Note: Descriptions are shown in the official language in which they were submitted.
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EMULSIFICATION WITH MAGNETIC HYDROGELS
Technical Field
This disclosure relates to sample preparation tools that include hydrogels
comprising
magnetic nanoparticles.
Background
The analysis of single cells results in numerous advantages not available
using cells in
bulk. Single-cell RNA sequencing, for example, is used to uncover
relationships between genetic
mutations and disease, identify novel therapeutic agents, and assess the
effectiveness of those
therapeutic agents in real time. Unfortunately, the wide-spread implementation
of such
approaches is constrained by the costs associated with isolating single cells
and preparing
sequencing libraries.
Methods for isolating single cells generally require microfluidic devices that
are
expensive to use and complicated to operate. Moreover, even once single cells
are isolated, the
subsequent sequencing reactions require extracted RNA to be purified from
crude cell extracts
by a long series of centrifugation steps, which is laborious, time-consuming,
and often loses
valuable material. The current single cell analysis workflow is prohibitively
expensive and
difficult to access which severely hampers the pace at which new, life-saving
discoveries can be
made.
Summary
The present invention provides high-throughput systems and methods for sample
preparation. The invention provides an emulsion comprising a hydrogel matrix
containing
magnetic nanoparticles. The emulsion further includes binding elements
integrated into the
hydrogel matrix. Binding elements are grafted onto the hydrogel matrix and
associated with the
magnetic nanoparticles. When exposed to a sample, the binding elements bind
with their partners
(i.e., target analyte). The emulsion forms partitions (e.g., droplets) that
sequester analyte. The
partitions are manipulated using a magnetic field applied to the vessel
containing the emulsion.
This allows for the separation of analyte from other, non-analyte material in
the sample, with the
result being a reduction of background and concentration of the desired
analyte. The inclusion of
magnetic nanoparticles inside the hydrogel matrix also allows for the
manipulation of analyte
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during sample preparation even after partitions are broken. Among other
things, this allows
quick and efficient separation of analyte from crude sample material without
centrifugation. As
such, systems and methods of the invention reduce costs and time associated
with sample
preparation while improving sample quality.
Methods of the invention eliminate centrifugation steps during sample
preparation.
Instead of centrifugation, methods of the invention utilize magnetic fields to
manipulate and
isolate analyte bound with the hydrogel matrix. The manipulation of analyte is
accomplished by
applying a magnetic field to the emulsion described above to move and
sequester analyte as
desired. After that, any sample material that is not attached to a binder can
be removed by, for
example, pipetting. Thus, methods of the invention are useful for isolating
analyte directly from
samples that are often difficult to process, such as blood or tissue
homogenates. Moreover,
because methods and systems of the invention eliminate centrifugation steps,
they significantly
reduce the time required to prepare samples for analysis and reduce any costs
associated with
maintaining centrifuge devices. Systems and methods of the invention are
particularly well-
suited for liquid-handling robotic applications which, due to cost and size
constraints, struggle to
incorporate centrifuges inside their assemblies.
Methods and systems of the invention can simultaneously segregate analyte into
distinct
partitions and then process the analyte (e.g., by adding barcodes) inside each
of the partitions
concurrently within a single reaction tube. In some instances, the target
analyte is intracellular. In
those instances, cells are combined with the hydrogel emulsion. The emulsion
is agitated (e.g.,
vortexed) to create partitions and single cells are segregated inside the
partitions, wherein cells
are lysed, and the desired analyte is detected. As such, methods of the
invention provide a
massively parallel analytical workflow that is inexpensive to use for
preparing libraries from
single cells.
By isolating analyte in separate partitions and processing the analyte
separately inside
those partitions, methods of sample preparation avoid cross contamination.
Such methods are
particularly useful for preparing nucleic acid libraries from single cells
and/or amplifying rare
nucleic acids while reducing amplification bias. Accordingly, methods include
adding barcodes
to analyte such that the analyte can be tracked through an assay or procedure.
The barcodes are
preferably provided via molecular binders (e.g., oligos) that are attached to
hydrogels for binding
analyte, such that, binding of analyte to the molecular binder effectively
results in the analyte
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being barcoded. The oligos can include any combination of barcodes but
preferably include at
least one partition-specific barcode and at least one molecule-specific
barcode (e.g., a unique
molecular identifier). Thus, upon sequencing, each read can be traced back to
a unique molecule
in a unique partition.
In one aspect, the disclosure provides methods for sample preparation in which
hydrogels
(i.e., hydrogel beads) are decorated with molecular binders (e.g., oligos,
proteins) and combined
in an emulsion with a sample containing analytes to be measured. Each of the
hydrogel beads is
made of a hydrogel scaffold and contains magnetic nanoparticles. Methods
further include
partitioning the sample inside the vessel with the hydrogel beads.
Partitioning can be achieved by
vortexing the vessel and/or by adding partitioning reagents. Upon vortexing,
partitions are
formed that generally include one or zero hydrogel beads and a single portion
of sample
containing analyte. Inside the partitions, analyte binds with the molecular
binders, thereby
tethering the analyte to the hydrogel beads and thus associating analyte with
magnetic
nanoparticles. Methods further include separating bound analyte from an
unbound portion of the
sample by applying a magnetic field (which may be as simple as exposing the
sample to a
magnet in proximity thereto). The unbound portion can be crude sample
material, contaminants,
or any other undesired portion of sample.
Many analytical processes, such as sequencing, target capture, detection,
amplification,
etc., require separation of analyte from unwanted material. Methods and
systems of the invention
provide useful approaches for separating analyte from unwanted material. In
preferred
embodiments, analyte separation comprises contacting an exterior surface of a
vessel or tube
containing the sample in emulsion, causing bound analyte to associate near the
surface. Methods
may further involve pipetting the unbound portion of the sample out from the
vessel while
contacting the magnet with the surface, or, alternatively, separating bound
analyte from the
unbound portion of the sample by moving, with the magnet, the bound analyte
away from the
unbound portion.
The hydrogel emulsions include molecular binders for capturing analyte. The
molecular
binders can be oligonucleotides, with capture ligands for binding analyte or
may be nucleic acid
binding proteins, antibodies, or modified nucleic acids (e.g., locked nucleic
acids, peptide nucleic
acids and the like). The oligos may be linked to the hydrogel beads via
acrydite linkages.
Because the hydrogel beads contain magnetic nanoparticles, the capture of
analyte with a binder
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effectively associates the captured analyte with magnetic nanoparticles, and
thus enables the
captured analyte to be manipulated by applying a magnetic field.
Analytes may be any capture target, including nucleic acid, protein,
polysaccharides, and
others. In some embodiments, the analyte being characterized is mRNA, which is
useful for
assessing gene expression. As such, the molecular binders can be oligos with
capture ligands,
wherein the capture ligands are portions of the oligos comprising sequences of
nucleotides
complementary to the RNA, e.g., mRNA. For example, the complementary sequences
may
comprise poly-T sequences for binding poly-A tails of mRNA. Alternatively, the
complementary
sequences may be sequences complementary to specific mRNAs, such as, mRNA of
genes
differentially regulated in certain diseases, such as cancer. In other
embodiments, the capture
ligands may comprise sequences of nucleic acids complementary to portions of
DNA. The
portions of DNA may be any portion of DNA that is of interest. For example,
the capture ligands
may be designed to bind with specific portions of DNA associated with certain
cancer mutations.
In other embodiments, the capture ligands may be designed to bind with target
protein. For
example, the capture ligands may comprise antibodies. In some embodiments, the
beads may
comprise a plurality of different types of capture ligands for capturing
different types of analyte,
such as, capturing at least two of DNA, RNA, and protein.
In preferred instances, each of the molecular binders include a barcode. For
example, the
molecular binders can include barcodes that are unique to each partition. Such
that, each analyte
captured inside a partition is barcoded by the partition in which the analyte
is captured. The
molecular binders may further include barcodes that are unique to each
distinct analyte or
molecule, e.g., a unique molecular identifier (UMI), so that library
preparation yields library
members in which analyte, or sequences reads from analyte, contain barcodes
specific for each
input molecule, and barcode specific for each "partition" (or cell that was
isolated in a partition),
by virtue of combinations of at least two distinct barcodes.
In some instances, the hydrogel beads further include an agent that improves
visibility of
the beads inside the vessel. This allows, for example, fluids to be removed
during sample
preparation from a vessel with increased precision by allowing the researcher
or clinician to
visualize the hydrogel beads while separating the bound analyte from an
unbound portion of
sample. The agent may be a dye or a contrasting colored material.
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In another aspect, this disclosure provides a reagent for use during sample
preparation.
The reagent includes a plurality of hydrogel beads. Each one of the hydrogel
beads includes a
hydrogel scaffold with magnetic nanoparticles embedded therein. Each hydrogel
bead further
includes molecular binders attached to the hydrogel scaffold. The molecular
binders may be
covalently attached to the hydrogel scaffold. The molecular binders may
include capture ligands
for binding nucleic acid or protein. The molecular binders preferably include
barcodes. In
preferred instances, the molecular binders include at least two distinct
barcodes, wherein one
barcode is unique to a hydrogel bead, and another barcode is unique to a
molecular binder
associated with the hydrogel bead. The magnetic nanoparticles contained in the
hydrogel
scaffold may be ferromagnetic or paramagnetic. Preferably the magnetic
nanoparticles are
ferromagnetic. The hydrogel beads may be of any size that is sufficient to
partition an emulsion
and also capture target analyte. As such, in preferred embodiments the beads
are at least 10
micrometers in diameter, and preferably greater. For example, the hydrogel
beads may comprise
a diameter of between 10-200 micrometers. The magnetic nanoparticles are
preferably less than
1 micrometer in size, and more preferably, approximately 10 nanometers.
Magnetic particles may be associated with hydrogel beads in a manner that does
not
compromise partition uniformity, maintains magnetic particle association with
each hydrogel,
and does not compromise enzyme or reagent function within a partition or
within the mixture
(e.g., reverse transcription reagents or PCR amplification reagents).
Accordingly, methods of the invention may comprise combining hydrogel beads
and
magnetic particles in a mixture, thereby associating the magnetic particles
with a plurality of the
hydrogel beads. The magnetic particles are thereby embedded in the hydrogel
bead mixture
itself. The hydrogel beads may be designed to associate with the magnetic
particles. For
example, the hydrogel beads may comprise pores and the magnetic particles may
become
disposed within the pores of the hydrogel beads. The hydrogel beads and
magnetic particles may
also be associated using charge interactions between the hydrogel beads and
magnetic particles.
For example, the hydrogel beads may comprise oligonucleotides disposed on
their surface. The
oligonucleotides may be designed to carry a charge that attracts the magnetic
particles to the
hydrogel beads, for example using specific nucleotide modifications. Magnetic
particles may be
less than about 5 micrometers and also may be silica-coated in order to
facilitate association with
the hydrogel beads. The association of hydrogel particles and magnetic
particles may be
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mediated by van der Waals interaction between silica coated magnetic particles
and
oligonucleotide modifications on the surface of the hydrogel particles.
Once the magnetic particles are associated with the hydrogel beads, the
hydrogel beads
may be combined, in a vessel, with a sample comprising an analyte. The vessel
may be a
microcentrifuge tube. The hydrogel beads may then act as template particles to
generate a
plurality of uniform partitions near-instantly that encapsulate a single one
of the hydrogel beads
and a portion of the sample to form pre-templated instant partitions (PIPs).
The analyte is then
bound to the magnetic particles inside the partitions and the bound analyte
separated from the
sample using a magnet.
Brief Description of Drawings
FIG. 1 diagrams a method of sample prep.
FIG. 2 shows an illustration of a hydrogel bead.
FIG. 3 shows a hydrogel emulsion.
FIG. 4 shows the separation of analyte from a portion of sample inside a
vessel.
FIG. 5 illustrates a reagent containing hydrogel beads.
FIG. 6A-6D shows the separation of analyte from a portion of sample.
Detailed Description
This disclosure relates generally to methods and systems for preparing samples
for
analyte detection. Systems and methods of the invention use hydrogels to
create partitions (e.g.,
droplets) in an emulsion and segregate analyte to be detected in partitions
within the emulsion.
The hydrogels are made of a polymer matrix embedded with magnetic
nanoparticles. The
hydrogels include binding elements that specifically bind target analyte. The
binding elements
are preferably grafted onto the matrix. The binding elements attach the target
analyte to the
matrix. An applied magnetic field is then used to sequester (e.g., move,
capture, or detect)
analyte. It is an insight of the invention that the inclusion of the magnetic
hydrogels inside
templated partitions offers a useful mechanism for manipulating emulsions
during sample
preparation.
For example, after partitioning, a magnetic field is applied to attract and/or
aggregate
partitions. Preferably, the partitions are aggregated to a bottom surface of a
sample tube. The
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aggregation of partitions containing hydrogels at the bottom of the tube
displaces any partitions
that do not contain hydrogels. Accordingly, any partitions devoid of
hydrogels, such as, broken
partitions, and any other sample material, such as, excess oil, are displaced
towards a top of the
sample prep tube. This allows users to separate intact partitions having
analyte-binding
hydrogels from other unwanted sample material (e.g., broken partitions) by
moving the intact
partitions with a magnet. The unwanted sample material is easily removed
during sample
preparation simply by pipetting the material from the sample tube to thereby
reduce or eliminate
background or contamination.
According to systems and methods of the invention, hydrogels (referred to
herein as
hydrogel beads) comprise a hydrogel scaffold comprising magnetic inclusions.
In particular, the
hydrogel scaffold comprises a network of cross-linked polymer chains forming a
hydrogel
matrix. The magnetic inclusions (e.g., magnetic nanoparticles) are embedded
within the hydrogel
matrix. In practicing methods of the invention, the hydrogels serve as
templates to cause
aqueous-in-oil emulsion droplets to form when combined in water with oil and
mixed (e.g.,
vortexed). For example, an aqueous mixture can be prepared in a reaction tube
that includes the
hydrogels and a sample (e.g., water, saline, buffer, blood, tissue lysate,
etc.) having analyte. An
oil may be added to the tube, and the tube can be mixed or agitated. The
hydrogels act to
template the formation of droplets and segregate analyte (e.g., nucleic acid
contained in single
cells) inside the templated droplets. As such, hydrogels of the invention
comprise a size that is
sufficient to at least template the formation of a droplet around a cell. For
example, the hydrogels
may comprise a diameter of at least 10 micrometers, for example, between 10-
200 micrometers.
Magnetic inclusions contained within the hydrogel scaffold may be
substantially smaller,
preferably less than one micrometer in diameter, and more preferably
approximately 10
nanometers in diameter.
Methods and systems of the disclosure may be conducted with hydrogels by
using, for
example, the particle-templated emulsification technology described in Hatori
et. al., Anal.
Chem., 2018 (90):9813-9820, which is incorporated by reference. Essentially,
micron-scale
hydrogel beads or "template particles" having hydrogel scaffolds containing
magnetic inclusions
are used to define isolated fluid droplets surrounded by an immiscible
partitioning fluid. The
hydrogel beads, by virtue of being inside the droplets, allow droplets and
analyte to be easily
handled during sample preparation with a magnetic field.
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FIG. 1 diagrams a method 101 of sample preparation. The method 101 includes
combining 103 hydrogel beads with sample comprising analyte inside a vessel.
The hydrogel
beads comprise a hydrogel scaffold embedded with magnetic inclusions (e.g.,
magnetic
nanoparticles). The beads also include molecular binders. Once combined 103,
the method 101
involves partitioning 105 the sample inside the vessel. After partitioning
105, the methods
include binding 107 analyte to the molecular binders inside the partitions,
and separating 109,
with a magnet, bound analyte from an unbound portion of the sample.
While the hydrogel beads and sample may be combined 103 or added in any order
to a
vessel, it may be useful to provide the vessel with the hydrogel beads
included therein, and to
add the sample comprising analyte directly onto the hydrogel beads. For
example, the hydrogel
beads may be manufactured as a custom-made reagent provided to a researcher or
clinician
inside the vessel for receiving the sample directly therein.
As such, in some embodiments the hydrogel beads are provided in the vessel for
performing steps of the method 101. Any suitable vessel may be used. For
example, a sample
vessel may be, for example, a 0.5 to 1.5 milliliter microcentrifuge tube, such
as those sold under
the trademark EPPENDORF. The sample vessel may be a blood collection tube such
as the
collection tube sold under the trademark VACUTAINER. The tube may be a conical
centrifuge
tube sold under the trademark FALCON by Corning Life Science. In preferred
embodiments of
the method, the hydrogel beads are provided in the vessel within an aqueous
media such as a
buffer, nutrient broth, saline, or water.
The sample may be added directly into the vessel, e.g., directly upon
obtaining the
sample or after some minimal sample preparation step. The sample that contains
the analyte may
be from any biological source. Suitable samples include environmental,
clinical, library
specimen, or other samples with known or unknown analyte present. Suitable
samples may
include whole or parts of blood, plasma, cerebrospinal fluid, saliva, tissue
aspirate, microbial
culture, uncultured microorganisms, swabs, or any other suitable sample. For
example, in some
embodiments, a blood sample is obtained (e.g., by phlebotomy) in a clinical
setting. Whole blood
may be used, or the blood may be spun down to isolate a component of interest
from the blood,
such as peripheral blood monocytes (PBMCs). Preferably an oil is added to the
tube (which will
typically initially overlay the aqueous mixture). A surfactant may also be
added as discussed
further below.
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The analyte can be any chemical species, substance, or chemical constituent
that is of
interest. In some embodiments, the analyte comprises DNA. For example, analyte
may comprise
genomic DNA taken from a nucleus of a cell. In other instances, the DNA may be
cell free DNA,
such as, circulating tumor DNA, cell free mitochondrial DNA, or cell free
fetal DNA. For
example, the DNA may comprise cell free DNA that is present at elevated levels
in blood in a
subject having cancer, such as, breast cancer.
In some instances, the analyte comprises RNA. The RNA may comprise one or more
of
messenger RNA, transfer RNA, ribosomal RNA, micro RNA, or the like. The RNA
may be
isolated from a cell or extracellular vesicle.
In some instances, the target analyte comprises protein or protein fragments.
The analyte
may comprise, for example, a chain of amino acids that code for a portion of a
protein.
The method 101 further includes partitioning 105 the sample inside the vessel
with the
hydrogel beads. Partitioning 105, in a general sense, involves the action of
dividing the
combined mixture of beads and sample into parts. The parts, i.e., partitions,
preferably comprise
aqueous droplets that are substantially monodispersed within the vessel. The
partitions (i.e.,
droplets) are all formed at the moment of vortexing, essentially instantly, as
compared to the
formation of droplets by flowing two fluids through a junction on a
microfluidic chip, which is
limited by time as each droplet must be formed separately. Each droplet thus
provides an
aqueous partition, surrounded by oil.
Partitioning 105 can be performed by agitating the vessel containing the
sample, hydrogel
beads, and oil combined therein. Upon agitating, the hydrogel beads serve as
"templates" while
the shear forces generated from agitating the vessel causes the formation of
water-in-oil
partitions with, ideally, a single hydrogel bead and a portion of sample
comprising analyte inside
each partition. Agitating can be performed by pipetting the mixture to shear
the fluid causing
partitioning 105. Alternatively, partitioning may be performed by contacting
the vessel with a
standard lab-bench vortexer. It may be found that during the vortexing, the
mixture partitions
into the aqueous droplets within about 5 to about 50 seconds, resulting in
sample comprising
analyte to be segregated inside said partitions with the hydrogel beads. In
yet other embodiments,
partitioning 105 may be performed by adding reagents that cause the mixture to
shear.
Upon partitioning 105, a substantial portion of the resultant droplets will
contain a single
hydrogel bead and a portion of sample comprising analyte. Partitions formed
according to
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methods of the disclosure are generally monodisperse, meaning that the vast
majority of the
droplets will include one hydrogel bead and the vast majority of hydrogel
beads will template
into one partition, i.e., droplet. Said another way, monodisperse means that
comparing the
number of hydrogel beads initially provided in the aqueous mixture to the
number of droplets
produced by vortexing, the smaller number will be at least 90% of the larger
number, and in
practice usually at least 95%, more preferably 98% or 99%.
Partitions containing more than one or zero beads can be removed, destroyed,
or
otherwise ignored. For example, in preferred embodiments, methods of the
invention include
manipulating the emulsion by interacting with templated partitions (i.e.,
those that were
templated by hydrogel beads). For example, in some embodiments, a magnet can
be used to
interact directly with intact, templated partitions to aggregate or pellet
those partitions containing
hydrogel beads to a bottom of the vessel. Partitions devoid of hydrogel beads
are consequently
pushed upwards towards the opening of the vessel. A pipette can then easily be
inserted into the
vessel to remove or destroy partitions lacking a hydrogel. Advantageously, by
removing
partitions devoid of hydrogel beads, background noise produced during
sequencing reactions is
substantially reduced.
In some embodiments, analyte is segregated into the partitions as single
cells. In such
embodiments, the single cells are lysed to thereby release the contents of the
single cells,
including analyte, inside the partitions. An important insight of the
disclosure is that the hydrogel
beads may themselves contain reagents that promote useful reactions inside the
partitions, such
as cell lysis. For example, reagents, detergents, enzymes, and cations that
induce cell lysis may
be provided by the hydrogel beads. Such material may be provided by the
hydrogel beads via
internal compartments inside the hydrogel. In some embodiments, lysing may
involve heating
the partitions to a temperature sufficient to release lytic reagents, such as,
divalent cations,
contained inside the hydrogels into the partitions. Lysing may be accomplished
using
mechanical, chemical, or enzymatic means, the addition of heat, divalent
cations (e.g., Mn2+
and/or Mg2+), or any combination thereof.
Each of the hydrogel beads may include a plurality of molecular binders for
binding
analyte. The hydrogel beads may include hundreds to thousands to millions of
distinct molecular
binders for binding analyte. The molecular binders may be designed to bind
with the identical
analyte, for example, gene transcripts of an identical gene, or the molecular
binders may be
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designed to bind with distinct analyte, such as, gene transcripts coded by
different genes. In some
instances, the molecular binders may be made to bind with different types of
analyte (i.e., DNA,
RNA, or protein). For example, each hydrogel bead may include molecular
binders that capture
more than of DNA, RNA, or protein. Accordingly, methods and systems of the
invention can
provide for single-cell analysis, including single-cell multi-omic analysis,
of the transcriptome,
proteome, and genetic material of single cells, which has obvious advantages
of thorough,
efficient, full-characterization of cell function.
Preferably, the molecular binders are grafted onto the hydrogel scaffolds of
the hydrogel
beads. The molecular binders may be any biological material with an attractive
force towards the
target analyte. Preferably, the molecular binders are oligos, also known as
oligonucleotides,
which comprise contiguous strings of nucleic acids. At least a portion of the
oligos include a
capture ligand. The capture ligand is the portion of the molecular binder,
e.g., oligo, which is
made to have an affinity for target analyte, such as, by having a
complementary nucleotide
sequence.
After sample comprising analyte has been segregated into the partitions,
target analyte
binds with the molecular binders inside the partitions. In some embodiments,
target analyte may
comprise DNA, and portions of the molecular binders may comprise sequences
that are
complementary with portions of the target DNA. Binding of analyte with
molecular binders
inside the partitions may occur via Watson-Crick base pairing resulting in
hybridization of the
analyte with portions of the molecular binders. As such, methods may include
incubating a
vessel at a temperature for a period of time sufficient for hybridization to
occur. While the exact
temperatures and time periods will vary depending on specific sequence
compositions of analyte
and binders, it may be found that incubating the vessel at 37 degrees Celsius
for 1 hour is
sufficient for the target analyte to bind with the molecular binders.
In some instances, the target analyte is RNA. As such, the molecular binders
may
comprise oligos comprising base pair sequences that are complementary to
target RNA. The
target RNA may comprise a subset of RNA released by a single cell. For
example, the subset of
RNA may be genes known to be differentially regulated during disease or
pathogenic infection.
The subset of RNA can be bound with hydrogel beads on account of a portion of
the molecular
binders comprising sequences complementary to at least a portion of the subset
of RNA. In some
instances, it may be desirable to profile total messenger RNA released by a
single cell. In such
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instances, each hydrogel bead may include molecular binders, wherein at least
a portion of the
binders comprise a poly-T sequences for binding with poly-A tails of the
messenger RNA.
In some instances, the target analyte is protein. Where the target analyte is
protein,
molecular binders of the invention may comprise oligos with an antibody or a
portion of an
antibody attached thereto. The antibody may be attached to oligos via methods
known in the art,
such as those discussed in Wiener, 2020, Preparation of single- and double-
oligonucleotide
antibody conjugates and their application for protein analytics, Scientific
Reports 10 (1457),
which is incorporated herein by reference.
Methods of the invention involve separating bound analyte from an unbound
portion of
the sample by applying a magnetic field. The magnetic field may be applied
with a magnet. The
magnet can be any material or object that produces a magnetic field. Because
analyte is bound
with hydrogel beads containing magnetic nanoparticles (e.g., ferromagnetic
nanoparticles)
analyte can be separated from the rest of the sample with a magnet. For
example, separating may
comprise contacting the magnet to an exterior surface of the vessel thereby
causing the magnetic
nanoparticles embedded inside hydrogel beads, and consequently, bead-bound
analyte, to
associate with a surface inside the vessel that is adjacent the surface on
which the magnet is
positioned. Separating may further include pipetting any unbound portion of
the sample from the
vessel while contacting the magnet with the surface of the vessel to thereby
separate the unbound
portion of the sample from the bound analyte. Alternatively, separating bound
analyte may
involve moving, with the magnet, the bound analyte away from the unbound
portion of the
sample.
FIG. 2 shows an illustration of a hydrogel bead 201 with magnetic
nanoparticles 207. In
particular, illustrated is a hydrogel bead 201 comprising a hydrogel scaffold
203 with magnetic
nanoparticles 207 embedded within the scaffold. The hydrogel scaffold 203
comprises a
hydrogel polymer. The hydrogel polymer can be any suitable material, such as,
for example,
polyacrylamide (PAA), bis-acrylamide, agarose, poly-ethylene-glycol (PEG), or
polystyrene.
In some embodiments, the hydrogel beads 201 are premade and purchased from a
vendor.
In some embodiments, the hydrogel beads 201 are made inhouse. The scaffolds
203 of the
hydrogel beads 201 may comprise, for example, 6.2% acrylamide (Sigma-Aldrich),
0.18% N,N'-
methylene-bis-acrylamide (Sigma-Aldrich), and 0.3% ammonium persulfate (Sigma-
Aldrich),
which are used for PAA scaffold generation. A total of 14% (w/v) 8-arm PEG SH
(Creative
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PEGworks) in 100 mM NaHCO3 and PEGDA (6 kDa, Creative PEGworks) in 100 mM
NaHCO3 may be used for PEG scaffold generation. Or, a 1% low melting
temperature agarose
(Sigma-Aldrich), which may be used for agarose scaffold generation.
Agarose and PEG scaffold solutions may be injected into a droplet generation
device
with oil (HFE-7500 fluorinated oil supplemented with 5% (w/w) deprotonated
Krytox 157 FSH)
using syringe pumps (New Era, NE-501). The PAA scaffold solution may be
injected into the
droplet generation device with the fluorinated oil supplemented with 1% TEMED.
The hydrogel
solution and oil are preferably loaded into separate 1 mL syringes (BD) and
injected at 300 and
500 microliters, respectively, into the droplet generation device using
syringe pumps.
A desired quantity of magnetic nanoparticles can be added into the droplet
generation
device to thereby create hydrogel with magnetic particles embedded therein.
The magnetic
nanoparticles are preferably ferromagnetic nanoparticles, e.g., iron or iron
oxide, for example,
such as the magnetic particles sold under the trade name Ferrotec. The
hydrogel beads may be
made to contain a magnetite nanoparticle content with as high as 30% by weight
or more.
To improve biocompatibility, it may be helpful to coat the magnetic
nanoparticles with a
synthetic or biological polymer before adding the magnetic nanoparticles to
the droplet
generation device. The magnetic nanoparticles may be coated with, for example,
PEG. The PAA
and PEG droplets are collected and incubated for 1 hour at room temperature
for gelation. The
agarose droplets are incubated on ice for gelation. After gelation, the gelled
droplets are
transferred to an aqueous carrier by destabilizing them in oil with the
addition of an equal
volume of 20% (v/v) perfluoro-l-octanol in HFE-7500. The particles are washed
twice with
hexane containing 2% Span-80 (Sigma-Aldrich) to remove residual oil. Following
the hexane
wash, the particles are washed with sterile water until all oil is removed.
For further discussion on making hydrogel beads according to aspects of the
invention,
see Berensmeier, 2006, Magnetic particles for the separation and purification
of nucleic acids,
Appl Microbiol Biotechnol, 73(3): 495-504; Philippova, 2011, Magnetic polymer
beads: Recent
trends and developments in synthetic design and applications, European Polymer
Journal, 47 (4):
542-559; Suh, 2012, Synthesis of magnetic hydrogel microparticles for
bioassays and tweezer
manipulation in microwells, Microfluidics and Nanofluidics, 13: 665-674, Bong,
2011,
Magnetic Barcoded Hydrogel Microparticles for Multiplexed Detection, Langmuir
26(11):
8008-8014, each of which is incorporated herein by reference.
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Preferably, the hydrogel scaffold 203 includes a concentration of hydrogel
that
effectively prevents the magnetic nanoparticles from seeping from the hydrogel
beads. The
concentration of the hydrogel may be about 0.009 ¨ 1.0 percent hydrogel, and
more preferably,
about 0.01-0.5 percent hydrogel.
In some embodiments, the hydrogels may be disulfide soluble hydrogels.
Disulfide
soluble hydrogels can allow for release of functional hydrogels after
partitioning.
The hydrogel beads 201 further include molecular binders 209 for binding
analyte. Any
number of molecular binders 209 may be included. The molecular binders 209 may
be designed
to bind with the identical analyte, for example, gene transcripts of an
identical gene, or the
molecular binders may be designed to bind with distinct analyte, such as, gene
transcripts coded
by different genes. In some instances, the molecular binders may be made to
bind with different
types of analyte (i.e., DNA, RNA, or protein). Accordingly, hydrogel beads 201
of the invention
can be used for single-cell analysis, including single-cell multi-omic
analysis, to study the
transcriptome, proteome, and genome of single cells.
The molecular binders 209 are preferably oligos. The oligos may comprise
capture
ligands for binding target analyte. For example, at least a portion of the
oligo may comprise a
sequence of nucleotides that is complementary to at least a portion of target
analyte. The
molecular binders 209 are preferably attached to an exterior surface of the
hydrogel scaffold 203.
In some instances, the molecular binders are attached to an exterior surface
of the hydrogel
scaffold via acrydite linkages.
In some instances, the hydrogel bead 201 may include an agent that improves
visibility of
the bead 201, for example, when the bead is being used during sample
preparation. The agent
may be any natural or synthetic substance that, when added to the hydrogel,
changes the color of
the hydrogel. By changing the color, the hydrogel beads may be easier to see
inside the vessel.
The advantage of improved visibility is that it makes it easier to separate
hydrogel beads having
bound analyte away from any other portion of the sample since the researcher
or clinician can
observe, in real time, whether material that is being moved away from a tube
includes the beads
based on the visualization of the agent, such as a dye.
Because methods of the disclosure are useful for isolating analyte into
partitions, and then
preparing libraries of large numbers of molecules in each partition, some
methods of the
invention include barcoding analyte such that the analyte can be tracked
through an assay.
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Accordingly, aspects of the invention provide reliable methods for "barcoding"
analyte inside
partitions. The barcodes are preferably provided by the molecular binders,
such that, binding of
analyte to the molecular binder effectively results in the analyte being
barcoded. The term
barcode should be understood to mean any number of barcodes, index or index
sequence, or
UMIs, which are unique, i.e., distinguishable from other barcodes. The
sequences may be of any
suitable length which is sufficient to distinguish the barcode, or index,
sequence from other
barcode sequences. A barcode, or index, sequence may have a length of 4,5, 6,
7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides, or more.
Specifically, each of the molecular binders are preferably designed to include
one or
more distinct barcodes. Preferably, the one or more barcodes include at least
one partition-
specific barcode (i.e., a barcode that is unique to each hydrogel bead) and an
analyte specific
barcode (i.e., a barcode that is unique to each molecule, e.g., a unique
molecular identifier
(UMI). The partition-specific barcode is advantageous because, for example,
during single cell
analysis, a researcher or clinician can determine which analytes were
contained together within a
single cell based on the presence of identical barcodes.
UMIs are a type of barcode that may be provided to a sample to make each
nucleic acid
molecule, together with its barcode, unique, or nearly unique. This may be
accomplished by
adding one or more UMIs to one or more molecular binders of the present
invention. By
selecting an appropriate number of UMIs, every molecule of analyte in the
sample, together with
its UMI, will be unique or nearly unique.
UMIs are advantageous in that they can be used to count transcripts in a
sample from
sequence data. Because each transcript is tagged with an essentially unique
UMI barcode,
sequencing the transcripts will create sequence read data that is unique for
each transcript. This is
valuable when multiple transcripts present in a cell have identical sequences.
Attaching a UMI to
each allows one to count the number of identical transcripts in the original
cell (regardless of the
presence of other barcode information such a cellular barcodes and/or
sequencing instrument
index sequences). When a transcript with an attached UMI is amplified and then
sequenced, the
resultant sequence read data will have an arbitrary number of identical
sequence reads, produced
by sequencing clonal amplicons of the transcript created during amplification.
To count the
transcripts present in the sample, one "de-duplicates" the sequence read data
with the UMI
sequences included (sometimes called collapsing reads) and counts the
remaining unique
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sequences. Transcript counting may be performed with existing software tools
for deduplicating
reads based on UMIs. See Islam, 2014, Quantitative single-cell RNA-seq with
unique molecular
identifiers, Nat Meth 11(2):163-6 and Liu, 2019, Algorithms for efficiently
collapsing reads
with Unique Molecular Identifiers, Peer J 7:e8275, both incorporated by
reference.
UMIs are advantageous in that they can be used to correct for errors created
during
amplification, such as amplification bias or incorrect base pairing during
amplification. For
example, when using UMIs, because every nucleic acid molecule in a sample
together with its
UMI or UMIs is unique or nearly unique, after amplification and sequencing,
molecules with
identical sequences may be considered to refer to the same starting nucleic
acid molecule,
thereby reducing amplification bias. Methods for error correction using UMIs
are described in
Karlsson et al., 2016, Counting Molecules in cell-free DNA and single cells
RNA", Karolinska
Institutet, Stockholm Sweden, incorporated herein by reference.
FIG. 3 shows a hydrogel emulsion. In particular, shown is a vessel 301
containing sample
303 and hydrogel beads 307 combined inside an emulsion. Upon mixing the
emulsion, the
hydrogel beads 307 template partitions and segregate analyte therein. Any
templated partition
includes one of the hydrogel beads 307. The partitions containing the hydrogel
beads 307 can be
manipulated, for example, moved within the vessel, by applying a magnetic
field to the vessel on
account of the magnetic nanoparticles contained within the hydrogel.
FIG. 4 shows the separation of analyte from a portion of sample 409 inside a
vessel 301.
In particular, the figure shows hydrogel beads 307 bound with analyte being
separated from a
portion of sample 409 using a magnet 411. The magnet 411 is attached to a
magnetic station 413
for holding the vessel during sample preparation. Specifically, once the
vessel is inserted into the
magnetic station 413, the presence of the magnet 411 near a side of the vessel
301 causes
magnetic nanoparticles embedded in the hydrogel beads 307 to move towards the
side of the
vessel 301 nearest the magnet 411. As such, hydrogel beads 307 bound with
analyte are
aggregated inside the vessel 301. Once the aggregated, the portion of the
sample 409 not
comprising the hydrogel beads can be easily and effectively removed from the
vessel 301 using a
pipette, or by dumping the contents of the vessel, without centrifugation.
FIG. 5 illustrates a reagent 501 comprising hydrogel beads 503. The hydrogel
beads 503
may be provided in an aqueous solution (e.g., water, saline, or buffer) or may
be provided in
dried format. Making reference to FIG. 2, each of the hydrogel beads is
preferably made of a
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hydrogel scaffold with magnetic nanoparticles embedded inside the scaffold.
The hydrogel beads
503 may further include molecular binders (e.g., oligos). The molecular
binders are preferably
grafted onto the hydrogel scaffold. The molecular binders may include capture
ligands for
binding one of nucleic acid or protein. The capture ligands are preferably
portions of the oligos
that code for nucleic acid sequences that are complementary to target analyte.
The nucleic acid
sequences can be designed using online tools that are well known in the art,
for example, as
described in Jayaraman, 2019, AnthOligo: Automating the design of
oligonucleotides for
capture/enrichment technologies, bioRxiv, the contents of which are
incorporated by reference.
The molecular binders are made to include one or more barcodes. Preferably,
the one or
more barcodes include at least one barcode that is unique to all the binders
attached to a
particular hydrogel bead. The one or more barcodes further include an analyte
specific barcode,
i.e., a barcode that is unique to each molecule, e.g., a UMI. The hydrogel-
specific barcode is
advantageous because, for example, during single cell analysis, a researcher
or clinician can
determine which analytes were contained together within a particular
partition, and thus, from a
single cell based on the presence of identical barcodes.
According to some aspects of the invention, this disclosure provides methods
for
preparing libraries from single cells. The libraries may be used for
sequencing by next-
generation sequencing devices. The hydrogel beads embedded with magnetic
nanoparticles may
be combined with aqueous liquid and cells and other reagents are introduced
(reagents, such as
lysis reagents, may be delivered within the hydrogel beads). An oil is
overlaid, optionally with a
surfactant (discussed in greater detail above), and the mixture is sheared or
vortexed, which
causes the beads to act as templates to form monodisperse emulsions, which may
be referred to
as pre-templated instant partitions, or "PIPs". In general, each partition
includes one or zero
hydrogel beads, sometimes referred to as a template particle, and a volume of
partitioned fluid,
and a surfactant stabilized shell or surface. To reduce background, it may be
desirable to remove
or destroy partitions devoid of hydrogel.
Cells may be lysed inside partitions to release target analyte for binding
with binders. In
some instances, lysis reagents may diffuse from the hydrogels into the aqueous
partitions of the
emulsion. In some embodiments, nucleic acids are fragmented to create
diversity within the pool
of nucleic acids so as to uniquely identify analyte without using UMIs, for
example, as described
in co-pending Application No. 63/109,035, which is incorporated herein by
reference.
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After lysis, analyte, e.g., RNA that is expelled by the single cells binds
with molecular
binders inside the partitions. Preferably, the molecular binders are attached,
via covalent bonds,
to a surface of the hydrogel, thereby tethering the RNA to the hydrogel beads.
However, in some
instances, it may be advantageous to package the molecular binders within a
compartment of the
.. hydrogels and release the molecular binders from the hydrogels via an
external stimulus (e.g.,
heat), inside the droplets. It may be found that by releasing the binders from
an internal
compartment that certain undesirable intra- or inter-molecular binder
interactions occurring
during binding of analyte, such as, RNA to the binders may be avoided. In
other embodiments, it
is preferable to have the molecular binders attached to the hydrogels so as to
allow for precise
handling of analyte during sample preparation by magnetism after rupturing the
partitions.
In some embodiments, a poly-T end of the molecular binder hybridizes to and
captures
mRNA via a poly-A tail of the mRNA. After binding the mRNA with the molecular
binder, the
partitions may be broken. Any emulsions can be freely broken, and products
pooled due to the
barcodes provided by the molecular binders from the hydrogel beads.
After the emulsions are broken, a magnet may be contacted with the vessel to
pellet
hydrogels inside the vessel. One or more wash steps may be used to rid the
sample of unwanted
cell debris and any other contaminants. After which, reverse transcription
(RT) may be
performed. The reverse transcriptase copies the bound mRNA into complementary
cDNA. In
some embodiments, the reverse transcriptase adds untemplated C bases during
RT. Preferably,
the oligos are attached to beads and after RT each extends to include a cDNA
sequence followed
by several terminal C bases. A template switching oligo (TSO) may be
introduced and
hybridized to the Cs. The TSO can be used to add a common sequence to the cDNA
that is used
downstream for library creation. Polymerase copies the TSO thereby extending
the oligos on the
bead. The TSO may include a preferred sequencing adaptor, such as, the
Illumina P5 adaptor.
The final product may optionally include indexed sequencing adaptors and may
be amplified
using, for example, known platform-specific sequencing amplification primers
such as Illumina
forward and reverse primers.
Sequencing yields genetic sequences that can be de-multiplexed informatically
by
referencing the information introduced by the ligation barcodes. Embodiments
of the ligated
barcodes of this disclosure are useful in methods for reverse transcribing
mRNA into
complementary DNA (cDNA) from cells isolated within aqueous partitions.
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In certain aspects, the disclosure provides a library preparation method for
RNA-
sequencing. The method includes preparing a mixture that includes cells and
reagents for reverse
transcription (RT) and vortexing or optionally pipetting the mixture. During
the vortexing (or
pipetting), the mixture partitions into aqueous-in-oil droplets that each
essentially include zero or
one cell, the cells are lysed to release mRNA into the droplets, and reverse
transcriptase copies
the mRNA into cDNAs. The method preferably further includes amplifying the
cDNAs into a
library of amplicons. Preferably the mixture includes beads that template the
formation of the
droplets upon vortexing. The beads may be gels that include paramagnetic or
preferably
ferromagnetic nanoparticles therein. The mixture may be aqueous and the method
may include
adding an oil onto the mixture prior to the vortexing/pipetting. The method
may include heating
the mixture to a temperature that promotes activity of the reverse
transcriptase (e.g., between
about forty and about fifty degrees C). The mixture is preferably sheared by
any suitable
mechanism or device, such as a benchtop vortexer or shaker, a pipette (e.g.,
micropipette), a
magnetic or other stirrer or similar. The beads may be linked to molecular
binders, sometimes
referred to as capture oligos, that have a free, 3' poly-T region. The beads
may also include
cDNA capture oligos that have 3' portions that hybridize to cDNA copies of the
mRNA. The 3'
portions of the cDNA capture oligos may include gene-specific sequences or
oligomers. The
oligomers may be random or "not-so-random" (NSR) oligomers (NSR0s), such as
random
hexamers or NSR hexamers. The beads may be linked to capture oligos that
include one or more
handles such as primer binding sequences cognate to PCR primers that are used
in the
amplification step or the sequences of NGS sequencing adaptors. The cDNA
capture oligos may
include template switching oligos (TS0s), which may include poly-G sequences
that hybridize to
and capture poly-C segments added during reverse transcription.
In some embodiments, emulsions of the invention include one or more
surfactants.
Inclusion of a surfactant may improve stability of the emulsion. Exemplary
surfactants are
described in published application W02020069298A1, which is incorporated by
reference.
Because emulsions generated by systems and methods of the invention allow
users to
interact with intact partitions during sample preparation, for example, by
moving intact partitions
within a tube and separating intact partitions from broken partitions, these
methods are
particularly well suited for droplet PCR applications (dPCR) to segregate
target analyte (i.e.,
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nucleic acids) from unwanted material and directly quantify and clonally
amplify the target
analyte.
For example, the method may involve partitioning a PCR solution containing
hydrogel
and analyte into tens of thousands of nano-liter sized droplets. The solution
may include
components of a TaqMan assay, e.g., fluorescence-quencher probes, primers, a
PCR master mix
(DNA polymerase, dNTPs, MgCl2), and reaction buffers at optimal
concentrations. The PCR
solution can be divided into smaller reactions and then undergo PCR
individually. After multiple
PCR amplification cycles, the samples are checked for fluorescence with a
binary readout of "0"
or "1". The fraction of fluorescent droplets can be recorded and used to
quantify nucleic acid.
.. According to aspects of the invention, the partitions can be manipulated,
e.g., moved, sorted,
captured, etc., based on the presence of magnetic nanoparticles incorporated
inside hydrogel. In
some instances, this is useful for accurate and efficient quantification of
droplets using
fluorescence because magnets can be used to separate intact partitions from
broken partitions
before quantification of fluorescence to improve background signal and
generate more reliable
.. data.
Hydrogels embedded with magnetic nanoparticles offer precise methods for
interacting
with both analyte and intact partitions during sample prep. They also
eliminate the need for
centrifugation steps, which are required for the isolation and purification of
analyte by prior art
methods. The precision of these methods, and the replacement of centrifuges,
makes them well
suited for automatic sample preparation applications. For example, robotic
devices can be made
that prepare analyte for analysis by separating analyte from other portions of
sample using
magnetic stations. The magnetic stations may substantially resemble the
magnetic station shown
in FIG. 4. The automated system may, for example, prepare samples with
emulsions according to
steps described above, and automatically place the sample tubes, without
direct human
interaction, containing the sample into magnetic stations to thereby separate
analyte from other
sample material for processing.
FIG. 6A-6D shows the separation of analyte from a portion of sample 605 inside
a vessel
601. In particular, the figure shows hydrogel beads 607 bound with analyte
being separated from
a portion of sample 605 using a magnet 611. The sample comprising the analyte
605 and
.. hydrogels 607 comprising magnetic particles are combined and near-instantly
and
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simultaneously separated into partitions 609 comprising a single hydrogel and
a portion of the
sample. The analyte is bound to the magnetic particles within each partition
from the hydrogel.
The presence of the magnet 611 near a side of the vessel 601 causes the
magnetic particles to
move towards the side of the vessel 601 nearest the magnet 611. As such,
hydrogel beads 607
bound with analyte are aggregated inside the vessel 601. Once aggregated, the
portion of the
sample 605 not comprising the hydrogel beads can be easily and effectively
removed from the
vessel 601, for example using a pipette, or by dumping the contents of the
vessel, without
centrifugation.
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