U.S. patent application number 17/471603 was filed with the patent office on 2022-03-31 for instrument systems for integrated sample processing.
The applicant listed for this patent is 10x Genomics, Inc.. Invention is credited to Rajiv BHARADWAJ, Benjamin HINDSON, Donald A. MASQUELIER, Kevin NESS.
Application Number | 20220097045 17/471603 |
Document ID | / |
Family ID | 1000006025789 |
Filed Date | 2022-03-31 |
United States Patent
Application |
20220097045 |
Kind Code |
A1 |
MASQUELIER; Donald A. ; et
al. |
March 31, 2022 |
INSTRUMENT SYSTEMS FOR INTEGRATED SAMPLE PROCESSING
Abstract
An integrated system for processing and preparing samples for
analysis may include a microfluidic device including a plurality of
parallel channel networks for partitioning the samples including
various fluids, and connected to a plurality of inlet and outlet
reservoirs, at least a portion of the fluids comprising reagents, a
holder including a closeable lid hingedly coupled thereto, in which
in a closed configuration, the lid secures the microfluidic device
in the holder, and in an open configuration, the lid is a stand
orienting the microfluidic device at a desired angle to facilitate
recovery of partitions or droplets from the partitioned samples
generated within the microfluidic device, and an instrument
configured to receive the holder and apply a pressure differential
between the plurality of inlet and outlet reservoirs to drive fluid
movement within the channel networks.
Inventors: |
MASQUELIER; Donald A.;
(Tracy, CA) ; HINDSON; Benjamin; (Pleasanton,
CA) ; NESS; Kevin; (Pleasanton, CA) ;
BHARADWAJ; Rajiv; (Pleasanton, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
10x Genomics, Inc. |
Pleasanton |
CA |
US |
|
|
Family ID: |
1000006025789 |
Appl. No.: |
17/471603 |
Filed: |
September 10, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16274134 |
Feb 12, 2019 |
11135584 |
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17471603 |
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15958391 |
Apr 20, 2018 |
10245587 |
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16274134 |
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14934044 |
Nov 5, 2015 |
9975122 |
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15958391 |
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62075653 |
Nov 5, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 9/527 20130101;
B01L 2300/10 20130101; B01L 2300/0609 20130101; B01L 2300/18
20130101; B01L 2200/10 20130101; B01L 2300/0636 20130101; F16K
2099/0084 20130101; B01L 2400/0605 20130101; B01L 2200/027
20130101; C12Q 1/6874 20130101; B01L 2300/0864 20130101; B01L
3/50273 20130101; B01L 2300/14 20130101; B01L 2200/0673 20130101;
B01L 2300/043 20130101; F16K 99/0057 20130101; B01L 2400/0487
20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; F16K 99/00 20060101 F16K099/00; B01L 9/00 20060101
B01L009/00; C12Q 1/6874 20060101 C12Q001/6874 |
Claims
1-20. (canceled)
21. A method for measurement of parameters of fluid in samples for
analysis in a microfluidic device, the method comprising:
positioning an optical detector in optical communication with a
channel segment of at least one fluid channel of the microfluidic
device; imaging, by the optical detector, in a detection line
across the channel segment; and processing images of particulate or
droplet based materials of the samples as the materials pass
through the detection line to determine parameters of the
fluid.
22. The method of claim 21, wherein the optical detector and the
detection line are angled from an axis perpendicular to the channel
segment.
23. The method of claim 22, wherein the optical detector is angled
from 5-80 degrees from an axis perpendicular to the channel
segment.
24. The method of claim 21, wherein the optical detector comprises
at least one line scan sensor.
25. The method of claim 21, further comprising determining the
shape or size of the materials in the fluid.
26. The method of claim 21, wherein the channel segment is
downstream of a partitioning segment of the fluid channel of the
microfluidic device.
27. The method of claim 21, wherein the channel segment is upstream
of a partitioning segment of the fluid channel of the microfluidic
device.
28. An optical detection system for measurement of parameters of
fluid in samples for analysis in a microfluidic device, the system
comprising an optical detector configured to image a fluid in a
detection line across a channel segment of a first channel of the
microfluidic device and to measure at least a parameter of the
fluid in the channel segment; and a stage for holding the
microfluidic device.
29. The system of claim 28, further comprising the microfluidic
device.
30. The system of claim 29, wherein the first channel comprises a
partitioning segment, a pre-partitioning segment, and a post
partitioning segment.
31. The system of claim 30, wherein the channel segment is the
post-partitioning segment.
32. The system of claim 30, wherein the channel segment is the
pre-partitioning segment.
33. The system of claim 28, wherein the optical detector comprises
at least one line scan sensor.
34. The system of claim 28, further comprising one or more light
sources to direct illumination at the channel segment.
35. The system of claim 28, wherein the optical detector comprises
an objective lens.
36. The system of claim 28, wherein the optical detector comprises
a spectral filter.
37. The system of claim 28, wherein the optical detector comprises
a dichroic mirror.
38. The system of claim 28, wherein the optical detector comprises
a light microscope.
39. The system of claim 28, wherein the optical detector detects
fluorescence.
40. The system of claim 28, wherein the optical detector is angled
from an axis perpendicular to the channel segment.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/958,391, filed Apr. 20, 2018, which is a
continuation of U.S. patent application Ser. No. 14/934,044, filed
Nov. 5, 2015, now U.S. Pat. No. 9,975,122, issued May 22, 2018,
which claims priority to U.S. Provisional Patent Application No.
62/075,653, filed Nov. 5, 2014, each of which applications is
entirely incorporated herein by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] Not Applicable
BACKGROUND OF THE INVENTION
[0003] The field of life sciences has experienced dramatic
advancement over the last two decades. From the broad
commercialization of products that derive from recombinant DNA
technology, to the simplification of research, development and
diagnostics, enabled by the invention and deployment of critical
research tools, such as the polymerase chain reaction, nucleic acid
array technologies, robust nucleic acid sequencing technologies,
and more recently, the development and commercialization of high
throughput next generation sequencing technologies. All of these
improvements have combined to advance the fields of biological
research, medicine, diagnostics, agricultural biotechnology, and
myriad other related fields by leaps and bounds.
[0004] Many of these advances in biological analysis and
manipulation require complex, multi-step process workflows, as well
as multiple highly diverse unit operations, in order to achieve the
desired result. Nucleic acid sequencing, for example requires
multiple diverse steps in the process workflow (e.g., extraction,
purification, amplification, library preparation, etc.) before any
sequencing operations are performed. Each workflow process step and
unit operation introduces the opportunity for user intervention and
its resulting variability, as well as providing opportunities for
contamination, adulteration, and other environmental events that
can impact the obtaining of accurate data, e.g., sequence
information.
[0005] The present disclosure describes systems and processes for
integrating multiple process workflow steps in a unified system
architecture that also integrates simplified sample processing
steps.
BRIEF SUMMARY OF THE INVENTION
[0006] Provided are integrated systems and processes for use in the
preparation of samples for analysis, and particularly for the
preparation of nucleic acid containing samples for sequencing
analysis.
[0007] According to various embodiments of the present invention,
an integrated system for processing and preparing samples for
analysis comprises a microfluidic device including a plurality of
parallel channel networks for partitioning the samples including
various fluids, and connected to a plurality of inlet and outlet
reservoirs, at least a portion of the fluids comprising reagents, a
holder including a closeable lid hingedly coupled thereto, in which
in a closed configuration, the lid secures the microfluidic device
in the holder, and in an open configuration, the lid comprises a
stand orienting the microfluidic device at a desired angle to
facilitate recovery of partitions or droplets from the partitioned
samples generated within the microfluidic device. The integrated
system may further include an instrument configured to receive the
holder and apply a pressure differential between the plurality of
inlet and outlet reservoirs to drive fluid movement within the
channel networks.
[0008] In some embodiments, the desired angle at which the
microfluidic device is oriented by the lid ranges from 20-70
degrees, 30-60 degrees, 40-50 degrees.
[0009] In some embodiments, the desired angle at which the
microfluidic device is oriented by the lid is 45 degrees.
[0010] In some embodiments, the instrument comprises a retractable
tray supporting and seating the holder, and slidable into out of
the instrument, a depressible manifold assembly configured to be
actuated and lowered to the microfluidic device and to sealaby
interface with the plurality of inlet and outlet reservoirs, at
least one fluid drive component configured to apply the pressure
differential between the plurality of inlet and outlet reservoirs,
and a controller configured to operate the at least one drive fluid
component to apply the pressure differential depending on a mode of
operation or according to preprogrammed instructions.
[0011] In some embodiments, at least one of the parallel channel
networks comprises a plurality of interconnected fluid channels
fluidly communicated at a first channel junction, at which an
aqueous phase containing at least one of the reagents is combined
with a stream of a non-aqueous fluid to partition the aqueous phase
into discrete droplets within the non-aqueous fluid, and the
partitioned samples are stored in the outlet reservoirs for
harvesting, or stored in at least one product storage vessel.
[0012] In some embodiments, the plurality of interconnected fluid
channels comprises a microfluidic structure having intersecting
fluid channels fabricated into a monolithic component part.
[0013] In some embodiments, the integrated system further comprises
a gasket coupled to the holder and including a plurality of
apertures, in which when the lid is in the closed configuration,
the gasket is positioned between the reservoirs and the manifold
assembly to provide the sealable interface, and the apertures allow
pressure communication between at least one of the outlet and the
inlet reservoirs and the at least one fluid drive component.
[0014] In some embodiments, the integrated system further comprises
springs to bias the manifold assembly in a raised position, and a
servo motor to actuate and lower the manifold assembly.
[0015] In some embodiments, the integrated system further comprises
at least one monitoring component interfaced with at least one of
the plurality of channel networks and configured to observe and
monitor characteristics and properties of the at least one channel
network and fluids flowing therein. The at least one monitoring
component is selected from the group consisting of: a temperature
sensor, a pressure sensor, and a humidity sensor.
[0016] In some embodiments, the integrated system further comprises
at least one valve to control flow into a segment of at least one
channel of the plurality of parallel channel networks by breaking
capillary forces acting to draw aqueous fluids into the channel at
a point of widening of the channel segment in the valve.
[0017] In some embodiments, the at least one valve comprises a
passive check valve.
[0018] In some embodiments, at least one of the plurality of
parallel channel networks comprises a first channel segment fluidly
coupled to a source of barcode reagents, a second channel segment
fluidly coupled to a source of the samples, the first and second
channel segments fluidly connected at a first channel junction, a
third channel segment connected to the first and second channel
segments at the first channel junction, a fourth channel segment
connected to the third channel segment at a second channel junction
and connected to a source of partitioning fluid, and a fifth
channel segment fluidly coupled to the second channel junction and
connected to a channel outlet, The at least one fluid driving
system is coupled to at least one of the first, second, third,
fourth, and fifth channel segments, and is configured to drive flow
of the barcode reagents and the reagents of the sample into the
first channel junction to form a reagent mixture in the third
channel segment and to drive flow of the reagent mixture and the
partitioning fluid into the second channel junction to form
droplets of the first reaction mixture in a stream of partitioning
fluid within the fifth channel segment.
[0019] According to various embodiments of the present invention, a
holder assembly comprises a holder body configured to receive a
microfluidic device, the microfluidic device including a plurality
of parallel channel networks for partitioning various fluids, and a
closeable lid hingedly coupled to the holder body. In a closed
configuration, the lid secures the microfluidic device in the
holder body, and in an open configuration, the lid comprises a
stand to orient the microfluidic device at a desired angle to
facilitate recovery of partitions or droplets from the partitioned
fluids without spilling the fluids.
[0020] In some embodiments, the desired angle at which the
microfluidic device is oriented by the lid ranges from 20-70
degrees, 30-60 degrees, 40-50 degrees.
[0021] In some embodiments, the desired angle at which the
microfluidic device is oriented by the lid is 45 degrees.
[0022] According to various embodiments of the present invention, a
method for measurement of parameters of fluid in samples for
analysis in a microfluidic device of an integrated system comprises
positioning a line camera in optical communication with a segment
of at least one fluid channel of the microfluidic device, imaging,
by the at least one line scan camera, in a detection line across
the channel segment, and processing, by the at least one line scan
camera, images of particulate or droplet based materials of the
samples as the materials pass through the detection line, to
determine shape, size and corresponding characteristics of the
materials, and angling the at least one line camera and the
corresponding detection line across the channel segment to increase
a resolution of resulting images across the channel segment. An
angle at which the at least one line camera and the corresponding
detection line are angled across the channel segment ranges from
5-80 degrees from an axis perpendicular to the channel segment.
[0023] In some embodiments, the method for measurement further
comprises optically communicating the line camera with a post
partitioning segment of at least one fluid channel of the
microfluidic device, to monitor formed partitions emanating from a
partitioning junction of the microfluidic device.
[0024] In some embodiments, the method for measurement further
comprises optically communicating the line camera with a post
partitioning segment of at least one fluid channel of the
microfluidic device, to monitor formed partitions emanating from a
partitioning junction of the microfluidic device.
[0025] In some embodiments, the method for measurement further
comprises optically coupling at least one line scan sensor to one
or more of a particle inlet channel segment to monitor materials
being brought into a partitioning junction to be
co-partitioned.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 schematically illustrates a first level of system
architecture as further described herein.
[0027] FIG. 2 is an exemplary illustration of a consumable
microfluidic component for use in partitioning sample and other
materials.
[0028] FIGS. 3A, 3B, and 3C illustrate different components of a
microfluidic control system.
[0029] FIG. 4 schematically illustrates the structure of an example
optical detection system for integration into overall instrument
systems described herein.
[0030] FIG. 5 schematically illustrates an alternate detection
scheme for use in imaging materials within microchannels.
[0031] FIG. 6 illustrates an exemplary processing workflow, some or
all of which may be integrated into a unified system
architecture.
[0032] FIG. 7 schematically illustrates the integration of a
nucleic acid size fragment selection component into a microfluidic
partitioning component.
[0033] FIG. 8 illustrates a monitored pressure profile across a
microfluidic channel network for use in controlling fluidic flows
through the channel network.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention is directed to devices and systems for
use in apportioning reagents and other materials into extremely
large numbers of partitions in a controllable manner. In
particularly preferred aspects, these devices and systems are
useful in apportioning multiple different reagents and other
materials, including for example, beads, particles and/or
microcapsules into large numbers of partitions along with other
reagents and materials. In particularly preferred aspects, the
devices and systems apportion reagents and other materials into
droplets in an emulsion in which reactions may be carried out in
relative isolation from the reagents and materials included within
different partitions or droplets. Also included are systems that
include the above devices and systems for conducting a variety of
integrated reactions and analyses using the apportioned reagents
and other materials. Thus, the systems and processes of the present
invention can be used with any devices and any systems such as
those outlined in U.S. Provisional Patent Application No.
62/075,653, the full disclosure of which is expressly incorporated
by reference in its entirety for all purposes, specifically
including the Figures, Legends and descriptions of the Figures and
components therein.
I. Partitioning Systems
[0035] The systems described herein include instrumentation,
components, and reagents for use in partitioning materials and
reagents. In preferred aspects, the systems are used in the
delivery of highly complex reagent sets to discrete partitions for
use in any of a variety of different analytical and preparative
operations. The systems described herein also optionally include
both upstream and downstream subsystems that may be integrated with
such instrument systems.
[0036] The overall architecture of these systems typically includes
a partitioning component, which is schematically illustrated in
FIG. 1. As shown, the architecture 100, includes a fluidics
component 102 (illustrated as an interconnected fluid conduit
network 104), that is interfaced with one or more reagent and/or
product fluid storage vessels, e.g., vessels 106-116. The fluidics
component includes a network of interconnected fluid conduits
through which the various fluids are moved from their storage
vessels, and brought together in order to apportion the reagents
and other materials into different partitions, which partitions are
then directed to the product storage vessel(s), e.g., vessel
116.
[0037] The fluidics component 102 is typically interfaced with one
or more fluid drive components, such as pumps 118-126, and/or
optional pump 128, which apply a fluid driving force to the fluids
within the vessels to drive fluid flow through the fluidic
component. By way of example, these fluid drive components may
apply one or both of a positive and/or negative pressure to the
fluidic component, or to the vessels connected thereto, to drive
fluid flows through the fluid conduits. Further, although shown as
multiple independent pressure sources, the pressure sources may
comprise a single pressure source that applies pressure through a
manifold to one or more of the various channel termini, or a
negative pressure to a single outlet channel terminus, e.g., pump
128 at reservoir 116.
[0038] The instrument system 100 also optionally includes one or
more environmental control interfaces, e.g., environmental control
interface 130 operably coupled to the fluidic component, e.g., for
maintaining the fluidic component at a desired temperature, desired
humidity, desired pressure, or otherwise imparting environmental
control. A number of additional components may optionally be
interfaced with the fluidics component and/or one or more of the
reagent or product storage vessels 106-116, including, e.g.,
optical detection systems for monitoring the movement of the fluids
and/or partitions through the fluidic component, and/or in the
reagent and or product reservoirs, etc., additional liquid handling
components for delivering reagents and/or products to or from their
respective storage vessels to or from integrated subsystems, and
the like.
[0039] The instrument system also may include integrated control
software or firmware for instructing the operation of the various
components of the system, typically programmed into a connected
processor 132, which may be integrated into the instrument itself,
or maintained on a directly or wirelessly connected, but separate
processor, e.g., a computer, tablet, smartphone, or the like, for
controlling the operation of, and/or for obtaining data from the
various subsystems and/or components of the overall system.
II. Fluidics Component
[0040] As noted above, the fluidics component of the systems
described herein is typically configured to allocate reagents to
different partitions, and particularly to create those partitions
as droplets in an emulsion, e.g., an aqueous droplet in oil
emulsion. In accordance with this objective, the fluidic component
typically includes a plurality of channel or conduit segments that
communicate at a first channel junction at which an aqueous phase
containing one or more of the reagents is combined with a stream of
a non-aqueous fluid, such as an oil like a fluorinated oil, for
partitioning the aqueous phase into discrete droplets within the
flowing oil stream. While any of a variety of fluidic
configurations may be used to provide this channel junction,
including, e.g., connected fluid tubing, channels, conduits or the
like, in particularly preferred aspects, the fluidic component
comprises a microfluidic structure that has intersecting fluid
channels fabricated into a monolithic component part. Examples of
such microfluidic structures have been generally described in the
art for a variety of different uses, including, e.g., nucleic acid
and protein separations and analysis, cell counting and/or sorting
applications, high throughput assays for, e.g., pharmaceutical
candidate screening, and the like.
[0041] Typically, the microfluidics component of the system
includes a set of intersecting fluid conduits or channels that have
one or more cross sectional dimensions of less than about 200 um,
preferably less than about 100 um, with some cross sectional
dimensions being less than about 50 um, less than about 40 um, less
than about 30 um, less than about 20 um, less than about 10 um, and
in some cases less than or equal to about 5 um. Examples of
microfluidic structures that are particularly useful in generating
partitions are described herein and in co-pending U.S. Provisional
Patent Application No. 61/977,804, filed Apr. 4, 2014, the full
disclosure of which is incorporated herein by reference in its
entirety for all purposes.
[0042] FIG. 2 shows an exemplary microfluidic channel structure for
use in generating partitioned reagents, and particularly for use in
co-partitioning two or more different reagents or materials into
individual partitions. As shown, the microfluidic component 200
provides one or more channel network modules 250 for generating
partitioned reagent compositions. As shown, the channel network
module 250 includes a basic architecture that includes a first
channel junction 210 linking channel segments 202, 204 and 206, as
well as channel segment 208 that links first junction 210 to second
channel junction 222. Also linked to second junction 222 are
channel segments 224, 226 and 228.
[0043] As illustrated, channel segment 202 is also fluidly coupled
to reservoir 230, that provides, for example, a source of
additional reagents such as microcapsules, beads, particles or the
like, optionally including one or more encapsulated or associated
reagents, suspended in an aqueous solution. Each of channel
segments 204 and 206 are similarly fluidly coupled to reagent
storage vessel or fluid reservoir 232, which may provide for
example, a source of sample material as well as other reagents to
be partitioned along with the microcapsules. As noted previously,
although illustrated as both channel segments 204 and 206 being
coupled to the same reservoir 232, these channel segments are
optionally coupled to different reservoirs for introducing
different reagents or materials to be partitioned along with the
reagents from reservoir 230.
[0044] As shown, each of channel segments 202, 204 and 206 are
provided with optional additional fluid control structures, such as
passive fluid valve 236. These valves optionally provide for
controlled filling of the overall devices by breaking the capillary
forces that draw the aqueous fluids into the device at the point of
widening of the channel segment in the valve structure. Briefly,
aqueous fluids are introduced first into the device in reservoirs
230 and 232, at which point these fluids will be drawn by capillary
action into their respective channel segments. Upon reaching the
valve structure, the widened channel will break the capillary
forces, and fluid flow will stop until acted upon by outside
forces, e.g., positive or negative pressures, driving the fluid
into and through the valve structure. These structures are also
particularly useful as flow regulators for instances where beads,
microcapsules or the like are included within the reagent streams,
e.g., to ensure a regularized flow of such particles into the
various channel junctions.
[0045] Also shown in channel segment 202 is a funneling structure
252, that provides reduced system failure due to channel clogging,
and also provides an efficient gathering structure for materials
from reservoir 230, e.g., particles, beads or microcapsules, and
regulation of their flow. As also shown, in some cases, the
connection of channel segment 202 with reservoir 230, as well as
the junctions of one or more or all of the channel segments and
their respective reservoirs, may be provided with additional
functional elements, such as filtering structures 254, e.g.,
pillars, posts, tortuous fluid paths, or other obstructive
structures to prevent unwanted particulate matter from entering or
proceeding through the channel segments.
[0046] First junction 210 is fluidly coupled to second junction
222. Also coupled to channel junction 222 are channel segments 224
and 226 that are, in turn fluidly coupled to reservoir 234, which
may provide, for example, partitioning fluid that is immiscible
with the aqueous fluids flowing from junction 210. Again, channel
segments 224 and 226 are illustrated as being coupled to the same
reservoir 234, although they may be optionally coupled to different
reservoirs, e.g., where each channel segment is desired to deliver
a different composition to junction 222, e.g., partitioning fluids
having different make up, including differing reagents, or the
like.
[0047] In exemplary operation, a first fluid reagent, e.g.,
including microcapsules or other reagents, that is provided in
reservoir 230 is flowed through channel segment 202 into first
channel junction 210. Within junction 210, the aqueous first fluid
reagent solution is contacted with the aqueous fluids, e.g., a
second reagent fluid, from reservoir 232, as introduced by channel
segments 204 and 206. While illustrated as two channel segments 204
and 206, it will be appreciated that fewer (1) or more channel
segments may be connected at junction 210. For example, in some
cases, junction 210 may comprise a T junction at which a single
side channel meets with channel segment 202 in junction 210.
[0048] The combined aqueous fluid stream is then flowed through
channel segment 208 into second junction 222. Within channel
junction 222, the aqueous fluid stream flowing through channel
segment 208, is formed into droplets within the immiscible
partitioning fluid introduced from channel segments 224 and 226. In
some cases, one or both of the partitioning junctions, e.g.,
junction 222 and one or more of the channel segments coupled to
that junction, e.g., channel segments 208, 224, 226 and 228, may be
further configured to optimize the partitioning process at the
junction.
[0049] Further, although illustrated as a cross channel
intersection at which aqueous fluids are flowed through channel
segment 208 into the partitioning junction 222 to be partitioned by
the immiscible fluids from channel segments 224 and 226, and flowed
into channel segment 228, as described elsewhere herein,
partitioning structure within a microfluidic device of the
invention may comprise a number of different structures.
[0050] As described in greater detail below, the flow of the
combined first and second reagent fluids into junction 222, and
optionally, the rate of flow of the other aqueous fluids and/or
partitioning fluid through each of junctions 210 and 222, are
controlled to provide for a desired level of partitioning, e.g., to
control the number of frequency and size of the droplets formed, as
well as control apportionment of other materials, e.g.,
microcapsules, beads or the like, in the droplets.
[0051] Once the reagents are allocated into separate partitions,
they are flowed through channel segment 228 and into a recovery
structure or zone, where they may be readily harvested. As shown,
the recovery zone includes, e.g., product storage vessel or outlet
reservoir 238. Alternatively, the recovery zone may include any of
a number of different interfaces, including fluidic interfaces with
tubes, wells, additional fluidic networks, or the like. In some
cases, where the recovery zone comprises an outlet reservoir, the
outlet reservoir will be structured to have a volume that is
greater than the expected volume of fluids flowing into that
reservoir. In its simplest sense, the outlet reservoir may, in some
cases, have a volume capacity that is equal to or greater than the
combined volume of the input reservoirs for the system, e.g.,
reservoirs 230, 232 and 234.
[0052] In certain aspects, and as alluded to above, at least one of
the aqueous reagents to be co-partitioned will include a
microcapsule, bead or other microparticle component, referred to
herein as a bead. As such, one or more channel segments may be
fluidly coupled to a source of such beads. Typically, such beads
will include as a part of their composition one or more additional
reagents that are associated with the bead, and as a result, are
co-partitioned along with the other reagents. In many cases, the
reagents associated with the beads are releasably associated with,
e.g., capable of being released from, the beads, such that they may
be released into the partition to more freely interact with other
reagents within the various partitions. Such release may be driven
by the controlled application of a particular stimulus, e.g.,
application of a thermal, chemical or mechanical stimulus. By
providing reagents associated with the beads, one may better
control the amount of such reagents, the composition of such
reagents being co-partitioned, and the initiation of reactions
through the controlled release of such reagents.
[0053] By way of example, in some cases, the beads may be provided
with oligonucleotides releasably associated with the beads, where
the oligonucleotides represent members of a diverse nucleic acid
barcode library, whereby an individual bead may include a large
number of oligonucleotides, but only a single type of barcode
sequence included among those oligonucleotides. The barcode
sequences are co-partitioned with sample material components, e.g.,
nucleic acids, and used to barcode portions of those sample
components. The barcoding then allows subsequent processing of the
sequence data obtained, by matching barcodes as having derived from
possibly structurally related sequence portions. The use of such
barcode beads is described in detail in U.S. patent application
Ser. No. 14/316,318, filed Jun. 26, 2014, and incorporated herein
by reference in its entirety for all purposes.
[0054] The microfluidic component is preferably provided as a
replaceable consumable component that can be readily replaced
within the instrument system, e.g., as shown in FIG. 2. For
example, microfluidic devices or chips may be provided that include
the integrated channel networks described herein, and optionally
include at least a portion of the applicable reservoirs, or an
interface for an attachable reservoir, reagent source or recovery
component as applicable. Fabrication and use of microfluidic
devices has been described for a wide range of applications, as
noted above. Such devices may generally be fabricated from organic
materials, inorganic materials, or both. For example, microfluidic
devices may be fabricated from organic materials, such as
polyethylene or polyethylene derivatives, such as cyclic olefin
copolymers (COC), polymethylmethacrylate (PMMA),
polydimethylsiloxane (PDMS), polycarbonate, polystyrene,
polypropylene, or the like, or they may be fabricated in whole or
in part from inorganic materials, such as silicon, or other silica
based materials, e.g., glass, quartz, fused silica, borosilicate
glass, or the like. Particularly useful microfluidic device
structures and materials are described in Provisional U.S. Patent
Application No. 61/977,804, filed Apr. 4, 2014, previously
incorporated herein by reference.
III. Flow Controllers
[0055] As noted with reference to FIG. 1, above, typically, such
replaceable microfluidics structures are integrated within a larger
instrument system that, as noted above, includes a number of other
components for operation of the system, as well as optional
additional system components used for monitoring system operation,
and/or for processes in a workflow that sit upstream and/or
downstream of the partitioning processes.
[0056] In particular, as noted above, the overall system typically
includes one or more fluid driving systems for driving flow of the
fluid reagents through the channel structures within the fluidic
component(s). Fluid driving systems can include any of a variety of
different fluid driving mechanisms. In preferred aspects, these
fluid driving systems will include one or more pressure sources
interfaced with the channel structures to apply a driving pressure
to either push or pull fluids through the channel networks. In
particularly preferred aspects, these pressure sources include one
or more pumps that are interfaced with one or more of the inlets or
outlets to the various channel segments in the channel network.
[0057] As will be appreciated, in some cases, fluids are driven
through the channel network through the application of positive
pressures by applying pressures to each of the inlet reservoirs
through the interconnected channel segments. In such cases, one or
more pressure sources may be interfaced with each reservoir through
an appropriate manifold or connector structure. Alternatively, a
separately controllable pressure source may be applied to each of
one or more of the various different inlet reservoirs, in order to
independently control the application of pressure to different
reservoirs. Such independent control can be useful where it is
desired to adjust or modify of flow profiles in different channel
segments over time or from one application to another. Pressure
pumps, whether for application of positive or negative pressure or
both, may include any of a variety of pumps for application of
pressure heads to fluid materials, including, for example,
diaphragm pumps, simple syringe pumps, or other positive
displacement pumps, pressure tanks or cartridges along with
pressure regulator mechanisms, e.g., that are charged with a
standing pressure, or the like.
[0058] As noted, in certain cases, a negative pressure source may
be applied to the outlet of the channel network, e.g., by
interfacing the negative pressure source with outlet reservoir 238
shown in FIG. 2. By applying a negative pressure to the outlet, the
ratios of fluid flow within all of the interconnected channels is
generally maintained as relatively constant, e.g., flow within
individual channels are not separately regulated through the
applied driving force. As a result, flow characteristics are
generally a result of one or more of the channel geometries, e.g.,
cross section and length which impact fluidic resistance through
such channels, fluid the properties within the various channel
segments, e.g., viscosity, and the like. While not providing for
individual flow control within separate channel segments of the
device, it will be appreciated that one can program flow rates into
a channel structure through the design of the channel network,
e.g., by providing varied channel dimensions to impact flow rates
under a given driving force. Additionally, use of a single vacuum
source coupled to the outlet of the channel network provides
advantages of simplicity in having only a single driving force
applied to the system.
[0059] In alternative or additional aspects, other fluid driving
mechanisms may be employed, including for example, driving systems
that are at least partially integrated into the fluid channels
themselves, such as electrokinetic pumping structures, mechanically
actuated pumping systems, e.g., diaphragm pumps integrated into the
fluidic structures, centrifugal fluid driving, e.g., through rotor
based fluidic components that drive fluid flow outward from a
central reservoir through a radially extending fluidic network, by
rapidly spinning the rotor, or through capillary force or wicking
driving mechanisms.
[0060] The pump(s) are typically interfaced with the channel
structures by a sealed junction between the pump, or conduit or
manifold connected to the pump, and a terminus of the particular
channel, e.g., through a reservoir or other interfacing component.
In particular, with respect to the device illustrated in FIG. 2, a
pump outlet may be interfaced with the channel network by mating
the pump outlet to the opening of the reservoir with an intervening
gasket or sealing element disposed between the two. The gasket may
be an integral part of the microfluidic structure, the pump outlet,
or both, or it may be a separate component that is placed between
the microfluidic structure and the pump outlet. For example, an
integrated gasket element may be molded over the top layer of the
microfluidic device, e.g., as the upper surface of the reservoirs,
as a second deformable material, e.g., a thermoplastic elastomer
molded onto the upper lip of the reservoir that is molded from the
same rigid material as the underlying microfluidic structure.
Although described with reference to pressed interfaces of pump
outlets to reservoirs on microfluidic devices, it will be
appreciated that a variety of different interface components may be
employed, including any of a variety of different types of tubing
couplings (e.g., barbed, quick connect, press fit, etc.) to
interface pressure sources to channel networks. Likewise, the
pressure sources may be interfaced to upstream or downstream
process components and communicated to the channel networks through
appropriate interface components between the fluidic component in
the partitioning system and the upstream or downstream process
component. For example, where multiple integrated components are
fluidically coupled together, application of a pressure to one end
of the integrated fluidic system may be used to drive fluids
through the conduits of each integrated component as well as to
drive fluids from one component to another.
[0061] In some cases, both positive and negative pressures may be
employed in a single process run. For example, in some cases, it
may be desirable to process a partitioning run through a
microfluidic channel network. Upon conclusion of the run, it may be
desirable to reverse the flow through the device, to drive some
portion of the excess non-aqueous component back out of the outlet
reservoir back through the channel network, in order to reduce the
amount of the non-aqueous phase that will be present in the outlet
reservoir when being accessed by the user. In such cases, a
pressure may be applied in one direction, either positive or
negative, during the partitioning run to create the droplets
through the microfluidic device, e.g., device 200 in FIG. 2, that
accumulate in reservoir 238 along with excess non-aqueous phase
material, which will remain at the bottom of the reservoir, e.g.,
at the interface with the channel 228. By then reversing the
direction of pressure, either positive or negative, one may drive
excess non-aqueous material back into the channel network, e.g.,
channel 228.
[0062] Additional control elements may be included coupled to the
pumps of the system, including valves that may be integrated into
manifolds, for switching applied pressures as among different
channel segments in a single fluidic structure or between multiple
channel structures in separate fluid components. Likewise, control
elements may also be integrated into the fluidics components. For
example, valving structures may be included within the channel
network to controllably interrupt flow of fluids in or through one
or more channel segments. Examples of such valves include the
passive valves described above, as well as active controllable
valve structures, such as depressible diaphragms or compressible
channel segments, that may be actuated to restrict or stop flow
through a given channel segment.
[0063] FIGS. 3A-3C illustrate components of an exemplary
instrument/system architecture for interfacing with microfluidic
components, as described above. As shown in FIG. 3A, a microfluidic
device 302 that includes multiple parallel channel networks all
connected to various inlet and outlet reservoirs, e.g., reservoirs
304 and 306, is placed into a secondary holder 310 that includes a
closeable lid 312, to secure the device within the holder. Once the
lid 312 is closed over the microfluidic device 302 in the secondary
holder 310, an optional gasket 314 may be placed over the top of
the reservoirs, e.g., reservoirs 304 and 306, protruding from the
top of the secondary holder 310. As shown, gasket 314 includes
apertures 316 to allow pressure communication between the
reservoirs, e.g., reservoirs 304 and 306, and an interfaced
instrument, through the gasket. As shown, gasket 314 also includes
securing points 318 that are able to latch onto complementary hooks
or other tabs 320 on the secondary holder to secure the gasket 314
in place. Also as shown, secondary holder 310 is assembled such
that when the lid portion 312 is fully opened, it creates a stand
for the secondary holder 310 and a microfluidic device, e.g.,
microfluidic device 302, contained therein, retaining the
microfluidic device 302 at an appropriate orientation, e.g., at a
supported angle, for recovering partitions or droplets generated
within the microfluidic device 302. Typically, the supported angle
at which the microfluidic device 302 is oriented by the lid 312
will range from about 20-70 degrees, more typically about 30-60
degrees, preferrably 40-50 degrees, or in some cases approximately
45 degrees. Though recited in terms of certain ranges, it will be
understood that all ranges from the lowest of the lower limits to
the highest of the upper limits are included, including all
intermediate ranges or specific angles, within this full range or
any specifically recited range. Such angles provide an improved or
optimized configuration for recovering the partitions or droplets
generated within the microfluidic device 302 while minimizing or
preventing spillage of the fluids within the microfluidic device
302.
[0064] FIG. 3B shows a perspective view of an instrument system 350
while FIG. 3C illustrates a side view of the instrument system 350.
As shown, and with reference to FIG. 3A, a microfluidic device 302
may be placed into a secondary holder 310 that is, in turn, placed
upon a retractable tray 322, that moves is slidable into and out of
the instrument system 350. The retractable tray 322 is positioned
on guide rails 324 that extend in a horizontal direction of the
instrument system 350 (as shown by the arrows in FIG. 3C) and allow
the retractable tray 322 to slide into and out of a slot formed in
the housing 354 when driven by a driving mechanism. In some
embodiments, the driving mechanism may include a motor part (not
shown) to transmit rotation power, and a moving link part (not
shown) extending from the motor part towards the guide rails 324,
such that the moving link part is connected to the guide rails 324
to slide the guide rails 324 in the horizontal direction when the
motor part is operated. Pinion gears (not shown) may be formed on
the moving link part and rack gears (not shown) extending in the
horizontal direction may be formed on the guide rails 324 such that
the pinion gears are engaged with the rack gears, and when the
motor part is operated, the moving link part is rotated and the
pinion gears are rotated and moved along the rack gears to slide
the retractable tray 322, positioned on the guide rails 324, into
and out of the housing 354.
[0065] Once secured within the instrument system 350, a depressible
manifold assembly 326 is lowered into contact with the reservoirs,
e.g., reservoirs 304 and 306 in the microfluidic device 302, making
sealed contact between the manifold assembly 326 and the reservoirs
304 and 306 by virtue of intervening gasket 314. Depressible
manifold assembly 326 is actuated and lowered against the
microfluidic device 302 through incorporated servo motor 328 that
controls the movement of the manifold assembly 326, e.g., through a
rotating cam (not shown) that is positioned to push the manifold
assembly 326 down against microfluidic device 302 and gasket 314,
or through another linkage. The manifold assembly 326 is biased in
a raised position by springs 330. Once the manifold assembly 326 is
securely interfaced with the reservoirs, e.g., reservoirs 304 and
306, on the microfluidic device 302, pressures are delivered to one
or more reservoirs, e.g., reservoirs 304 and 306, within each
channel network within the microfluidic device 302, depending upon
the mode in which the system is operating, e.g., pressure or vacuum
drive. The pressures are supplied to the appropriate conduits
within the manifold 326 from one or both of pumps 332 and 334.
Operation of the system is controlled through onboard control
processor, shown as circuit board 356, which is programmed to
operate the pumps in accordance with preprogrammed instructions,
e.g., for requisite times or to be responsive to other inputs,
e.g., sensors or user inputs. Also shown is a user button 338 that
is depressed by the user to execute the control of the system,
e.g., to extend and retract the tray 322 prior to a run, and to
commence a run. Indicator lights 340 are provided to indicate to
the user the status of the instrument and/or system run. The
instrument components are secured to a frame 352 and covered within
housing 354.
IV. Environmental Control
[0066] In addition to flow control components, the systems
described herein may additionally or alternatively include other
interfaced components, such as environmental control components,
monitoring components, and other integrated elements.
[0067] In some cases, the systems may include environmental control
elements for controlling parameters in which the channel networks,
reagent vessels, and/or product reservoirs are disposed. In many
cases, it will be desirable to maintain controlled temperatures for
one or more of the fluidic components or the elements thereof. For
example, when employing transient reactants, it may be desirable to
maintain cooler temperatures to preserve those reagents. Likewise,
in many cases partitioning systems may operate more optimally at a
set temperature, and maintaining the system at such temperature
will reduce run-to-run variability. Temperature controllers may
include any of a variety of different temperature control systems,
including simple heaters and coolers, fans or radiators, interfaced
with the fluidics component portion of the system. In preferred
aspects, temperature control may be provided through a
thermoelectric heater/cooler that is directly contacted with the
device, or a thermal conductor that is contacted with the device,
in order to control its temperature. Thermoelectric coolers are
widely available and can generally be configured to apply
temperature control to a wide variety of different structures and
materials. The temperature control systems will typically be
included along with temperature sensing systems for monitoring the
temperature of the system or key portions of it, e.g., where the
fluidics components are placed, so as to provide feedback control
to the overall temperature control system.
[0068] In addition to temperature control, the systems may likewise
provide control of other environmental characteristics, such as
providing a controlled humidity level within the instrument, and/or
providing a light or air sealed environment, e.g., to prevent light
damage or potential contamination from external sources.
V. Monitoring and Detection
[0069] The systems described herein also optionally include other
monitoring components interfaced with the fluidics components. Such
monitoring systems include, for example, pressure monitoring
systems, level indicator systems, e.g., for monitoring reagent
levels within reservoirs, and optical detection systems, for
observing fluids or other materials within channels within the
fluidics components.
[0070] A. Pressure
[0071] A variety of different monitoring systems may be included,
such as pressure monitoring systems that may allow identification
of plugged channels, air bubbles, exhaustion of one or more
reagents, e.g., that may signal the completion of a given
operation, or real time feedback of fluid flows, e.g., indicating
viscosity by virtue of back pressures, etc. Such pressure
monitoring systems may often include one or more pressure sensors
interfaced with one or more fluidic channels, reservoirs or
interfacing components, e.g., within the lines connecting the pumps
to the reservoirs of the device, or integrated into other conduits
coupled to other reservoirs. By way of example, where a positive
pressure is applied to multiple inlet reservoirs, pressure sensors
coupled to those inlet reservoirs can allow the detection of a
channel clog which may be accompanied by a pressure increase, or
injection of air through a channel which may accompany exhaustion
of one or more reagents from a reservoir, which may be accompanied
by a pressure drop. Likewise, pressure sensors coupled to a
reservoir to which a negative pressure is applied may similarly
identify perturbations in pressure that may be indicative of
similar failures or occurrences. With reference to FIG. 1, pressure
sensors may be optionally integrated into one or more of the lines
connecting the pumps 118-128 (shown as dashed lines), or integrated
directly into the reservoirs 106-116, disposed at the termini of
the various channel segments in the fluidic channel network 104.
The sensors incorporated into the instrument may typically be
operably coupled to the controller that is integrated into the
instrument, e.g., on circuit board 356 shown in FIG. 3B.
Alternatively or additionally, the sensors may be linked, e.g.,
through appropriate connectors, to an external processor for
recording and monitoring of signals from those sensors.
[0072] As will be appreciated, when in normal operation, it would
be expected that the pressure profiles at the one or more sensors
would be expected to remain relatively steady. However, upon a
particular failure event, such as aspiration of air into a channel
segment, or a blockage at one or more channel segments or
intersections, would be expected to cause a perturbation in the
steady state pressure profiles. For example, for a system as shown
in FIG. 1, that includes an applied negative pressure at an outlet
reservoir, e.g., reservoir 116 with an integrated pressure sensor,
normal operation of the system would be expected to have a
relatively steady state of this negative pressure exhibited at the
reservoir. However, in the event of a system disturbance, such as
exhaustion of a reagent in one or more of reservoirs 106-114, and
resulting aspiration of air into the channels of the system, one
would expect to see a reduction in the negative pressure (or an
increase in pressure) at the outlet reservoir resulting from the
sudden decrease in fluidic resistance in the channel network
resulting from the introduction of air. By monitoring the pressure
profile, the system may initiate changes in operation in response
to such perturbations, including, e.g., shut down of the pumps,
triggering of alarms, or other measures, in order to void damaging
failure events, e.g., to the system or the materials being
processed therein. As will be appreciated, pressure profiles would
be similarly monitorable when using individually applied pressures
at multiple reservoirs/channel termini. For example, for positive
applied pressures, introduction of air into channels would be
expected to cause a drop in pressure at an inlet reservoir, while
clogs or obstructions would be expected to result in increases in
pressures at the inlets of a given clogged channel or channels.
[0073] In some cases, one or more pressure sensors may be
integrated within the manifold or pressure lines that connect to
one or more of the reservoirs or other channel termini, as
described herein. A variety of pressure sensor types may be
integrated into the systems described herein. For example, small
scale solid state pressure sensors may be coupled, in-line, with
pressure or vacuum lines connected to the reservoirs of the fluidic
components, in order to measure pressure within those lines and at
those reservoirs. As with the pumps described herein, pressure
sensors may be integrated with one or more of the reservoirs,
including the outlet and inlet reservoirs, as applicable. In some
cases, each pressure conduit connected to a reservoir within a
device may include a pressure sensor for monitoring pressures at
such reservoirs.
[0074] In operation, the pressure sensing system is used to
identify pressure perturbations that signal system failures or
end-of-run events, such as channel clogs, air aspiration through
channels, e.g., from reagent exhaustion, or the like. In
particular, the pressure sensing system is used to trigger system
operations when the steady state pressures measured by the pressure
sensing system deviate above or below a threshold amount. Upon
occurrence of such a perturbation, the system may be configured to
shut down, or reduce applied pressures, or initiate other
mitigation measures to avoid adulterating the overall system, e.g.,
by drawing fluids into the pumping system, or manifold. In certain
aspects, the system will be configured to shut down or reduce
applied pressures when the steady state pressure measured in one or
more channel segments deviates from the steady state pressure by
more than 10%, more than 20%, more than 30%, more than 40%, more
than 50%, more than 60%, or more.
[0075] In addition to or as an alternative to the pressure sensors
described above, one or more flow sensors may also be integrated
into the system, e.g., within the manifold or flow lines of the
system, in order to monitor flow through the monitored conduit. As
with the pressure sensors, these flow sensors may provide
indications of excessive flow rates within one or more of the
conduits feeding the fluidic device, as well as provide indications
of perturbations in that flow resulting from system problems or
fluidics problems, e.g., resulting from channel occlusions or
constrictions, exhaustion of one or more fluid reagents, etc.
[0076] B. Optical Monitoring and Detection
[0077] In addition to pressure sensors, the systems described
herein may also include optical sensors for measurement of a
variety of different parameters within the fluid components of the
system, as well as within other parts of the system. For example,
in at least one example, an optical sensor is positioned within the
system such that it is in optical communication with one or more of
the fluid channels in the fluid component. The optical sensor is
typically positioned adjacent one or more channels in the fluid
component, so that it is able to detect the passage of material
through the particular channel segment. The detection of materials
may be by virtue of the change in optical properties of the fluids
flowing through the channel, e.g., light scattering, refractive
index, or by virtue of the presence of optically detectable
species, e.g., fluorophores, chromophores, colloidal materials, or
the like, within the fluid conduits.
[0078] In many cases, the optical detection system optionally
includes one or more light sources to direct illumination at the
channel segment. The directed light may enhance aspects of the
detection process, e.g., providing contrasting light or excitation
light in the illumination of the contents of the channel. In some
cases, the light source may be an excitation light source for
exciting fluorescent components within the channel segment that
will emit fluorescent signals in response. These fluorescent
signals are then detected by the optical sensor.
[0079] FIG. 4 schematically illustrates an example of an optical
detection system for monitoring materials within fluidic channels
of the fluidics component of the systems described herein. As
shown, the optical detection system 400 typically includes an
optical train 402 placed in optical communication with one or more
channel segments within the fluidic component, e.g., channel
segment 404. In particular, optical train 402 is placed within
optical communication with channel segment 404 in order to
optically interrogate the channel segment and/or its contents,
e.g., fluid 406 and particles or droplets 408. Generally, the
optical train will typically include a collection of optical
components used for conveying the optical signals from the channel
segments to an associated detector or detectors. For example,
optical trains may include an objective lens 410 for receiving
optical signals from the fluid channel 404, as well as associated
optical components, e.g., lenses 412 and 414, spectral filters and
dichroics 416 and 418, and spatial filters, e.g., filter 420, for
directing those optical signals to a detector or sensor 422 (and
one or more optional additional sensors, e.g., sensor 424), such as
a CCD or CMOS camera, PMT, photodiode, or other light detecting
device.
[0080] In some cases, the optical detection system 400 may operate
as a light microscope to detect and monitor materials as they pass
through the channel segment(s) in question. In such cases, the
optical train 402 may include spatial filters, such as confocal
optics, e.g., filter 420, as well as an associated light source
426, in order to increase contrast for the materials within the
channel segment.
[0081] In some cases, the optical detection system may
alternatively, or additionally be configured to operate as a
fluorescence detection microscope for monitoring fluorescent or
fluorescently labeled materials passing through the channel
segments. In the case of a fluorescence detection system, light
source 426 may be an excitation light source, e.g., configured to
illuminate the contents of a channel at a wavelength that excites
fluorescence from the materials within the channel segment. In such
cases, the optical train 402, may additionally be configured with
filter optics to allow the detection of fluorescent emissions from
the channel without interference from the excitation light source
426. This is typically accomplished through the incorporation of
cut-off or narrow band pass filters, e.g., filter 416 within the
optical train to filter out the excitation wavelength while
permitting light of the wavelengths emitted by the fluorescent
species to pass and be detected.
[0082] In particularly preferred aspects, the optical sensor is
provided optically coupled to one or more of a particle inlet
channel segment (through which beads or other particles are
injected into the partitioning region of the fluidic component of
the system), e.g., channel segment 202 of FIG. 2, to monitor the
materials being brought into the partitioning junction, e.g.,
monitoring the frequency and flow rates of particles that are to be
co-partitioned in the partitioning junction. Alternatively or
additionally, the optical detector may be positioned in optical
communication with the post partitioning channel segment of the
fluidic component, e.g., channel segment 228, to allow the
monitoring of the formed partitions emanating from the partitioning
junction of the fluidic device or structure. In particular, it is
highly desirable to be able to monitor and maintain control of the
flow of particles that are being introduced into the partitioning
region, and to monitor and control the flow and characteristics of
partitions as they are being generated in order to ensure the
proper flow rates and generation frequencies for the partitions, as
well as to understand the efficiency of the partitioning
process.
[0083] In a particular example, the optical sensor is used to
monitor and detect partitions as they pass a particular point in
the channel segment. In such cases, the optical sensor may be used
to measure physical characteristics of the partitions, or their
components, as they pass the position in the channel, such as the
size, shape, speed or frequency of the partitions as they pass the
detector. In other cases, the optical detector or sensor 422 may be
configured to detect some other characteristics of the partitions
as they pass the detector or sensor 422, e.g., relating to the
contents of the partitions.
[0084] As noted above, in some cases, the optical detection system
will be configured to monitor aspects of the contents of the
created partitions. For example, in some cases, materials that are
to be co-partitioned into individual partitions may be monitored to
detect the relative ratio of the co-partitioned materials. By way
of example, two fluid borne materials, e.g., a reagent, and a bead
population, may each be differentially optically labeled, and the
optical detection system is configured to resolve the contribution
of these materials in the resulting partitions.
[0085] In an example system, two optically resolvable fluorescent
dyes may be separately suspended into each of the first reagent and
the second reagents that are to be co-partitioned. The relative
ratio of the first and second reagents in the resulting partition
will be ascertainable by detecting the fluorescent signals
associated with each fluorescent dye in the resulting partition.
Accordingly, the optical detection system will typically be
configured for at least two-color fluorescent optics. Such two
color systems typically include one or more light sources that
provide excitation light at the appropriate wavelengths to excite
the different fluorescent dyes in the channel segment. These
systems also typically include optical trains that differentially
direct the fluorescent emissions from those dyes to different
optical detectors or regions on the same detector. With reference
to FIG. 4, for example, two optically distinguishable fluorescent
dyes may be co-partitioned into droplets, e.g., droplets 408 within
channel segment 404. Upon excitation of those fluorescent dyes by
light source 426, two optically resolvable fluorescent signals are
emitted from the droplets 408, shown as solid arrow 428. The mixed
fluorescent signals, along with transient excitation light are
collected through objective 410 and passed through optical train
402. Excitation light is filtered out of the signal path by
inclusion of an appropriate filter, e.g., filter 416, which may
include one or more cut-off or notch filters that pass the
fluorescent light wavelengths while rejecting the excitation
wavelengths. The mixed fluorescent signals are then directed toward
dichroic mirror 420, which allows one of the fluorescent signals
(shown by arrow 430) to pass through to a first detector 422, while
reflecting a second, spectrally different fluorescent signal (shown
by arrow 432), to second detector 424.
[0086] The intensities of each fluorescent signals associated with
each dye, are reflective of the concentration of those dyes within
the droplets. As such, by comparing the ratio of the signal from
each fluorescent dye, one can determine the relative ratio of the
first and second fluids within the partition. Further, by comparing
the detected fluorescence to known extinction coefficients for the
fluorescent dyes, as well as the size of observed region, e.g., a
droplet, one can determine the concentration of each component
within a droplet. As will be appreciated, where looking to
partition particle based reagents into droplets, when using a
fluorescently labeled particle, these systems also will allow one
to ascertain the relative number of particles within a partition,
as well as identifying partitions that contain no particles.
[0087] In other aspects, the optical detection systems may be used
to determine other characteristics of the materials, particles,
partitions or the like, flowing through the channel segments,
including, for example, droplet or particle size, shape, flow rate,
flow frequency, and other characteristics. In at least one aspect,
optical detectors provided are configured to better measure these
characteristics. In one aspect, this is achieved through the
incorporation of a line scan camera or detector, e.g., camera 510,
into the optical system, that images across a channel segment in a
detection line in order to process images of the materials as they
pass through the detection line. This is schematically illustrated
in FIG. 5, top panel. As shown, a channel segment 502 is provided
wherein materials, and particularly particulate or droplet based
materials are being transported. The optical detection system
images a line across the channel segment 502 (indicated as image
zone 504). Because the line scan camera employs a line scanner,
rather than a two-dimensional array of pixels associated with other
camera types, it results in substantially less image processing
complexity, allowing greater flexibility of operation.
[0088] In addition to using a line scan camera system, in some
cases, it is desirable to provide higher resolution imaging using
such camera systems by angling the detection line across the
channel segment 502, as shown in FIG. 5, bottom panel. In
particular, assuming a linear, one-dimensional array of pixels in a
line scan camera (schematically illustrated as pixels 506 in camera
508), one would expect an image that is reflective of those pixels
(schematically illustrated as image 510). Typically, the angle
.theta. at which the detection line (indicated as image zone 504)
is angled across the channel segment 502 will range from about 5-80
degrees from an axis Y perpendicular to the channel segment 502,
more specifically 15-75 degrees, 20-70 degrees, 25-65 degrees,
30-60 degrees, 35-55 degrees, 40-50 degrees, or in some cases
approximately 45 degrees. Though recited in terms of certain
ranges, it will be understood that all ranges from the lowest of
the lower limits to the highest of the upper limits are included,
including all intermediate ranges or specific angles, within this
full range or any specifically recited range. By angling the camera
and the detection line/image zone 504, one achieves an effective
closer spacing of the pixels as they image flowing materials. The
resulting image thus is of higher resolution across the channel, as
shown by image 512, than for the perpendicularly oriented image
zone, as shown by image 510. By providing higher resolution, one is
able to obtain higher quality images of the particles, droplets or
other materials flowing through the channel segments of the device,
and from that, derive the shape, size and other characteristics of
these materials.
[0089] As will be appreciated, as the optical detection systems may
be used to monitor flow rates within channel segments of a device,
these detection systems may, as with the pressure monitoring
systems described above, identify perturbations in the operation of
the system. For example, where a reagent well is exhausted,
allowing air to be passed through the channels of he device, while
leading to a pressure drop across the relevant channel segments, it
will also result in an increase in flow rate through that channel
segment resulting from the lower fluidic resistance in that
channel. Likewise, an obstructed channel segment will in many
cases, lead to a reduced flow rate in downstream channel segments
connected to the obstructed channel segment. As such, perturbations
in flow rates measured optically, may be used to indicate system
failures or run completions or the like. In general, perturbations
of at least 5% in the optically determined flow rate, at least 10%,
at least 20%, at least 30%, at least 40%, or at least 50%, will be
indicative of a problem during a processing run, and may result in
a system adjustment, shutdown or the like.
[0090] FIG. 8 illustrates optical monitoring processes and systems
as described herein for use in identifying perturbations in flow
within channels of a fluidic network. As shown, a single a
microfluidic device, e.g., as shown in FIG. 2, is run under applied
pressures at each of the various inlet reservoirs, e.g., reservoirs
230, 232 and 234, under constant pressure. The flow rate of
droplets is measured within an outlet channel segment, e.g.,
channel segment 228 using an optical imaging system. The flow rate
of a normally operating channel segment is plotted in the first
portion 302 of the flow rate plot shown in FIG. 8. Upon exhaustion
of one reagent, e.g., the oil in reservoir 234, air is introduced
into the channel network, resulting in a reduced fluidic
resistance, causing an increase in the flow rate, as shown in the
second portion 304 of the plot.
VI. Reagent Detection
[0091] In addition to the components described above, in some
cases, the overall systems described herein may include additional
components integrated into the system, such components used to
detect the presence and amount of reagents present in any reagent
vessel component of the system, e.g., in a reservoir of a
microfluidic device, an amplification tube, or the like. A variety
of components may be used to detect the presence and/or amount of
reagents in any vessel, including, for example, optical detection
systems, that could include light transmission detectors that
measure whether light is altered in passing through a reservoir
based upon presence of a fluid, or machine vision systems that
image the reservoirs and determine whether there is fluid in the
reservoir and even the level of fluid therein. Such detection
systems would be placed in optical communication with the
reservoirs or other vessels of the system. In other cases,
electrical systems may be used that insert electrodes into a
reservoir and measure changes in current flow through those
electrodes based upon the presence or absence of fluid within the
reservoir or vessel.
VII. Additional Sensors/Monitoring
[0092] In addition to the sensing systems described above, a number
of additional sensing systems may also be integrated into the
overall systems described herein. For example, in some cases, the
instrument systems may incorporate bar-code reader systems in one
or more functional zones of the system. For example, in some cases,
a barcode reader may be provided adjacent a stage for receiving one
or more sample plates, in order to record the identity of the
sample plat and correlate it to sample information for that plate.
Likewise, barcode readers may be positioned adjacent a microfluidic
device stage in a partitioning zone, in order to record the type of
microfluidic device being placed on the stage, as reflected by a
particular barcode placed on the device. By barcoding and reading
the specific device, one could coordinate the specifics of an
instrument run that may be tailored for different device types. A
wide variety of barcode types and readers are generally used in
research instrumentation, including both one dimensional and two
dimensional barcode systems.
[0093] Other detection systems that are optionally integrated into
the systems described herein include sensors for the presence or
absence of consumable components, such as microfluidic devices,
sample plates, sample tubes, reagent tubes or the like. Typically,
these sensor systems may rely on one or more of optical detectors,
e.g., to sense the presence or absence of a physical component,
such as a plate, tube, secondary holder, microfluidic chip, gasket,
etc., or mechanical sensors, e.g., that are actuated by the
presence or absence of a plate, microfluidic device, secondary
holder, tube, gasket, etc. These sensor systems may be integrated
into one or more tube slots or wells, plate stages or microfluidic
device stages. In the event a particular component is missing, the
system may be programmed to provide an alert or notification as
well as optionally or additionally preventing the start of a system
run or unit operation.
II. Integrated Workflow Processes
[0094] The instrument systems described above may exist as
standalone instruments, or they may be directly integrated with
other systems or subsystems used in the particular workflow for the
application for which the partitioning systems are being used. As
used herein, integration of systems and subsystems denotes the
direct connection or joining of the systems and/their respective
processes into an integrated system or instrument architecture that
does not require user intervention in moving a processed sample or
material from a first subsystem to a second subsystem. Typically,
such integration denotes two subsystems that are linked into a
common architecture, and include functional interactions between
those subsystems, or another subsystem common to both. By way of
example, such interconnection includes exchange of fluid materials
from one subsystem to another, exchange of components, e.g.,
plates, tubes, wells, microfluidic devices, etc., between two
subsystems, and additionally, may include integrated control
components between subsystems, e.g., where subsystems are
controlled by a common processor, or share other common control
elements, e.g., environment control, fluid transport systems,
etc.
[0095] For ease of discussion, these integrated systems are
described with respect to the example of nucleic acid applications.
In this example, the partitioning instrument systems may be
integrated directly with one or more sample preparation systems or
subsystems that are to be used either or both of upstream and/or
downstream in the specific overall workflow. Such systems may
include, for example, upstream process systems or subsystems, such
as those used for nucleic acid extraction, nucleic acid
purification, and nucleic acid fragmentation, as well as downstream
processing systems, such as those used for nucleic acid
amplification, nucleic acid purification and nucleic acid
sequencing or other analyses.
[0096] For purposes of illustration, the integration of the
partitioning process components described above, with upstream
and/or downstream process workflow components is illustrated with
respect to a preferred exemplary nucleic acid sequencing workflow.
In particular, the partitioning systems described herein are
fluidly and/or mechanically integrated with other systems utilized
in a nucleic acid sequencing workflow, e.g., amplification systems,
nucleic acid purification systems, cell extraction systems, nucleic
acid sequencing systems, and the like.
[0097] FIG. 6 schematically illustrates an exemplary process
workflow for sequencing nucleic acids from sample materials and
assembling the obtained sequences into whole genome sequences,
contig sequences, or sequences of significantly large portions of
such genomes, e.g., fragments of 10 kb or greater, 20 kb or
greater, 50 kb or greater, or 100 kb or greater, exomes, or other
specific targeted portions of the genome(s).
[0098] As shown, a sample material, e.g., comprising a tissue or
cell sample, is first subjected to an extraction process 602 to
extract the genomic or other nucleic acids from the cells in the
sample. A variety of different extraction methods are commercially
available and may vary depending upon the type of sample from which
the nucleic acids are being extracted, the type of nucleic acids
being extracted, and the like. The extracted nucleic acids are then
subjected to a purification process 604, to remove extraneous and
potentially interfering sample components from the extract, e.g.,
cellular debris, proteins, etc. The purified nucleic acids may then
be subjected to a fragmentation step 606 in order to generate
fragments that are more manageable in the context of the
partitioning system, as well as optional size selection step, e.g.,
using a SPRI bead clean up and size selection process.
[0099] Following fragmentation, the sample nucleic acids may be
introduced into the partitioning system, which is used to generate
the sequenceable library of nucleic acid fragments. Within the
partitioning system larger sample DNA fragments are co-partitioned
at step 608, along with barcoded primer sequences, such that each
partition includes a particular set of primers representing a
single barcode sequence. Additional reagents may also be
co-partitioned along with the sample material, including, e.g.,
release reagents for releasing the primer/barcode oligonucleotides
from the beads, DNA polymerase enzyme, dNTPs, divalent metal ions,
e.g., Mg2+, Mn2+, and other reagents used in carrying out primer
extension reactions within the partitions. These released
primers/barcodes are then used to generate a set of barcoded
overlapping smaller fragments of the larger sample nucleic acid
fragments at amplification step 610, where the smaller fragments
include the barcode sequence, as well as one or more additional
sequencing primer sequences.
[0100] Following generation of the sequencing library, additional
process steps may be carried out prior to introducing the library
onto a sequencer system. For example, as shown, the barcoded
fragments may be taken out of their respective partitions, e.g., by
breaking the emulsion, and be subjected to a further amplification
process at step 612 where the sequenceable fragments are amplified
using, e.g., a PCR based process. Either within this process step
or as a separate process step, the amplified overlapping barcoded
fragments may have additional sequences appended to them, such as
reverse read sequencing primers, sample index sequences, e.g., that
provide an identifier for the particular sample from which the
sequencing library was created.
[0101] In addition, either after the amplification step (as shown)
or prior to the amplification step, the overlapping fragment set
may be size selected, e.g., at step 614, in order to provide
fragments that are within a size nucleotide sequence length range
that is sequenceable by the sequencing system being used. A final
purification step 616 may be optionally performed to yield a
sequenceable library devoid of extraneous reagents, e.g., enzymes,
primers, salts and other reagents, that might interfere with or
otherwise co-opt sequencing capacity of the sequencing system. The
sequencing library of overlapping barcoded fragments is then run on
a sequencing system at step 618 to obtain the sequence of the
various overlapping fragments and their associated barcode
sequences.
[0102] In accordance with the instant disclosure, it will be
appreciated that the steps represented by the partitioning system,
e.g., step 606, may be readily integrated into a unified system
with any one or more of any of steps 602-606 and 610-618. This
integration may include integration on the subsystem level, e.g.,
incorporation of adjacent processing systems within a unified
system architecture. Additionally or alternatively, one or more of
these integrated systems or components thereof, may be integrated
at the component level, e.g., within one or more individual
structural components of the partitioning subsystem, e.g., in an
integrated microfluidic partitioning component.
[0103] As used herein, integration may include a variety of types
of integration, including for example, fluidic integration,
mechanical integration, control integration, electronic or
computational integration, or any combination of these. In
particularly preferred aspects, the partitioning instrument systems
are fluidly and/or mechanically integrated with one or more
additional upstream and/or downstream processing subsystems.
[0104] A. Fluidic Integration
[0105] In the case of fluidic integration, it will be understood
that such integration will generally include fluid transfer
components for transferring fluid components to or from the inlets
and outlets, e.g., the reservoirs, of the fluidic component of the
partitioning system. These fluid transfer components may include
any of a variety of different fluid transfer systems, including,
for example, automated pipetting systems that access and pipette
fluids to or from reservoirs on the fluidic component to transfer
such fluids to or from reservoirs, tubes, wells or other vessels in
upstream or downstream subsystems. Such pipetting systems may
typically be provided in the context of appropriate robotics within
an overall system architecture, e.g., that move one or both of the
fluidics component and/or the pipetting system relative to each
other and relative to the originating or receiving reservoir, etc.
Alternatively, such systems may include fluidic conduits that move
fluids among the various subsystem components. Typically, hard
wired fluidic conduits are reserved for common reagents, buffers,
and the like, and not used for sample components, as they would be
subject to sample cross contamination.
[0106] In one example, a fluid transfer system is provided for
transferring one or more fluids into the reservoirs that are
connected to the channel network of the fluidics component. For
example, in some cases, fluids, such as partitioning oils, buffers,
reagents, e.g., barcode beads or other reagents for a particular
application, may be stored in discrete vessels, e.g., bottles,
flasks, tubes or the like, within the overall system. These storage
vessels would optionally be subject to environmental control
aspects as well, to preserve their efficacy, e.g., refrigeration,
low light or no light environments, etc.
[0107] Upon commencement of a system run, those reagent fluids
would be transported to the reservoirs of a fluidic component,
e.g., a microfluidic device, that was inserted into the overall
system. Again, reagent transport systems for achieving this may
include dispensing systems, e.g., with pipettors or dispensing
tubes positioned or positionable over the reservoirs of the
inserted device, and which are connected to the reagent storage
vessels and include pumping systems.
[0108] Likewise, fluid transport systems may also be included to
transfer the partitioned reagents from the outlet of the fluidic
component, e.g., reservoir 238 in FIG. 2, and transported to
separate locations within the overall system for subsequent
processing, e.g., amplification, purification etc.
[0109] In other cases, the partitions may be maintained within the
outlet reservoir of the fluidic component, which is then directly
subjected to the amplification process, e.g., through a thermal
controller placed into thermal contact with the outlet reservoir,
that can perform thermal cycling of the reservoir's contents. This
thermal controller may be a component of the mounting surface upon
which the microfluidic device is positioned, or it may be a
separate component that is brought into thermal communication with
the microfluidic device or the reservoir.
[0110] However, in some cases, fully integrated systems may be
employed, e.g., where the transfer conduits pass the reagents
through thermally cycled zones to effect amplification. Likewise,
alternative fluid transfer systems may rely upon the piercing of a
bottom surface of a reservoir on a given device to allow draining
of the partitions into a subsequent receptacle for
amplification.
[0111] B. Mechanical Integration
[0112] In cases of mechanical integration, it will be understood
that such integration will generally include automated or
automatable systems for physically moving system components, such
as sample plates, microfluidic devices, tubes, vials, containers,
or the like, from one subsystem to another subsystem. Typically,
these integrated systems will be contained within a single unified
structure, such as a single casing or housing, in order to control
the environments to which the various process steps, carried out by
the different system components, are exposed. In some cases,
different subsystem components of the overall system may be
segregated from other components, in order to provide different
environments for different unit operations performed within the
integrated system. In such cases, pass-throughs may be provided
with closures or other movable barriers to maintain environmental
control as between subsystem components.
[0113] Mechanical integration systems may include robotic systems
for moving sample containing components from one station to another
station within the integrated system. For example, robotic systems
may be employed within the integrated system to move lift and move
plates from one station in a first subsystem, to another station in
another subsystem.
[0114] Other mechanical integration systems may include conveyor
systems, rotor table systems, inversion systems, or other
translocation systems that move, e.g., a partitioning microfluidic
device, tubes, or multiwall plate or plates, from one station to
another station within the unified system architecture, e.g.,
moving a microfluidic device from its control station where
partitions are generated to a subsequent processing station, such
as an amplification station or fluid transfer station.
[0115] C. Examples of Integration
[0116] A number of more specific simple examples of integration of
the aforementioned process components are described below.
[0117] In some cases, the up front process steps of sample
extraction and purification may be integrated into the systems
described herein, allowing users to input tissue, cell, or other
unprocessed samples into the system in order to yield sequence data
for those samples. Such systems would typically employ integrated
systems for lysis of cell materials and purification of desired
materials from non-desired materials, e.g., using integrated filter
components, e.g., integrated into a sample vessel that could be
integrated onto a microfluidic device inlet reservoir following
extraction and purification. These systems again would be driven by
one or more of pressure or vacuum, or in some cases, by gravitation
al flow or through centrifugal driving, e.g., where sample vessels
are positioned onto a rotor to drive fluid movements.
[0118] In some cases, it may be desirable to have sample nucleic
acids size-selected, in order to better optimize an overall sample
preparation process. In particular, it may be desirable to have one
or more selected starting fragment size ranges for nucleic acid
fragments that are to be partitioned, fragmented and barcoded,
prior to subjecting these materials to sequencing. This is
particularly useful in the context of partition-based barcoding and
amplification where larger starting fragment sizes may be more
desirable. Examples of available size selection systems include,
e.g., the Blue Pippen.RTM. system, available from Sage Sciences
(See also U.S. Pat. No. 8,361,299), that relies upon size
separation through an electrophoretic gel system, to provide
relatively tightly defined fragment sizes.
[0119] In accordance with the present disclosure, systems may
include an integrated size selection system for generating nucleic
acid fragments of selected sizes. While in some cases, these size
selection components may be integrated through fluid transport
systems that transport fragments into the inlet reservoirs of the
fluidic components, e.g., pipetting systems, in certain cases, the
size selection system may be integrated within the fluidic
component itself, such that samples of varied fragment sizes may be
input into the device by the user, followed by an integrated size
separation process whereby selected fragment sizes may be allocated
into inlet reservoirs for the fluidic components of the device.
[0120] For example, and as shown in FIG. 7, a size selection
component 700 including a capillary or separation lane 702, is
integrated into a microfluidic device. An electrophoretic
controller is coupled to the separation lane via electrodes 704,
706 and 708 that apply a voltage differential across the separation
matrix in lane 702 in order to drive the size-based separation of
nucleic acid samples that are introduced into well 710. In
operation, a separation voltage differential is applied across the
separation lane by applying the voltage differential between sample
reservoir 710 and waste reservoir 712. At the point in the
separation at which the desired fragment size enters into junction
714, the voltage differential is applied between reservoir 710 and
elution reservoir 716, by actuation of switch 718. This switch of
the applied voltage differential then drives the desired fragment
size into the elution reservoir 716, which also doubles as the
sample inlet reservoir for the microfluidic device, e.g., reservoir
232 in FIG. 2. Once sufficient time has passed for direction of the
desired fragment into reservoir 716, the voltage may again be
switched as between reservoir 710 and waste reservoir 712.
[0121] Upon completion of the separation, fragments that have been
driven into the sample elution reservoir/sample inlet reservoir,
may then be introduced into their respective microfluidic
partitioning channel network, e.g., channel network 720, for
allocation into partitions for subsequent processing. As will be
appreciated, in cases where an electrophoretic separation component
is included within the system, e.g., whether integrated into the
microfluidic device component or separate from it, the systems
described herein will optionally include an electrophoretic
controller system that delivers appropriate voltage differentials
to the associated electrodes that are positioned in electrical
contact with the content of the relevant reservoirs. Such systems
will typically include current or voltage sources, along with
controllers for delivering desired voltages to specified electrodes
at desired times, as well as actuation of integrated switches.
These controller systems, either alone, or as a component of the
overall system controller, will typically include the appropriate
programming to apply voltages and activate switches to drive
electrophoresis of sample fragments in accordance with a desired
profile.
[0122] As will be appreciated, a single microfluidic device may
include multiple partitioning channel networks, and as such, may
also include multiple size separation components integrated therein
as well. These size separation components may drive a similar or
identical size separation process in each of the different
components, e.g., to provide the same or similar sized fragments to
each different partitioning channel network. Alternatively, the
different size separation components may drive a different size
selection, e.g., to provide different sized fragments to the
different partitioning networks. This may be achieved through the
inclusion of gel matrices having different porosity, e.g., to
affect different separation profiles, or it may be achieved by
providing different voltage profiles or switching profiles to the
electrophoretic drivers of the system.
[0123] As will be appreciated, for microfluidic devices that
include multiple parallel arranged partitioning channel networks,
multiple separation channels may be provided; each coupled at an
elution zone or reservoir that operates as or is coupled to a
different inlet reservoir for the partition generating fluidic
network. In operation, a plurality of different separation channel
components maybe provided integrated into a microfluidic device.
The separation channels again are mated with or include associated
electrodes for driving electrophoresis of nucleic acids or other
macromolecular sample components, through a gel matrix within the
separation channels. Each of the different separation channels may
be configured to provide the same or differing levels of
separation, e.g., resulting in larger or smaller eluted fragments
into the elution zone/inlet reservoir of each of the different
partitioning channel networks. In cases where the separation
channels provide different separation, each of the different
channel networks would be used to partition sample fragments of a
selected different size, with the resulting partitioned fragments
being recovered for each channel network in a different outlet or
recovery reservoir, respectively.
[0124] 3. Amplification
[0125] In some cases, the systems include integration of one or
more of the amplification process components, e.g., steps 610 and
612, into the overall instrument system. In particular, as will be
appreciated, this integration may be as simple as incorporating a
temperature control system within thermal communication with the
product reservoir on the fluidic component of the system, e.g.,
reservoir 238 in FIG. 2, such that the contents of the reservoir
may be thermally cycled to allow priming, extension, melting and
re-priming of the sample nucleic acids within the partitions by the
primer/barcode oligonucleotides in order to create the overlapping
primer sequences template off of the original sample fragment.
Again, such temperature control systems may include heating
elements thermally coupled to a portion of the fluidic component so
as to thermally cycle the contents of the outlet reservoir.
[0126] Alternatively, the integration of the amplification system
may provide for fluid transfer from the outlet reservoir of the
fluidic component to an amplification reservoir that is positioned
in thermal contact with the above described temperature control
system, e.g., in a temperature controlled thermal cycler block,
within the instrument, that is controlled to provide the desired
thermal cycling profile to the contents taken from the outlet
reservoir. As described above, this fluid transfer system may
include, e.g., a pipetting system for drawing the partitioned
components out of the outlet reservoir of the microfluidic device
and depositing them into a separate reservoir, e.g., in a well of a
multiwall plate, or the like. In another alternative configuration,
fluid transfer between the microfluidic device and the
amplification reservoir may be directed by gravity or pressure
driven flow that is actuated by piercing a lower barrier to the
outlet reservoir of the microfluidic device, allowing the generated
partitions to drain or flow into a separate reservoir below the
microfluidic device that is in thermal communication with a
temperature control system that operates to thermally cycle the
resultant partitions through desired amplification thermal
profiles.
[0127] In a particular example, and with reference to the nucleic
acid analysis workflow set forth above, the generated partitions
from step 608 may be removed from the fluidics component by an
integrated fluid transfer system, e.g., pipettors, that withdraw
the created partitions form, e.g., reservoir 238 of FIG. 2, and
transport those partitions to an integrated thermal cycling system
in order to conduct an amplification reaction on the materials
contained within those partitions. Typically, the reagents
necessary for this initial amplification reaction (shown at step
610, in FIG. 6), will be co-partitioned in the partitions. In many
cases, the integrated thermal cycling systems may comprise separate
reagent tubes disposed within thermal cycling blocks within the
instrument, in order to prevent sample to sample cross
contamination. In such cases, the fluid transport systems will
withdraw the partitioned materials from the outlet reservoir and
dispense them into the tubes associated with the amplification
system.
[0128] 4. Size Selection of Amplification Products
[0129] Following amplification and barcoding step 610, the
partitioned reagents are then pooled by breaking the emulsion, and
subjected to additional processing. Again, this may be handled
through integrated fluid transfer systems that may introduce
reagents into the wells or tubes in which the sample materials are
contained, or by transferring those components to other tubes in
which such additional reagents are located. In some cases,
mechanical components may also be included within the system to
assist in breaking emulsions, e.g., through vortexing of sample
vessels, plates, or the like. Such vortexing may again be provided
within a set station within the integrated system. In some cases,
this additional processing may include a size selection step in
order to provide sequenceable fragments of a desired length.
[0130] 5. Additional Processing and Sequencing
[0131] Following further amplification, it may be desirable to
include additional clean up steps to remove any unwanted proteins
or other materials that may interfere with a sequencing operation.
In such cases, solid phase DNA separation techniques are
particularly useful, including, the use of nucleic acid affinity
beads, such as SPRI beads, e.g., Ampure.RTM. beads available from
Beckman-Coulter, for purification of nucleic acids away from other
components in fluid mixtures. Again, as with any of the various
unit operations described herein, this step may be automated and
integrated within the overall integrated instrument system.
[0132] In addition to integration of the various upstream processes
of sequencing within an integrated system, in some cases, these
integrated systems may also include an integrated sequencer system.
In particular, in some cases, a single integrated system may
include one, two, three or more of the unit process subsystems
described above, integrated with a sequencing subsystem, whereby
prepared sequencing libraries may be automatically transferred to
the sequencing system for sequence analysis. In such cases,
following a final pre-sequencing process, the prepared sequencing
library may be transferred by an integrated fluid transfer system,
to the sample inlet of a sequencing flow cell or other sequencing
interface. The sequencing flow cell is then processed in the same
manner as non-integrated sequencing samples, but without user
intervention between library preparation and sequencing.
[0133] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. For example, all the
techniques and apparatus described above can be used in various
combinations. For example, particle delivery can be practiced with
array well sizing methods as described. All publications, patents,
patent applications, and/or other documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication,
patent, patent application, and/or other document were individually
and separately indicated to be incorporated by reference for all
purposes.
* * * * *