Patent Application
20220323963
Example embodiments relate generally to sample processing devices that prepare a biological sample (e.g., a biological sample suspended in a liquid matrix) for DNA sequencing, in which the biological sample may be prepared for DNA sequencing with minimal or no interaction by a user.
Commercial sample preparation devices currently have a rather large footprint, multiple independent hardware components, and depend on a high degree of user interaction. Additionally, such devices are not designed to work outside of a laboratory environment.
Certain embodiments disclosed herein provide a multi-module sample preparation device (e.g., cartridge or chip) that includes a sample inlet for receiving a liquid sample comprising DNA, a sample outlet (e.g., to be delivered to a DNA sequencer), a waste outlet, and a plurality of operatively connected modules. The multi-module sample preparation device includes a first lyophilized chamber module comprising a plurality of lyophilized PCR primers and a lyophilized PCR master mix including one or more deoxynucleoside triphosphates (dNTPs), one or more buffers, and/or one or more polymerases. The first lyophilized chamber module may include a first lyophilized chamber inlet operatively connected to the sample inlet, and a first lyophilized chamber outlet. The multi-module sample preparation device may also include a first mixing module comprising a first mixing module inlet operatively connected to the first lyophilized chamber outlet, and a first mixing module outlet. The multi-module sample preparation device may also include an amplification module, such as a PCR module comprising a serpentine microfluidic channel and a plurality of discrete heaters in operative communication with a plurality of predetermined zones of the serpentine microfluidic channel oriented to produce one or more amplified target DNA regions. The PCR module, for example, may include a PCR inlet operatively connected to the first mixing module outlet, and a PCR outlet. The multi-module sample preparation device may also include a purification module comprising an active region including a solid phase configured to bind and release the one or more amplified target DNA regions, in which the purification module includes a purification inlet in operative communication with the PCR outlet, and a purification outlet being operatively and selectively connected with a first pathway from the purification outlet to the waste outlet and a second pathway from the purification outlet to a purified stream outlet. The multi-module sample preparation device may also include a second lyophilized chamber module comprising a plurality of lyophilized adapter sequences for enabling sequencing of the amplified target DNA regions, in which the second lyophilized chamber module includes a second lyophilized chamber module inlet operatively connected to the purified stream outlet, and a second lyophilized chamber module outlet. The multi-module sample preparation device may also include a third lyophilized chamber module comprising a lyophilized sequencing loading buffer composition, in which the third lyophilized chamber module includes a third lyophilized chamber module inlet operatively connected to a source of a reconstitution fluid, and a third lyophilized chamber module outlet. The multi-module sample preparation device may also include a second mixing module comprising one or more second mixing pools, in which the second mixing module includes one or more second mixing module inlets operatively connected to the second lyophilized chamber module outlet and the third lyophilized chamber module outlet, and a second mixing module outlet connected to the sample outlet.
In another aspect, the invention provides a system optionally including a liquid sample collection apparatus including a collection apparatus outlet and a multi-module sample preparation device, such as those described and disclosed herein, in which the sample inlet of the multi-module sample preparation device is in operative communication with the collection apparatus outlet. The system may also comprise a DNA sequencer, in which the DNA sequencer is in operative communication with the sample outlet of the multi-module sample preparation device. In certain example embodiments, the system may include a sequencer interface module located between and in operative communication with the sample outlet of the multi-module sample preparation device and the DNA sequencer.
In yet another aspect, the present invention provides a method of preparing a sample for DNA sequencing, in which the method may include the following: (a) optionally collecting a liquid sample, (b) feeding a liquid sample (e.g., lysed sample) into a multi-module sample preparation device, such as those described and disclosed herein; (c) flowing the liquid sample through a first lyophilized chamber module and reconstituting the plurality of lyophilized PCR primers and the lyophilized PCR mastermix; (d) flowing the liquid sample from the first lyophilized chamber into the first mixing module forming a homogenous PCR-ready liquid sample; (e) flowing the PCR-ready liquid sample from the first mixing module into and through the PCR module, and performing an amplification process within the PCR module and forming an amplified liquid sample; (f) flowing the amplified liquid sample from the PCR module into and through the purification module forming a purified liquid sample; (g) flowing the purified liquid sample into and through the second lyophilized chamber module and reconstituting the plurality of lyophilized adapter sequences and allowing attachment of the adapter sequences to the one or more amplified target DNA regions forming a sequence-able DNA sample; (h) flowing a reconstitution fluid into and through the third lyophilized chamber module and reconstituting the lyophilized sequencing loading buffer composition forming a liquid sequencing buffer solution; and (i) flowing the sequence-able DNA liquid sample into the second mixing module and flowing the liquid sequencing buffer solution into the second mixing module, and mixing the sequence-able DNA liquid sample and the liquid sequencing buffer solution forming a sequencing-ready liquid sample.
Example embodiments will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments are shown. Indeed, the technology described herein may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout, and wherein:
Some example embodiments will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all example embodiments are shown. Indeed, the examples described and pictured herein should not be construed as being limited to the scope, applicability, or configuration of the present disclosure. Rather, these example embodiments are provided so that this disclosure will satisfy applicable legal requirements. As used in the specification, and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise. Like reference numerals refer to like elements throughout.
Example embodiments herein relate generally to sequencing preparation for a variety of liquid samples, such as environmental DNA (eDNA) from water samples as only one example. A liquid sample for preparation for DNA sequencing, such as from an auto-sampler that may perform lysis of the cells in the sample to provide fresh lysate for being prepared for DNA sequencing. In this regard, lysate for DNA sequencing may generally require the amplification, such as by polymerase chain reaction (PCR) techniques, purification of the amplified target DNA regions, and library preparation prior to injection into a DNA sequencer. In accordance with certain example embodiments, a multi-module sample preparation device (e.g., embodied as a cartridge or a chip including the modules) may contain a plurality of interconnected but independent modules to accomplish each of the steps necessary for automating sample preparation for DNA sequencing with little or no user interaction. As different steps and/or modules within the multi-module sample preparation device may require a variety of reagents for accomplishing a particular step, such reagents may be preloaded in the appropriate module. Accordingly, the multi-module sample preparation device may enable a “hands-off” sample preparation process, which effectively eliminates user error and enables in-field or on-site sample preparation for DNA sequencing. In this regard, the multi-module sample preparation device may be beneficially employed in both laboratory environments and point-of-sample acquisition (e.g., in a field setting and/or underwater environments, such as submersible autonomous underwater vehicle (AUV). Individual modules, as discussed in more detail below, may be fabricated on a chip, such as described and disclosed herein, and in the case of the purification module and lyophilization modules, either packed, functionalized, or filled with the appropriate reagents before shelf-stabilization and sealing.
In example embodiments, a liquid sample including one or more lysates may be delivered to the multi-module sample preparation device from, by way of example only, an upstream sample collector. The liquid sample will reconstitute lyophilized primers and polymerase chain reaction (PCR) master mix in a first module, such as a first lyophilized chamber module, and then mix in a second module, such as a first mixing module. After reconstitution and mixing of the lyophilized primers and PCR master mix within the liquid sample, the resulting solution will then flow into an amplification module, such as a PCR module. In certain example embodiments, the PCR module may perform a fixed number of PCR cycles by passing the liquid sample through different temperature zones generated by heaters adhered (or alternatively operatively connected) to the multi-module sample preparation device, or alternatively a common “bus” or jig that this multi-module sample preparation device sits into and interfaces therewith. By way of example, the bus may include integrated heaters and allow for all of the necessary fluidic connections. After amplification, the now amplified liquid sample will flow into the purification module that removes at least a portion of undesired constituents (e.g., residual primer sequences, dNTPs, and salts). The purification module, for example, may perform a cleanup and purification of the amplified liquid sample using solid phase extraction (SPE) techniques. A waste outlet, in accordance with certain example embodiments, may be incorporated into the end of this module and allow for removal of excess PCR product and the reagents required for SPE. Following rinsing and prior to elution, for example, the exit port (e.g., waste outlet) from this module may be switched to connect to an inlet of the subsequent module, such as by using a simple valve. The purified liquid sample may then be passed to a second lyophilized chamber module filled with lyophilized sequencing adapters, for example, a rapid adapter from Oxford Nanpore Technologies, for sequencing preparation. While the purified liquid sample is incubating in a delay circuit of the second lyophilized chamber module, such as for about 5 minutes (which may be altered depending the desired incubation time), a third lyophilized chamber module including, for example, a lyophilized sequencing loading buffer and optionally loading solution may be reconstituted. Accordingly, two separate reagent pools (e.g., one in the second lyophilized chamber module and a second in the third lyophilized chamber module) may be mixed in a second mixing module to form a purified and sequencer-ready liquid sample, which may be transported off the multi-module sample preparation device (e.g., sample preparation chip) to a sequencer interface, such as multi-port rotary valve, for injection into a DNA sequencer.
As referenced above, the multi-module sample preparation device may be embodied as a single-use cartridge or chip housing the plurality of modules utilized in the sample preparation for DNA sequencing. For example, the multi-module sample preparation device may comprise a micro-fluidic chip in which each of the modules (e.g., micro-channels and/or chambers) may, by way of example only, have been 3D printed, or hot embossed, or etched and/or molded into a material (glass, silicon, or a plastic such as polydimethylsiloxane—PDMS or polymethyl methacrylate—PMMA, a polycarbonate, or pre-fluorinated polymers). In this regard, the micro-channels and/or chambers forming each of the modules of the micro-fluidic chip are connected together in order to achieve the desired features (e.g., reconstitution of lyophilized reagents, mixing, controlling residence time for a given portion of a given module, etc.). The micro-channels and/or chambers forming each of the modules of the micro-fluidic chip may have different inner diameters, for example, ranging from 5 to 2500 microns, such as at least about any of the following: 5, 10, 25, 50, 80, 100, 120, 150, 180, and 200 microns, and/or at most about any of the following: 2500, 2000, 1500, 1000, 800, 600, 500, 450, 400, 350, 300, 250, and 200 microns. The network of micro-channels and/or chambers formed into the micro-fluidic chip may be connected to the outside by inputs and outputs pierced through the chip or through the use of a flangeless connector interfaced to the network of micro-channels and/or chambers via one or more tapped ports or by the use of face compression and adhesive seals. It is through these holes that the liquids (or gases) may be injected and removed from the micro-fluidic chip (e.g., through tubing, syringe adapters or even simple holes in the chip) with external active systems (e.g., pressure controller, syringe-pump or peristaltic pump) or passive ways (e.g. hydrostatic pressure).
As illustrated in
In certain example embodiments, the first step in sample preparation requires combining the PCR primers and master mix with the lysate of the liquid sample. In this regard, the first lyophilized chamber module 120 includes a preloaded and lyophilized mixture of the PCR primers and master mix. For example, the PCR primers and master mix have been lyophilized and stored in the first lyophilized chamber module 120 prior to use. The liquid sample, for example, may enter into the first lyophilized chamber module 120 and reconstitute the lyophilized mixture of the PCR primers and master mix. In certain example embodiments, the first lyophilized chamber module 120 may comprise a capillary bed configuration that exerts a capillary force on the liquid sample that is greater than gravitational forces acting on the liquid sample. For example, the capillary bed configuration may include a plurality of posts or columns that may extend at least a portion (or completely) of the distance of the bed depth, in which the plurality of posts or columns may be provided in a high density. In this regard, the bed depth and the distance between the posts or columns in both the X and Y directions impact the filling properties and strength of the capillary force. In accordance with certain example embodiments, the capillary bed configuration may be formed with at least the following three (3) design criteria: (1) hold the correct volume; (2) maximize capillary force; and (3) fit within the footprint required for integration with an optional liquid sample collection apparatus. For example, the shape, number, and distance between individual posts or columns may be chosen to direct the flow path (e.g., distance between posts in one direction may be smaller than the distance between posts in the other direction to create choke-points) and increase capillary force (e.g., higher density, greater force), while still holding the necessary volume. For example, the capillary bed design of the first lyophilized chamber module 120 may have a large surface area due at least in part to the incorporation of a high density of the posts or columns, which may be tailored to encourage even and repeatable filling under conditions that may be seen in the field (e.g. rocking, jostling, etc.) as well as aid in cake formation during the lyophilization process. The large surface area along with the plurality of posts or columns encourages even and repeatable filing. For example, reliable filling in the field may be realized when the structure of the first lyophilized chamber module 120 exerts a higher capillary force on the filling fluid (e.g., liquid sample being prepared before subsequent DNA sequencing) than the other forces acting on the fluid including the gravitational force.
In certain example embodiments, the first lyophilized chamber module 120 comprises a microfluidic chamber having an average depth from about 5 to about 2500 microns, such as at least about any of the following: 5, 10, 25, 50, 80, 100, 120, 150, 180, and 200 microns, and/or at most about any of the following: 2500, 2000, 1500, 1000, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, and 200 microns.
As noted above, a first mixing module 130 is incorporated between the first lyophilized chamber module 120 and PCR module 140 to ensure even distribution of reagents to optimize performance in the PCR module 140. In accordance with certain example embodiments and as illustrated in
In certain example embodiments, the one or more first mixing pools 132 may have an average depth from about 50 to about 600 microns, such as at least about any of the following: 50, 100, 150, 200, 220, 250, 280, 300, 320, and 350 microns, and/or at most about any of the following: 600, 580, 550, 520, 500, 480, 460, 440, 420, 400, 380, and 350 microns.
After exiting the first mixing module 130, the liquid sample (now including the reconstituted primers and PCR master mix) enters the PCR module 140 for amplification. In accordance with certain example embodiments, the PCR module 140 may utilize a micro-fluidic PCR approach where temperature cycling can be achieved by continuously flowing the reaction mixture (i.e., the liquid sample including the reconstituted primers and PCR master mix) through different temperature zones in the serpentine path. The temperatures necessary to cycle through denaturation, annealing, and extension steps may be controlled via integrated heaters (e.g., strip heaters along with thermocouples or other temperature measurement devices that provide feedback of localized temperatures), and heating/cooling rates may be controlled by varying the cross-sectional area of the micro-fluidic channels, thickness of the base substrate, and/or reaction mixture flow rate. A software package may monitor and control multiple separate temperature zones. For example, software may monitor and control three separate temperature zones using the integrated heaters to provide zones of denaturing, annealing and extension steps. Modification of temperature set-points for separate temperature zones (e.g., three temperature zones for denaturing, annealing, and/or extension) alone or in combination with modifications to the geometry of the serpentine path allow this device to be optimized for a variety of PCR reactions. Such modifications, as briefly noted above, may include temperature changes, ramp rates, reaction times, and liquid volumes. In certain example embodiments, the surface of the PCR module 140 may be, for example, functionalized or otherwise treated to reduce non-specific protein binding and/or modify the hydrophobicity of the substrate. In this regard, for example, certain example embodiments include one or more (e.g., all) of the modules functionalized as noted above, for example, to prevent bio-fouling of the micro-fluidic chip surface. Additionally or alternatively, treatment of the PCR module 140 to reduce non-specific protein binding may include, for example, surface polishing to smooth the serpentine channel.
In certain example embodiments, the PCR module 140 may utilize ramp rates from hot to cold and vice versa that may be identical. In certain example embodiments, the PCR module 140 may utilize a gradual heating and rapid cooling of the liquid sample during continuous flow through the serpentine micro-fluidic channel. By way of example only, the liquid sample may be steadily heated through the serpentine micro-fluidic channel at a constant rate between about 1 and 4° C./s for sample extension and denaturing then rapidly cooled at a rate above 10°/s. In certain other example embodiments, the heating ramp rates may comprise about 1.75° C./s. These example heating and cooling rates can be controlled in a small form factor by, for example, varying the cross-sectional area of the serpentine micro-fluidic channel and/or the flow rate, and thus localized flow velocity, through an isothermal gradient. Such temperature rates, by way of example only, may be realized by resistive microstrip heaters held at 95° C. and 60° C. As noted above, the plurality of discrete heaters may be in operative communication, such a via a common bus as noted above, with a plurality of predetermined zones of the serpentine micro-fluidic channel and define denaturing zones, annealing zones, and extension zones along a length of the serpentine microfluidic channel. In this regard, the PCR module 140 may perform multiple cycles (e.g., 10-50 cycles) prior to the liquid sample leaving the PCR module 140. In accordance with certain example embodiments, the PCR module may also include an initial denature (e.g., 5 minutes) and final extension (e.g., 5 minutes) times that act to increase the efficiency of the reaction. By way of example only, the initial denature may be performed in the horizontal channel at the bottom of the PCR module 140 and final extend may be the pattern of smaller channels on the left-hand side of the PCR module 140.
In certain example embodiments, the serpentine micro-fluidic channel of PCR module 140, may comprise a uniform cross-section along a total length of the serpentine micro-fluidic channel. Alternatively, the serpentine micro-fluidic channel may comprise a variable cross-section along a total length of the serpentine micro-fluidic channel, including a first cross-section at a first location and a second cross-section at a second location in which the first cross-section is larger than the second cross-section. Alternatively, the serpentine micro-fluidic channel may have a constant cross-section. For example only, the serpentine micro-fluidic channel may have a constant width of about 250 microns (e.g., +/−about 5, 10, 15, 20, 25, 30, 40, or 50 microns), and a constant depth of about 300 microns (e.g., +/−about 5, 10, 15, 20, 25, 30, 40, or 50 microns). In accordance with certain example embodiments, the depth of the serpentine micro-fluidic channel may be about 10% to about 40% larger than the width of the serpentine micro-fluidic channel, such as at least about any of the following: 10, 15, 20, and 25% larger, and/or at most about any of the following: 40, 35, 30, and 25% larger.
In certain example embodiments, the serpentine micro-fluidic channel may have an average depth from about 200 to about 750 microns, such as at least about any of the following: 200, 220, 250, 280, 300, 320, and 350 microns, and/or at most about any of the following: 750, 700, 650, 600, 580, 550, 520, 500, 480, 460, 440, 420, 400, 380, and 350 microns.
Subsequent to amplification in the PCR module 140, the now amplified liquid sample enters the purification module 150. In this regard, the purification module 150 may extract and purify the amplified DNA present in the liquid sample. As noted above, the purification module 150 may include an active region followed by a valve (or similar control mechanism) that switches flow between a waste outlet and downstream modules in the multi-module sample preparation device (e.g., micro-fluidic chip). The active region, for example, may comprise of either a packed bead bed or periodic array of surface functionalized structures (e.g., pillars, beads, lengthwise columns, etc.) that will specifically bind to and release the amplified DNA regions upon exposure to various reagents and/or stimuli (e.g., temperatures, pH, light, etc.). Similar methods may be employed by which the amplified DNA is captured and purified with a series of wash and elution steps. One example procedure may involve washing the amplified DNA received from the PCR module 140 over a functionalized surface, rinsing the purification module with a wash buffer, and then eluting the amplified DNA to the downstream modules. For instance, one example procedure may involve washing the amplified DNA received from the PCR module 140 over a chitosan functionalized PMMA surface with an acidic solution (i.e. pH <6), rinsing the purification module 150 with an acidic wash buffer to waste, and then eluting the amplified DNA with a basic solution (e.g., pH >8) to the downstream modules. The waste outlet, for example, may shunt excess reagents to waste and then actively switch fluid paths using a pinch valve or similar on the waste outlet. In certain example embodiments, therefore, the purification module 150 may comprise one or more mobile phase inlets in operative communication with the active region. The purification module 150 also comprises a pinch valve or a diverter valve comprising a first orientation that defines the first pathway from the purification outlet to the waste outlet and a second orientation that defines a second pathway from the purification outlet to a purified stream outlet that leads to downstream modules.
As noted above, the solid phase of the purification module 150 may comprise a packing media having a functionalized surface configured to bind and release the one or more amplified target DNA regions. The packing media can include but is not limited to commercial beads optimized for DNA clean up (e.g., AMPure XP beads), standard common materials (e.g., silica beads), or functionalized substrates (e.g., PMMA functionalized with chitosan, hydroxypropylmethyl-cellulose, or poly(vinyl alcohol)).
Subsequent to the purification module 150, the purified liquid sample having the amplified DNA may be reacted with adapter sequences for enabling DNA sequencing of the amplified DNA regions. Accordingly, the next steps in sample processing may include reconstitution of lyophilized sequencing reagents to modify the purified sequencing reagents for sequencing. The adapter sequences and buffer components required for sequencing, such as by a MinION sequencer, may be less stable in liquid form for prolonged periods. As such, these adapter sequences and buffer components may be lyophilized in micro-fluidic lyophilized chamber modules modeled after capillary pumps and as described above. As noted above when discussing the first lyophilized chamber module, these designs enable the liquid reagents to be readily dispersed over a large surface area and dried down in a lyophilization chamber to form a lyophilized chamber. The large surface area also aids in quick reconstitution of reagents; however, such a design if too deep, is highly susceptible to inconsistent fluid flow and bubble entrapment when not operated on a level plane. Therefore, the lyophilized chambers may have a much shallower depth than the other modules, and features may be spaced-out such that fluid follows a general path in the lyophilized chambers, while still allowing for mixing therein. For example, the characteristic dimension (i.e., depth for the lyophilized chambers) has the greatest effect on capillary force and may in certain example embodiments be minimized to ensure a reliable filling pattern.
In certain example embodiments, the reagents in the lyophilized chambers modules (i.e., the second lyophilized chamber module 160 and the third lyophilized chamber module 170) can be reconstituted in parallel. For instance, reagents in the third lyophilization chamber module 170 may be reconstituted with a reconstituting fluid (e.g., water) while reagents in the second lyophilized chamber module 160 may be reconstituted with the purified DNA solution and then incubated in a long channel 162 (e.g., a delay circuit) as shown in
As noted above, the purified liquid sample leaving the purification module 150 may be directed to or passed into the second lyophilized chamber module 160. Similar to the first lyophilized chamber module 120, the second lyophilized chamber module 160 may comprise a capillary bed configuration that exerts a capillary force on the liquid sample that is greater than gravitational forces acting on the liquid sample. For example, the capillary bed configuration may include a plurality of posts or columns that may extend at least a portion (or completely) of the distance of the bed depth, in which the plurality of posts or columns may be provided in a high density. In this regard, the bed depth and the distance between the posts or columns in both the X and Y directions impact the filling properties and strength of the capillary force. In accordance with certain example embodiments, the capillary bed configuration may be formed with at least the following three (3) design criteria: (1) hold the correct volume; (2) maximize capillary force; and (3) fit within the footprint required for integration with an optional liquid sample collection apparatus. For example, the shape, number, and distance between individual posts or columns may be chosen to direct the flow path (e.g., distance between posts in one direction may be smaller than the distance between posts in the other direction to create choke-points) and increase capillary force (e.g., higher density, greater force), while still holding the necessary volume. For example, the capillary bed design of the first lyophilized chamber module 120 may have a large surface area due at least in part to the incorporation of a high density of the posts or columns, which may be tailored to encourage even and repeatable filling under conditions that may be seen in the field (e.g. rocking, jostling, etc.) as well as aid in cake formation during the lyophilization process. The large surface area along with the plurality of posts or columns encourages even and repeatable filing. For example, reliable filling in the field may be realized when the structure of the first lyophilized chamber module 120 exerts a higher capillary force on the filling fluid (e.g., liquid sample being prepared before subsequent DNA sequencing) than the other forces acting on the fluid including the gravitational force.
In certain example embodiments, the second lyophilized chamber module 160 comprises a microfluidic chamber having an average depth from about 5 to 2500 microns, such as at least about any of the following: 5, 10, 25, 50, 80, 100, 120, 150, 180, and 200 microns, and/or at most about any of the following: 2500, 2000, 1500, 1000, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, and 200 microns.
In certain example embodiments and as shown in
As noted above and as illustrated in
In certain example embodiments, the one or more second mixing pools 180 may have an average depth from about 50 to about 600 microns, such as at least about any of the following: 50, 100, 150, 200, 220, 250, 280, 300, 320, and 350 microns, and/or at most about any of the following: 600, 580, 550, 520, 500, 480, 460, 440, 420, 400, 380, and 350 microns.
In accordance with certain example embodiments, the multi-module sample preparation device 100 may further comprise and/or be operatively connected to a sequencer interface module, such as via the sample outlet 105. The sequencer interface module, for instance, may be operatively connected to the sample outlet 105 of the multi-module sample preparation device 100 and a DNA sequencer. Although the DNA sequencer may not be particularly limited,
The sequencer interface module 300, as illustrated by
In certain example embodiments, the priming buffer inlet 310, the sequencing-ready liquid sample inlet 320, and the waste outlet 330 are each operatively connected to a multi-port rotary valve 340 (best shown in
In certain example embodiments and as illustrated in
In accordance with certain example embodiments, one or more of the lyophilized chambers may instead include a blister system instead of lyophilized reagents. For example, the blister system may comprise self-contained fluid reservoirs that can be punctured or otherwise broken open to release their contents. By way of example only, the first lyophilized chamber may include a first plurality of blisters including the reagents associated with the first lyophilized chamber, the second lyophilized chamber may include a second plurality of blisters including the reagents associated with the second lyophilized chamber, and/or the third lyophilized chamber may include a third plurality of blisters including the reagents associated with the third lyophilized chamber. Blister systems, in accordance with certain example embodiments, may also be used for the reconstitution fluid and/or for the purification reagents.
In accordance with certain example embodiments, the multi-module sample preparation device (e.g., micro-fluidic chip) may include an integrated bubble trap. For example, the bubble trap may be incorporated into the microfluidic manifold. Bubble traps, for example, may comprise microporous membranes that will allow air to pass out of the device, but not fluids. By way of example only, the microporous membrane may comprise polytetrafluoroethylene (PTFE). The bubble trap, for example, may be integrated into the manifold of the sequencer interface as the last step before feeding the sample into the DNA sequencer. In certain example embodiments, the bubble trap may be located between the end of the injection loop 345b and injection port 315. In this regard, the bubble trap may protect the DNA sequencer from air bubbles hat may damage, for example, a nanopore array in the DNA sequencer.
In another aspect and as illustrated by
In yet another aspect and as illustrated by
These and other modifications and variations to embodiments may be practiced by those of ordinary skill in the art without departing from the spirit and scope, which is more particularly set forth in the appended claims. In addition, it should be understood that aspects of the various embodiments may be interchanged in whole or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and it is not intended to limit the invention as further described in such appended claims. Therefore, the spirit and scope of the appended claims should not be limited to the exemplary description of the versions contained herein.
This application claims the benefit of U.S. Provisional Application No. 63/172,842 filed on Apr. 9, 2021, the entire contents of which are hereby incorporated herein by reference
| Number | Date | Country | |
|---|---|---|---|
| 63172842 | Apr 2021 | US |
