversibly terminating
nucleotides are used to perform sequential sequencing. After
nucleotide incorporation, a laser is used to excite the
fluorophores, and an image is captured and the identity of the
first base is recorded. The 3' terminators and fluorophores from
each incorporated base are removed and the incorporation, detection
and identification steps are repeated.
[0066] In another embodiment, Ion Torrent sequencing can be used.
(See, e.g., U.S. patent application numbers 2009/0026082,
2009/0127589, 2010/0035252, 2010/0137143, 2010/0188073,
2010/0197507, 2010/0282617, 2010/0300559), 2010/0300895,
2010/0301398, and 2010/0304982), the content of each of which is
incorporated by reference herein in its entirety.) Oligonucleotide
adaptors are ligated to the ends of target nucleic acid molecules.
The adaptors serve as primers for amplification and sequencing of
the fragments. The fragments can be attached to a surface and is
attached at a resolution such that the fragments are individually
resolvable. Addition of one or more nucleotides releases a proton
(H+), which signal detected and recorded in a sequencing
instrument. The signal strength is proportional to the number of
nucleotides incorporated.
[0067] Embodiments of a typical fluidics based droplet digital
amplification platform generally include one or more instrument
elements employed to execute one or more process steps. FIG. 1
provides an illustrative example of droplet system 100 constructed
and arranged to generate droplets containing templates,
amplification of the templates, and detection of the amplified
products. In some embodiments, droplet system 100 includes droplet
generation instrument 110, thermocycler instrument 115, and droplet
detection instrument 120, although it will be appreciated that
operations may be combined into a single instrument depending on
the number and nature of process steps. Importantly, user 101 may
include any type of user of droplet amplification technologies.
[0068] Also in the same or alternative embodiments, droplet system
100 comprises sequencing instrument 130 that may include a
subsystem that operatively couples a reaction substrate to a
particular mode of data capture (i.e. optical, temperature, pH,
electric current, electrochemical, etc.), one or more data
processing elements, and a fluidic subsystem that enables execution
of sequencing reactions on the reaction substrate. For example,
some embodiments of detectors for fluorescence readout may include
conventional epifluorescence microscopy with a custom
microscope.
[0069] Further, as illustrated in FIG. 1, droplet system 100 may be
operatively linked to one or more external computer components,
such as computer 150 that may, for instance, execute system
software or firmware, such as application 155 that may provide
instructional control of one or more of the instruments, such as
droplet generation instrument 110, thermocycler instrument 115,
droplet detection instrument 120, sequencing instrument 130, and/or
signal processing/data analysis functions. Computer 150 may be
additionally operatively connected to other computers or servers
via network 180 that may enable remote operation of instrument
systems and the export of large amounts of data to systems capable
of storage and processing. Also in some embodiments network 180 may
enable what is referred to as "cloud computing" for signal
processing and/or data analysis functions. In the present example,
droplet system 100 and/or computer 130 may include some or all of
the components and characteristics of the embodiments generally
described herein.
[0070] FIG. 2 provides an illustrative example of droplet generator
200. Droplet generation instrument 110 typically includes one or
more embodiments of droplet generator 200, where in some
embodiments it is highly desirable to have multiple embodiments of
droplet generator 200 that operate in parallel to substantially
increase the rate of droplet generation. In the present example,
droplet generator 200 includes inlet channel 201, outlet channel
202, and two carrier fluid channels 203 and 204. Channels 201, 202,
203, and 204 meet at a junction 205. Inlet channel 201 flows sample
fluid to junction 205. Carrier fluid channels 203 and 204 flow a
carrier fluid that is immiscible with the sample fluid to junction
205. Inlet channel 201 narrows at its distal portion wherein it
connects to junction 205. Inlet channel 201 is oriented to be
perpendicular to carrier fluid channels 203 and 204. As described
elsewhere, droplets are formed as sample fluid flows from inlet
channel 201 to junction 205, where the sample fluid interacts with
flowing carrier fluid provided to the junction 205 by carrier fluid
channels 203 and 204. Outlet channel 202 receives the droplets of
sample fluid surrounded by carrier fluid.
[0071] An exemplary embodiment of a computer system for use with
the presently described invention may include any type of computer
platform such as a workstation, a personal computer, a server, or
any other present or future computer. It will, however, be
appreciated by one of ordinary skill in the art that the
aforementioned computer platforms as described herein are
specifically configured to perform the specialized operations of
the described invention and are not considered general purpose
computers, although the specialized computer platforms may also be
capable of performing operations typical of general purpose
computers. Computers typically include known components, such as a
processor, an operating system, system memory, memory storage
devices, input-output controllers, input-output devices, and
display devices. It will also be understood by those of ordinary
skill in the relevant art that there are many possible
configurations and components of a computer and may also include
cache memory, a data backup unit, and many other devices.
[0072] Display devices may include equipment that provides visual
information, this information typically may be logically and/or
physically organized as an array of pixels. An interface controller
may also be included that may comprise any of a variety of known or
future software programs for providing input and output interfaces.
For example, interfaces may include what are generally referred to
as "Graphical User Interfaces" (often referred to as GUI's) that
provides one or more graphical representations to a user.
Interfaces are typically enabled to accept user inputs using means
of selection or input known to those of ordinary skill in the
related art.
[0073] In the same or alternative embodiments, applications on a
computer may employ an interface that includes what are referred to
as "command line interfaces" (often referred to as CLI's). CLI's
typically provide a text based interaction between an application
and a user. Typically, command line interfaces present output and
receive input as lines of text through display devices. Those of
ordinary skill in the related art will appreciate that interfaces
may include one or more GUI's, CLI's or a combination thereof.
[0074] A processor may include a commercially available processor
or a processor that are or will become available. Some embodiments
of a processor may include what is referred to as Multi-core
processor and/or be enabled to employ parallel processing
technology in a single or multi-core configuration. For example, a
multi-core architecture typically comprises two or more processor
"execution cores". In the present example, each execution core may
perform as an independent processor that enables parallel execution
of multiple threads. In addition, those of ordinary skill in the
related will appreciate that a processor may be configured in what
is generally referred to as 32 or 64 bit architectures, or other
architectural configurations now known or that may be developed in
the future.
[0075] A processor typically executes an operating system that
interfaces with firmware and hardware in a well-known manner, and
facilitates the processor in coordinating and executing the
functions of various computer programs that may be written in a
variety of programming languages. An operating system, typically in
cooperation with a processor, coordinates and executes functions of
the other components of a computer. An operating system also
provides scheduling, input-output control, file and data
management, memory management, and communication control and
related services, all in accordance with known techniques.
[0076] System memory may include any of a variety of known or
future memory storage devices. Examples include any commonly
available random access memory (RAM), magnetic medium, such as a
resident hard disk or tape, an optical medium such as a read and
write compact disc, or other memory storage device. Memory storage
devices may include any of a variety of known or future devices,
including a compact disk drive, a tape drive, a removable hard disk
drive, USB or flash drive, or a diskette drive. Such types of
memory storage devices typically read from, and/or write to, a
program storage medium such as, respectively, a compact disk,
magnetic tape, removable hard disk, USB or flash drive, or floppy
diskette. Any of these program storage media, or others now in use
or that may later be developed, may be considered a computer
program product. As will be appreciated, these program storage
media typically store a computer software program and/or data.
Computer software programs, also called computer control logic,
typically are stored in system memory and/or the program storage
device used in conjunction with memory storage device.
[0077] In some embodiments, a computer program product is described
comprising a computer usable medium having control logic (computer
software program, including program code) stored therein. The
control logic, when executed by a processor, causes the processor
to perform functions described herein. In other embodiments, some
functions are implemented primarily in hardware using, for example,
a hardware state machine. Implementation of the hardware state
machine so as to perform the functions described herein will be
apparent to those skilled in the relevant arts.
[0078] Input-output controllers could include any of a variety of
known devices for accepting and processing information from a user,
whether a human or a machine, whether local or remote. Such devices
include, for example, modem cards, wireless cards, network
interface cards, sound cards, or other types of controllers for any
of a variety of known input devices. Output controllers could
include controllers for any of a variety of known display devices
for presenting information to a user, whether a human or a machine,
whether local or remote. In the presently described embodiment, the
functional elements of a computer communicate with each other via a
system bus. Some embodiments of a computer may communicate with
some functional elements using network or other types of remote
communications.
[0079] As will be evident to those skilled in the relevant art, an
instrument control and/or a data processing application, if
implemented in software, may be loaded into and executed from
system memory and/or a memory storage device. All or portions of
the instrument control and/or data processing applications may also
reside in a read-only memory or similar device of the memory
storage device, such devices not requiring that the instrument
control and/or data processing applications first be loaded through
input-output controllers. It will be understood by those skilled in
the relevant art that the instrument control and/or data processing
applications, or portions of it, may be loaded by a processor in a
known manner into system memory, or cache memory, or both, as
advantageous for execution.
[0080] Also, a computer may include one or more library files,
experiment data files, and an internet client stored in system
memory. For example, experiment data could include data related to
one or more experiments or assays such as detected signal values,
or other values associated with one or more experiments or
processes. Additionally, an internet client may include an
application enabled to accesses a remote service on another
computer using a network and may for instance comprise what are
generally referred to as "Web Browsers". Also, in the same or other
embodiments an internet client may include, or could be an element
of, specialized software applications enabled to access remote
information via a network such as a data processing application for
biological applications.
[0081] A network may include one or more of the many various types
of networks well known to those of ordinary skill in the art. For
example, a network may include a local or wide area network that
may employ what is commonly referred to as a TCP/IP protocol suite
to communicate. A network may include a network comprising a
worldwide system of interconnected computer networks that is
commonly referred to as the internet, or could also include various
intranet architectures. Those of ordinary skill in the related arts
will also appreciate that some users in networked environments may
prefer to employ what are generally referred to as "firewalls"
(also sometimes referred to as Packet Filters, or Border Protection
Devices) to control information traffic to and from hardware and/or
software systems. For example, firewalls may comprise hardware or
software elements or some combination thereof and are typically
designed to enforce security policies put in place by users, such
as for instance network administrators, etc.
Embodiments of the Presently Described Invention
[0082] As described above, embodiments of the described invention
relate to systems and methods for super-efficient reverse
transcription and detection of RNA species in a single target
molecule format. Importantly, it is the super-efficient nature of
the reverse transcription in the single molecule format that that
enables the reduction of user intervention required while reducing
background noise. In general, embodiments of the invention enable
accurate detection of multiple species of small RNA, mRNA, viral
RNA, and other RNA or DNA that may be digitally counted (or
detected by sequencing, arrays, or other methods) to provide a
quantitative measure of the presence and/or absence of RNA targets.
This further enables the generation of a profile of the RNA targets
to aid in disease identification, for pre-clinical and clinical
investigations.
[0083] The term "one step" as used herein generally refers to the
number of user intervention steps where, for example, in the
embodiments described herein user intervention is not required
after droplet generation in order to generate a detectable product
from RNA. Importantly, the one-step process compartmentalizes an
RNA template and reagents in a single compartment and without any
transfer steps that can introduce contamination and loss. FIG. 3
provides an illustrative example of a typical process that requires
multiple user intervention steps for reverse transcription and
amplification of miRNA.
[0084] As described above, some embodiments of the invention
provide systems and methods for quantifying multiple species of a
sample's RNA molecules (e.g. miRNA, siRNA, piRNA, ceRNA (competing
endogenous RNA), saRNA (small activating RNA), other non-coding
RNA, mRNA, viral RNA) by first compartmentalizing target RNA
molecules into a collection of fluid compartments, such that most
of the compartments contain either zero or one target molecules
(e.g. a `digital format` or `single target molecule format` or
`single-molecule-format`) together with the enzymes, primers,
probes, and reagents to perform one-step cDNA synthesis and
amplification and detection within each droplet. Typical droplets
are aqueous droplets surrounded by an immiscible fluid, such as
oil, fluorinated oil or other non-immiscible fluid.
[0085] Those of ordinary skill in the related art appreciate that
the embodiments of the presently described invention are amenable
for use with combinations of nucleic acid that may exist in the
same sample, such as for example RNA, cDNA, DNA, or other nucleic
acid or synthetic nucleic acid now known or developed in the
future.
[0086] In the embodiments described herein, RNA species are
compartmentalized in droplets, converted to first strand cDNA (in
some embodiments a second strand may also be synthesized to produce
a cDNA duplex molecule) and detected in the droplets and/or
released from the droplets and detected in bulk or additional
droplet single-molecule formatted assays. In some embodiments the
cDNA may be amplified in the droplets to produce many substantially
identical copies of the RNA that may increase the level of
detectable signal desirable for some applications such as
sequencing and/or dPCR. For example, the droplets may include an
oligonucleotide probe that is complementary to the cDNA made from a
small RNA (and/or the substantially identical DNA copies of the
small RNA) and includes a detectable reporter. In one embodiment a
detectable reporter may include what is generally referred to as a
TaqMan probe that comprises a quencher molecule and a fluorescent
reporter molecule, or in the same or alternative embodiments what
is generally referred to as an EvaGreen dye may be used. In the
example of TaqMan, the fluorescent reporter molecule may be
separated from the quencher molecule during an amplification
process where the fluorescent signal emitted in response to an
excitation light becomes detectable. Other embodiments utilize
detection by sequencing or any other method (e.g. array).
[0087] The embodiments described herein provide substantial
benefits over previous approaches due to the negligible background
signal produced and super-efficient reverse transcription of RNA
into `first-strand` cDNA, `second strand` cDNA synthesis,
amplification, and fluorescence generation (e.g. probe hydrolysis,
dye intercalation) of target RNA molecules accomplished in the
present invention by employing a one-step method in droplets. The
very low background signal produced is likely attributed to the
very small volumes provided by the droplets that reduce the
likelihood of self-priming by the RT or stem loop primers. This is
a very important aspect of the described embodiments due to the
fact that background noise is a significant source of error that
inhibits the ability to effectively discriminate a true signal.
Moreover, differences in RT or qPCR efficiencies do not affect the
quantification of target RNA species as long as a sufficient
difference between background and signal is present at the
amplification endpoint.
[0088] Also, full length transcripts generated from the initial
encapsulated single target RNA template are generated within the
isolated compartment, enabling any incomplete RT to complete (i.e.
falling off and subsequent re-hybridization) without the chance to
encounter a different target template molecule (no `strand
switching`). Thus there will be fidelity for any splice variants to
be quantified and detected. For example, the super-efficient
approach to cDNA synthesis from RNA in droplets as described herein
allows the generation of cDNA molecules that are representative of
the actual number of target RNA molecules. The cDNA molecules may
be directly counted and quantified or amplified to generate
substantially identical products that may be quantified or
sequenced.
[0089] In the described embodiments either the single or double
stranded cDNA synthesized from small RNA in the droplets are
present at a substantially 1:1 relationship to the original number
of small RNA targets and in some embodiments can be counted without
further amplification to produce a true digital representation of
the amount of small RNA present in the sample. For example, in some
embodiments an identifier, such as a barcode sequence, fluorescent
moiety, or other reporter known in the related art, may be
incorporated into the cDNA during the cDNA synthesis step. In the
same or alternative example, what is referred to as Radio Frequency
Identification (often referred to as RFID) that receives a signal
and transmits information over a short distance and may be used by
coupling an RFID tag to the cDNA molecule in a droplet, followed by
detection.
[0090] In the event that self-priming or other events distort the
apparent number of small RNA molecules, a droplet can be discarded
or ignored whereas in bulk analysis such events tend to contaminate
the entire sample. Thus, the described embodiments reduce biases
that are often introduced when carried out in a bulk reaction. Also
in the present example, components of total RNA, such as rRNA,
tRNA, or other RT or PCR inhibitor may additionally inhibit cDNA
generation and/or amplification efficiency by competing for
reagents and producing undesired products, where such effects are
similarly reduced by the droplet environment (e.g. volume).
[0091] Also as described above, in some embodiments exogenous or
endogenous controls are utilized to improve the accuracy of
quantification. As described elsewhere, an exogenous control may
include nucleic acid added by a user (e.g. "spiked-in") that may
not be naturally present in the sample or occurs at a level where
it is still useful as a reference. For example, an endogenous mRNA
target known to be present in the sample with the RNA targets may
be detected using the same method as used to detect the RNA. The
concentration of the mRNA present in the sample may be known or
unknown and used as a relative measure to the quantity of RNA
detected. In the present example, use of the endogenous mRNA as a
control may be advantageous for some diagnostic uses, such as what
is referred to as point of care diagnostics that in some cases may
not take place in a clinical laboratory environment where access to
laboratory equipment to effectively enable a spike-in method may be
limited.
[0092] In some of the embodiments described herein, aqueous
droplets are generated from a sample such that each droplet
contains one or fewer RNA molecules of interest on average, and
reagents necessary for cDNA synthesis and/or amplification (e.g.
some embodiments of non-sequencing quantification assays using the
combined cDNA synthesis with digital PCR amplification and counting
may be referred to as RT-dPCR). The RNA in the droplets is
subjected to a cDNA synthesis step to produce a single cDNA strand
that is a copy of the RNA. In some embodiments, synthesis of a
second cDNA strand may also be performed however it may not be
necessary in all embodiments. It will be appreciated by those of
ordinary skill that, as described above, the efficiency of the
conversion of RNA to cDNA can be very important to the accuracy of
the results where the synthesis of cDNA is super-efficient in the
volume of the droplet environment.
[0093] In some embodiments target specific primer species and/or
random hexamers constructed to amplify small RNA are employed in
the RT-dPCR process. Examples of primer species enabled to covert
small RNA to cDNA include what are referred to as stem-loop RT
primers (also referred to as "hairpin-loop" primers, available from
the Life Technologies division of Thermo Fisher Scientific). An
illustrative example of a stem-loop RT primer is provided in FIG.
3. An additional example of stem-loop primers for generating cDNA
is described in Chen, C. F. et al., Nucleic Acids Res. 33 (2005)
e179 (which is hereby incorporated by reference herein in its
entirety for all purposes). Those of ordinary skill in the art will
appreciate that other reagents and designs can be used to initiate
reverse transcription of small RNA to generate first-strand cDNA,
and therefore the use of stem-loop RT primers should not be
considered limiting.
[0094] In some embodiments, the stem loop primers may include a
barcode, such as a short length sequence of known or random
composition that may be used as an identifier of each small RNA in
a droplet that may be useful to later identify if experimentally
introduced error is present. For example, a random barcode may be
incorporated into a cDNA synthesized from a small RNA molecule,
where one or more base insertion/deletion events (often referred to
as "indels") may have occurred via polymerase error during
subsequent amplification of the cDNA. The barcode may be applied to
group the detected amplicons (e.g. via sequencing) together and
identify the frequency that a variation occurs, where a variation
that occurs at a low rate (e.g. .about.10% or less) may be
attributable to experimental error. Barcoding methods may be used
for sequencing `error correction`.
[0095] In some instances, the droplets containing the isolated RNA
may already include one or more barcodes that are incorporated into
the cDNA during reverse transcription, or hybridize to amplicons
produced in the droplets. The barcodes may be used in lieu of
fluorescent probes, to detect the presence of a target sequence, or
the barcodes can be used in addition to fluorescent probes, to
track a multitude of sample sources. A detectable barcode-type
label can be any barcode-type label known in the art including, for
example, barcoded magnetic beads (e.g., from Applied Biocode, Inc.,
Santa Fe Springs, Calif.), and nucleic acid sequences. Nucleic acid
barcode sequences typically include a set of oligonucleotides
ranging from about 4 to about 20 oligonucleotide bases (e.g., 8-10
oligonucleotide bases) and uniquely encode a discrete library
member without containing significant homology to any sequence in
the targeted sample.
[0096] The barcode sequence generally includes features useful in
sequencing reactions. For example, the barcode sequences are
designed to have minimal or no homopolymer regions, i.e., 2 or more
of the same base in a row such as AA or CCC, within the barcode
sequence. The barcode sequences are also designed so that they are
at least one edit distance away from the base addition order when
performing base-by-base sequencing, ensuring that the first and
last base do not match the expected bases of the sequence. In
certain embodiments, the barcode sequences are designed to be
correlated to a particular subject, allowing subject samples to be
distinguished. Designing barcodes is shown U.S. Pat. No. 6,235,475,
the contents of which are incorporated by reference herein in their
entirety.
[0097] In some instances, the primers used in the invention
(including, e.g., primers having targeting arms flanked with a
bridge section) may include barcodes such that the barcodes will be
incorporated into the amplified products. For example, the unique
barcode sequence could be incorporated into the 5' end of the
primer, or the barcode sequence could be incorporated into the 3'
end of the primer. In some embodiments, the barcodes may be
incorporated into the amplified products after amplification. For
example, a suitable restriction enzyme (or other endonuclease) may
be introduced to a sample, e.g., a droplet, where it will cut off
an end of an amplification product so that a barcode can be added
with a ligase.
[0098] Attaching barcode sequences to nucleic acids is shown in
U.S. Pub. 2008/0081330 and PCT/US09/64001, the content of each of
which is incorporated by reference herein in its entirety. Methods
for designing sets of barcode sequences and other methods for
attaching barcode sequences are shown in U.S. Pat. Nos. 6,138,077;
6,352,828; 5,636,400; 6,172,214; 6,235,475; 7,393,665; 7,544,473;
5,846,719; 5,695,934; 5,604,097; 6,150,516; RE39,793; U.S. Pat.
Nos. 7,537,897; 6,172,218; and 5,863,722, the content of each of
which is incorporated by reference herein in its entirety.
[0099] Some of the embodiments described herein also allow for
multiple species of RNA to be detected and digitally counted
simultaneously (e.g. multiplexing). For example, numerous one-step
reactions may be carried out in parallel in many droplets with
primers targeting multiple species of RNA molecule in each droplet.
For example, droplets are produced such that each droplet contains
either a single RNA target of interest or no targets of interest,
as well as multiple species of primer each in sufficient quantity
to recognize and produce a cDNA copy of one of the RNA target of
interest. In other words, each droplet comprises multiplexed primer
species each targeting a specific RNA target in order to detect a
plurality of RNA targets from a biological sample in multiple
droplets in order to detect a plurality of targets. The individual
counts from the detected targets or ratios of individual counts may
be used.
[0100] As discussed herein, digital PCR is ideal for detection and
quantification of small RNA targets. The sensitivity of digital PCR
is limited only by the number of independent amplifications that
can be analyzed. For example, by combining one-step methodologies
the embodiments of the invention enable highly accurate detection
with low background noise. In essence, embodiments of the invention
increase the accuracy, sensitivity, and selectivity of small RNA
detection by counting each RNA molecule after conversion to cDNA,
in a single compartment and without any transfer steps that can
introduce contamination, loss, or user error. Continuing with the
present example, to increase the accuracy of quantitation a
reference such as an endogenous or exogenous nucleic acid can be
used. For instance, an exogenous nucleic acid can be spiked into
the sample in a known concentration and analyzed simultaneously
with the small RNA molecules, or and endogenous species, such as
mRNA, can be counted simultaneously with one or more small RNA
species. The endogenous and exogenous references provide useful
normalizing comparators for the target small RNA in the sample.
[0101] Also as described above, some embodiments of the invention
provide systems and methods for quantifying species of viral RNA.
In particular an embodiment is described herein for quantification
of retroviral RNA transcribed from reservoirs of latent virus, such
as RNA from HIV.
[0102] In the embodiments described herein detection of RNA
transcribed from latent virus and/or proviruses represent a
particularly powerful option when compared to the current methods.
In particular embodiments, the methods can be used to determine
latent HIV viral load. With respect to HIV, the current "gold
standard" VOA assay detects cells that can, when activated, release
viruses capable of robust replication (i.e., are
replication-competent), and provides an estimate of the frequency
of latently infected cells that that must be eliminated to ensure
eradication of the HIV infection. However, the VOA assay is
expensive, time, and labor intensive, and requires large amounts of
blood (120-180 ml) from the HIV positive donor. It has also been
shown that the VOA assay tends to under-represent the true in vivo
frequency of the replication-competent latent viral reservoir where
the extent of the under-estimate may be up to 50-fold. In contrast,
assays which quantify specific viral nucleic acids, e.g., in
plasma, have been shown to overestimate the amount of
replication-competent virus. This overestimate may be the result of
detecting non-integrated or degraded viral nucleic acids.
[0103] Embodiments of a VOA assay are typically performed on highly
purified resting CD4+ T cells (e.g. cells containing latent HIV
provirus), which do not produce virus without extra stimulation. In
embodiments of the VOA assay, resting CD4+ T cells purified from a
peripheral blood sample extracted from an HIV positive donor are
stimulated with mitogen phytohemagglutinin (PHA) or with anti-CD3
plus anti-CD28 antibodies in the presence of irradiated peripheral
blood mononuclear cells (PBMC) from and HIV negative donor. These
stimuli induce global T cell activation, which reverses latency of
at least a fraction of cells carrying integrated HIV DNA, which
subsequently is transcribed into RNA. The cells also translate
necessary enzymes and proteins for packing the viruses into
infective virion particles. The virion particles are released from
these cells into the supernatant. In some embodiments, the virion
particles are incubated with PBMC cells from HIV negative donors or
with PBMC cells that have been irradiated to deactivate viruses.
The virion particles can then be analyzed after 2-3 weeks with an
ELISA assay for HIV p24 antigen in the supernatant, or by analyzing
the `naive` cells incubated with the test cells. As mentioned
previously, this VOA method is expensive and inefficient because of
the large volume of blood required, long timeline for test
completion, and significant manpower required for each analysis.
Embodiments of the presently described invention eliminate many of
the weaknesses of the VOA assay and provide an efficient means for
quantifying the amount of latent virus in HIV positive donors. For
example, in some embodiments the purified resting CD4+ T cells are
stimulated as described for the VOA assay, however there is no need
to culture the stimulated cells with the irradiated PBMC cells in
order to amplify the latent virus. Instead, the supernatant is
collected after sufficient time to allow for release of the virion
particles, and the virion particles concentrated (e.g. via
centrifugation and collection of the pellet) and/or purified (e.g.
using methods known to those of ordinary skill such as polyethylene
glycol (PEG) precipitation, or filtration), followed by direct
quantification without further incubation. (It will, however, be
appreciated that the stimulated CD4+ T cells can be cultured with
irradiated PBMC cells for a period in order to increase the number
of virion particles in the supernatant which may increase the
sensitivity of the droplet based assay described herein.)
[0104] In the presently described example, the
concentrated/purified virion particles may be introduced into an
emulsion of aqueous droplets so that individual droplets typically
contain no more than a single virion particle. The virion particles
are subsequently lysed within the droplets and super-efficient
reverse transcription of the RNA performed to produce a cDNA
product as described elsewhere in the description. The cDNA may
represent the entire RNA sequence. In some embodiments it may also
be desirable to amplify the cDNA within the droplet to produce
substantially identical DNA copies although it will be appreciated
that the cDNA may be released from the droplet for amplification.
In other embodiments, the cDNA can be directly sequenced, wherein
an identifying label, e.g., a barcode, is used to facilitate
identifying the original RNA molecules. The super-efficient reverse
transcription and/or amplification may be performed using primers
targeting the HIV gag region and/or HIV long terminal repeat region
(also referred to as LTR), although it will be appreciated that
other target regions may be employed. In some instances, the entire
cDNA sequence will be determined in order to identify whether the
RNA is replication-competent.
[0105] In an alternative embodiment the HIV virions may be lysed
together as a pool or partitioned into different wells and lysed.
The subsequent lysate may be processed further or put directly into
the aqueous droplets for super-efficient reverse transcription.
Once the sample has been partitioned the RNA or the enzymes or both
can be quantified in order to determine a latent viral load. In
some embodiments it may be advantageous to incorporate one or more
molecular barcodes into the cDNA product produced from the
super-efficient reverse transcription. For example, it may be
highly desirable to associate a specific barcode to each virion
particle in that can be used to identify sequence characteristics
specific to each virion. One method for associating a unique
barcode to each virion is to use a "merge" step where a droplet is
merged with another droplet or stream of aqueous fluid. For
instance, the formed droplets may contain primers with a barcode
that is unique from other droplets and merged with a stream of
aqueous fluid comprising the virion particles. The result of the
merge is a droplet with the primer/barcode combined with a virion
particle. In other embodiments each droplet may initially include
one or more unique (or semi-unique) barcodes that will become
incorporated into the cDNA, allowing later identification of the
original RNA. It is to be recognized that these same techniques may
additionally be used to uniquely identify enzymatic activity by
identifying enzymatic products, e.g., nucleic acids produced by the
action of individual enzymes that are isolated in a droplet.
[0106] In some of the embodiments described herein, it may be
desirable to release and sequence the cDNA from the droplets. In
some embodiments, the cDNA may be amplified. The sequence
composition of the virion particles may be used to identify
characteristics that enabled the latent virus to become replication
competent as well as targets for drug therapy. Further, the number
of replication competent CD4 T cells can be quantified which, as
described above, is very important to enable an effective cure of
HIV infection. In some embodiments, droplet digital PCR may be used
along with or in place of sequencing to identify the replication
competent population of latent HIV.
[0107] FIG. 11 provides an illustrative example of a workflow for
encapsulating barcode labels in droplets with single virions from a
sample extracted from a tissue or fluid (e.g. blood). In some
embodiments that barcode labels may be incorporated into the
droplets at the time of droplet formation, however it may be
desirable in some embodiments to merge the barcode labels into the
droplets comprising the HIV virions (e.g. droplet-droplet merging
or merging droplets with a stream of fluid) that may provide better
control of barcode distribution. For example, it may be highly
desirable that each droplet has a barcode label comprising sequence
composition that has a sufficient degree of uniqueness so that it
is easily distinguishable from barcode labels in other droplets. In
the present example, each droplet has a single species of barcode
label, however in some embodiments more than one species of barcode
label may be present in individual droplets so long as the barcode
species retain their uniqueness either alone or in combination.
[0108] In the example of FIG. 11, the virions are lysed within the
droplets and cDNA products produced that incorporate the barcode
label into each cDNA product. The cDNA products are subsequently
amplified (e.g. in the droplets, or released and amplified outside
the droplets such as in a bulk solution), typically to produce
clonal populations of substantially identical copies amenable for
sequencing, although it will be appreciated that clonal populations
are not necessary for all sequencing technologies. The amplified
products are sequenced to produce sequence composition of each
viral genome identified by the associated barcode sequence. The
sequence composition of each viral genome may then be analyzed for
a variety of purposes that include, but are not limited to, Viral
quantitation, identification of variation associated with
resistance to one or more drugs or drug combinations, replication
competency, and latent reservoir detection and
characterization.
[0109] As described above, some embodiments of the invention may
alternatively or additively include detection of an enzyme, e.g., a
reverse transcriptase or RNAseH, corresponding to the latent viral
load. FIG. 9 shows an illustration of the concept and workflow for
a digital droplet ELISA assay, one example of an upfront assay that
can be coupled to the digital reporter enzyme assay readout. When
protein concentrations are too low for standard detection methods
(typically low-sub-picomolar), the disclosed methods enables
protein quantification by counting individual protein molecules
with a fluorescent readout. Droplets containing a single molecule
(e.g. in an ELISA sandwich) will be fluorescent, and the number of
fluorescent droplets in a population of total droplets will yield a
digital count of molecules per volume (e.g. concentration) down to
a limit of detection dependent only on the number of droplets
examined.
[0110] FIG. 9 shows one example ELISA assay format and should not
be considered the only or preferred format (e.g. magnetic beads
could be added following antibody binding in solution). The
protein-containing sample (proteins shown as diamonds with the rare
target protein to be counted shown as solid diamonds) is combined
with the binding reagents and incubated for a sufficient time to
bind into productive complexes.
[0111] In the "ELISA Sandwich Formation" step, each target protein
molecule is bound to two affinity reagents (each binding separate
epitopes of the same target molecule), generating an immunoaffinity
"sandwich" complex. In the example shown, one of the affinity
reagents (e.g. antibody) is immobilized onto a magnetic bead while
the other biotinylated antibody is free in solution. In certain
embodiments, the number of magnetic beads (with immobilized
antibody) is significantly greater than the number of target
proteins in solution, so that single target proteins are bound by
single beads. If the second antibody is used at the same time, its
concentration should be greater than the number of target
molecules, but less than the number of immobilized antibodies.
Alternatively, the second antibody can be added following the first
binding step (ensuring that all target molecules are bound to the
immobilized antibody first).
[0112] After the target proteins are bound into sandwich complexes,
the magnetic beads are retained by a magnetic field to allow
removal of unbound non-target proteins and free antibodies, and
washed to remove non-specific binders. Addition of the reporter
enzyme (e.g. streptavidin-beta galactosidase) results in binding to
the second biotinylated antibody and assembly of the final ELISA
sandwich, which is again washed to remove unbound reporter enzyme.
The final material (see, e.g., FIG. 10A) is re-suspended in a small
volume, along with a fluorogenic substrate, for processing in the
digital droplet readout.
[0113] FIGS. 10A-10D show a number of different readout `modes` for
running the digital droplet readout, following the ELISA sandwich
complex construction. In FIG. 10A, more than one magnetic bead is
in each droplet, but only a single ELISA sandwich is in any single
droplet (e.g. in this case sub-micron magnetic beads are used).
[0114] FIG. 10B shows a mode where at most a single bead is in each
droplet, with at most one ELISA sandwich.
[0115] FIG. 10C shows a mode where the second antibody complexed to
the reporter enzyme has been eluted off of the magnetic bead, and
the droplets are loaded such that at most one antibody-reporter
complex is present in any droplet.
[0116] In FIG. 10D, the reporter enzyme itself is released off of
the magnetic bead, with droplets loaded such that at most one
enzyme molecule is present in any droplet.
[0117] Any suitable method can be used for releasing the enzyme
from the ELISA sandwich. Exemplary methods include: 1) competition
of a desthiobiotin-streptavidin interaction using biotin; 2)
reduction of a linker that contains a disulfide bond; 3) enzymatic
cleavage of a linker group. Other variations can be considered, and
Poisson and non-Poisson models can be used to enable high occupancy
loading while still providing quantitative counting. Thus, it is
possible to quantify an amount of latent viral load by
characterizing an amount of enzyme that is present as a result of
latent viral infection.
[0118] The methods for evaluating enzymatic activity are not
limited to ELISA-type detection, as described above, however. For
example, reverse transcriptase activity can be performed by
co-encapsulating enzymes resulting from a latent virus with a known
substrate (e.g. exogenous RNA) along with a detection assay (e.g.
qPCR assay for the exogenous RNA, or the use of a fluorogenic RNA
substrate). In droplets having active enzymes and complimentary
substrates, new products will be formed that can be detected, e.g.,
with fluorescent detection or by sequencing the resulting products.
Similarly, RNAseH detection would utilize coencapsulation of a
substrate (e.g. synthetic RNA/DNA hybrid) together with a detection
assay (e.g. qPCR assay for the exogenous DNA that is released from
the RNA/DNA hybrid after RNAseH activity or the use of a
fluorogenic RNA/DNA substrate). Again, the enzymatic products can
be detected and quantified with a variety of techniques, including
sequencing and fluorescent detection.
Examples
[0119] FIGS. 4-8 provide examples of data obtained from experiments
conducted to detect and quantify small RNA in droplets.
[0120] FIGS. 4 and 5 illustrate droplet counts plotted on VIC/FAM
scatter or cluster plots from an assay involving miR-16 and Xeno
synthetic RNA. As shown in FIG. 4, results of digital PCR duplex
assays for quantifying miR-16 and an exogeneous spiked in RNA
species are displayed (VIC intensity is shown on the y axis and FAM
intensity on the x-axis). At the end of the RT-PCR of a plurality
of droplets containing miR-16 and exogenous RNA, the fluorescence
emission from each droplet was determined and plotted on a scatter
(or cluster) plot based on its wavelength and intensity. Three
elliptical circles or gates are shown (many other gating methods
can be used) outlining either the cluster of droplets containing
species of miR-16, the Xeno RNA spike-in molecules, or droplets
containing no target molecules (Neg). All plots shown in FIG. 4 are
from the same experiment, with all samples having the same `duplex`
assay mixtures containing primers and probe species for detection
of Xeno molecules using VIC hydrolysis probes and miR-16 FAM
hydrolysis probes, with the different samples A-H having different
amounts of either the miR-16 RT primer or input nucleic acid.
Sample A shows the results of a positive control dPCR assay using
miR-16 cDNA input together with synthetic Xeno RNA, illustrating
that the miR16 assay reagents work together with Xeno RT-PCR assay
reagents. Sample B shows the results of the duplexed assay with no
miR-16 target input and Xeno RNA input, with no background seen for
the miR-16 cluster location, illustrating that the digital droplet
environment does not result in `background` signal from the RT
primer self-priming in the absence of its target (in the gated
region of the graph where miR-16 is detected in the positive
control, no significant background signal is seen). Sample D show
the results with both Xeno and miR-16 synthetic RNA inputs and no
miR-16 RT primer added, showing the specificity of the Xeno assay.
In Samples E-H the same duplex assay is used with miR-16 RT primer
added together with a constant amount of Xeno synthetic RNA and a
variable amount of total human RNA (commercial source from a
mixture of organs). The RT-dPCR (digital RT-PCR) assay count the
same number of Xeno molecule-containing droplets and a variable
amount of miR-16 that linearly correlates with the amount of input
total RNA added to the sample. Similar to the control sample D
compared to Sample C, when the RT primer for miR-16 is omitted from
the assay in Sample H there was no background signal detected where
miR-16 would be present on the graph (compare Sample H and Sample
G).
[0121] Additionally, FIG. 6 shows duplex assays quantifying the
amount of either miR-16, miR-21, or miR-92a, as well as Xeno RNA
spiked in for miR-16 or miR-21 cases (assays listed at the top of
each sample, with input RNA listed below each sample). Sample B
shows the specificity of the reaction (i.e. no miR-21 is detected
when only miR-16 is inputted into the assay). Comparing Samples D,
E, and F, no significant signal is detected within the gates for
miR-21 and miR-16 when the associated RT primers are omitted from
the assay, again illustrating a significant lack of background
signal for these species. Samples D and E show the specificity of
the reaction for either miR-21 or miR-92a, displaying little
background when either specific RT primer is omitted from the
reaction, when the input RNAs are the same.
[0122] FIG. 7 shows an example of expected results of a triplex
RT-dPCR reaction, with Sample A showing the results with both
miR-21 and miR-15 RT primers, Sample B showing specific detection
of only miR-21 when no miR-16 RT primer is added, and Sample C
showing specific detection of only miR-16 when no miR-21RT primer
is added. All three samples show the same amount of Xeno synthetic
RNA spiked-in, with the Xeno qPCR utilizing both a VIC-probe and
and FAM-probe to the same Xeno sequence (thus droplets with a
molecule of Xeno RNA hydrolyze both VIC and FAM probes, resulting
in these droplets appearing `off-axis` in the scatter plot).
[0123] FIG. 8 shows the results of a duplex assay for an endogenous
mRNA (POL2RA, on the VIC-axis) and miR-21 (on the FAM-axis), with
Sample B showing the lack of detection of miR-21 when miR-21 RT
primer is omitted from the assay.
[0124] Having described various embodiments and implementations, it
should be apparent to those skilled in the relevant art that the
foregoing is illustrative only and not limiting, having been
presented by way of example only. Many other schemes for
distributing functions among the various functional elements of the
illustrated embodiments are possible. The functions of any element
may be carried out in various ways in alternative embodiments.
INCORPORATION BY REFERENCE
[0125] References and citations to other documents, such as
patents, patent applications, patent publications, journals, books,
papers, web contents, have been made throughout this disclosure.
All such documents are hereby incorporated herein by reference in
their entirety for all purposes.
EQUIVALENTS
[0126] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein.
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