Patent Application
20230136755
The invention relates generally to biological sample preparation and analysis, and, more particularly, to devices for improving protocol compliance, reproducibility, and sensitivity in single-cell sequencing assays.
When performing any type of assay, it is important that the associated protocol and workflow be carried out with precision and in a timely manner. This is particularly critical when performing diagnostic assays. Rapid results with high sensitivity and specificity are key to a positive health outcome. In contrast, an inaccurate and/or late diagnosis can lead to a misdiagnosis and/or diagnosis only after late-stage disease has developed.
Transcriptional analysis of single cells by RNA sequencing is increasingly recognized as the gold standard for understanding complex cellular populations. Single-cell RNA sequencing can provide gene expression profiles of single cells and uncover heterogeneity hidden within a sample of different cellular phenotypes. As such, methods of single-cell RNA sequencing are incorporated into clinical practice to define complex pathologies, e.g., tumors, and characterize their pathogenesis for patient diagnosis and treatment.
For clinics using RNA sequencing, accurate and timely identification of differentially expressed genes is critical for informing patient health status and treatment monitoring. Although single-cell RNA sequencing has the potential to provide those services, the complexity of the workflows, high costs of specialized devices, and lack of highly skilled technicians are barriers to its widespread use outside of state-of-the-art laboratories.
The typical single-cell RNA sequencing workflow entails sample collection, preparation steps, nucleic acid extraction, cDNA library preparation, PCR amplification, sequencing, and data analysis. In particular, it is critical that certain procedures for preparing and processing a sample are executed in a precise, consistent, repeatable, and accurate manner. Without specialized devices and/or skilled workers, parts of the workflow are inefficient, overly laborious, and error prone. In particular, certain aspects of single-cell RNA sequencing require some form of manual processing, particularly with respect to sample collection and processing. However, manual sample preparation processes may increase the likelihood that a sample will become contaminated and/or improperly processed, thereby leading to an inaccurate result. Given that single-cell RNA sequencing is relatively expensive, and that the results can have a profound impact on patients’ lives, extreme caution must be exercised to avoid re-runs and delays, which can be too high a price to pay when patients are waiting for tailored treatments.
The present invention provides devices for improving assay protocol compliance, reproducibility, and sensitivity. In particular, devices of the present invention are tailored to certain single-cell sequencing assays, including single-cell RNA sequencing (scRNA-Seq) assays that utilize pre-templated instant partitions (PIPs). In such assays, PIPs templates are used to simultaneously segregate complex cell mixtures into partitions with barcoded template particles that can be easily processed for single cell applications, such as single-cell RNA sequencing. The devices of the present invention generally improve the manner with which a medical technologist (or other lab personnel) can carry out the various sample preparation steps involved in scRNA-Seq using PIPs template.
For example, performing a scRNA-Seq assay utilizing PIPs templates requires careful sample handling and manipulation of multiphase samples, which can be challenging for standard hand-held liquid handling pipettors. Devices of the present invention improve the reliability of liquid handling, which ultimately results in improved protocol compliance, improved assay performance, and improved assay reproducibility.
The typical workflow begins with sample preparation, in which a single cell suspension of interest is mixed with PIPs. The PIPs partition the mixture and isolate single cells inside compartments for conducting individual, parallel processes. The PIPs include template particles, which are generally hydrogel particles that function as templates, causing water-in-oil emulsion droplets to form when mixed inside a mixture of aqueous solution with oil and vortexed or sheared. The partitioning step involves vortexing, which is preferred for its ability to reliably generate partitions of a uniform size distribution. However, the PIPs templates have a tendency to settle and clump at the bottom of sample tubes. It has been found that standard vertical vortexing is inefficient and unreliable at disaggregating clumped hydrogel particles, and may ultimately result in non-uniform vortexing conditions required for reproducible PIPs partitioning.
Recognizing the limitations of vertical vortexing, the present invention provides a vortex adapter configured to mount to a standard benchtop vortex unit and to retain sample tubes in either a horizontal position or a vertical position, thereby allowing for horizontal or vertical vortexing as desired. In particular, the vortex adapter comprises a base including a proximal end releasably couplable to a hub of a standard vortex unit and a distal end to which a tube holder is mounted. The tube holder includes apertures or slots into which individual tubes are placed and retained via a friction fit, for example. The tube holder may generally be releasably mounted to the distal end of the base in either a vertical position (i.e., the cap of each tube is facing upwards and away from the vortex unit) or a horizontal position (i.e., the tubes are positioned on their sides relative to the vortex unit). By providing a vortex adapter that allows for a selectable position (i.e., vertical or horizontal position), a technician can perform both horizontal and vertical vortexing as needed and realize the benefits of each. In particular, horizontal vortexing generates more chaotic mixing and is effective at disaggregating settled PIPs templates while the improved vertical vortexing resulting from the invention is useful for providing uniform particle templated emulsification.
In some assays, magnetic nanoparticles may be used in partitioning and isolating target cells (via an associated binding element). Accordingly, when exposed to a sample, the binding elements bind with their partners (i.e., target analyte) and the emulsion forms partitions (e.g., droplets) that sequester the analyte. The partitions may then be manipulated using a magnetic field applied to the vessel containing the emulsion. However, magnetic particle-loaded PIPs templates are weakly magnetic, and, as a result, are unable to be efficiently collected via standard magnetic separators. Accordingly, the present invention provides a magnetic collector device that enables at least a two-stage magnetic separation by providing at least two orientations of a magnetic field relative to sample tubes. In particular, the magnetic collector device comprises a platform including multiple slots or apertures for holding sample tubes within and arranged in a row. The device further includes a magnet assembly comprising a set of magnets provided on a moveable guide and arranged in a row and aligned with respective apertures of the platform. The moveable guide is configured to transition between a bottom-most position and a top-most position relative to the aperture on the platform. When in the bottom-most position, each magnet is positioned adjacent to a bottom portion of a respective tube held within a respective slot. When in the top-most position, each magnet is positioned adjacent to a side portion of a respective tube held within a respective slot. In the bottom-most position, magnetic particles are attracted to the bottom of the sample tube, while in the top-most position, magnetic particles are attracted to a side of the sample tube. By providing at least two different orientations of a magnetic field, the magnetic collector device improves the ability with which magnetic particles can be collected from the sample tubes.
After agitation of the sample with the PIPs templates (e.g., vortexing), a plurality (e.g., thousands, tens of thousands, hundreds of thousands, one million, two million, ten million, or more) of aqueous partitions is formed simultaneously inside the tube. Vortexing causes the fluids to partition into a plurality of monodisperse droplets. A substantial portion of droplets will contain a single template particle and a single cell. Droplets containing more than one or none of a template particle or target cell can be removed, destroyed, or otherwise ignored.
The next steps in the workflow involves lysing the single cells and capturing released mRNA inside the partitions. First-strand cDNA is then generated via reverse transcription (RT) and amplified to create a cDNA library for each individual cell. These are then processed into sequencing libraries using standard library preparation methods and subsequently sequenced and analyzed. Such processes generally involve, and require, careful sample handling and manipulation of the multiphase samples, including washing and fluid handling. Recognizing the difficulties technicians face with standard hand-held liquid handling pipettors, the present invention provides a sample tube holder, generally in the form of a guide rack, that incorporates certain visual aids to enable precise removal of excess volume in washing and fluid handling steps. In particular, the guide rack includes a plurality of slots or apertures configured to receive and releasably retain sample tubes therein, and further includes a visual guide rod positioned relative to each slot or aperture. The visual guide rod may be at a fixed position that corresponds to a particular volume at which excess fluid can be removed from a given tube. Accordingly, the guide rack improves the ease with which a technician can work with the sample tubes, particularly during washing and fluid handling steps.
It is noted that certain steps (including washing steps) may further require the use of a centrifuge. For example, centrifuging a sample tube prior to performing PCR ensures that all reactants are in the bottom of the tube for proper concentrations and improved yields. A common issue encountered with a standard benchtop centrifuge is that sample tubes sit at a fixed and angled position, which results in formation of a slanted pellet within the tube, which is not optimal. Some centrifuges have been developed that are configured to receive microwell plates (i.e., typically 96-, 384-, or 1536-well plates) and swing the plates into a vertical position (whereas, the wells comprising the plates are oriented horizontally) during operation, thereby resulting in concentrating the resulting pellet into the well bottoms. However, such centrifuges (also referred to as microplate microcentrifuges) are limited to receiving microwell plates and are unable to accept individual sample tubes.
The present invention provides a centrifuge adapter allowing for standard benchtop microwell plate centrifuges to accommodate individual sample tubes and/or strip tube configurations. The adapter comprises a base portion shaped and/or sized to fit within a microwell plate holder of a microcentrifuge. The base portion is configured to receive and releasably retain sample tube holders thereto. In particular, the base portion includes multiple recesses shaped and/or sized to receive and retain portions of sample tube holders thereto. For example, in one embodiment, the base portion may include a recess defined on each end thereof in which portions of respective sample tube holders may be received and retained. A given sample tube holder includes a frame including apertures for receiving sample tubes within (i.e., 0.5 mL, 1.5 mL, 2.0 mL Eppendorf tubes). The frame may further include one or more gradation lines or other visual indicia adjacent the apertures for providing a technician with a visual indication of certain tube fill volumes (when a sample tube is placed within). In some embodiments, each sample tube holder may be configured to releasably attach to another sample tube holder via magnets or other connecting means that are integrated into sides of the sample tube holder, to thereby arrange the sample tubes in a row. Once loaded into a microwell plate holder, the base of the centrifuge adapter retains sample tube holders in a horizontal position such that individual tubes are also positioned in a horizontal orientation, in which the bottom of the tubes are facing an outward direction and the tops are facing an inward direction. Accordingly, operation of the centrifuge will result in a substantially level, and more concentrated, pellet to form in the bottom of the tubes. Accordingly, the centrifuge adapter is able to adapt various sample tube formats to a common microplate microcentrifuge, which was previously limited to only accepting microwell plates.
Accordingly, devices of the present invention improve upon protocol compliance, reproducibility, and sensitivity, particularly in single-cell sequencing assays. Such devices are tailored to single-cell RNA sequencing assays that utilize PIPs and will ultimately improve the manner with which a medical technologist (or other lab personnel) carry out the various sample preparation steps involved in single-cell RNA sequencing using PIPs templates, thereby reducing overall time required in performing a given assay and further reducing the risk of inaccurate diagnoses.
One aspect of the present invention includes a vortex adapter configured for use with a vortex mixer. The vortex adapter includes: a base comprising a proximal portion releasably couplable to a hub of a vortex mixer; and a tube holder releasably mounted to a distal portion of the base and comprising a plurality of apertures for receiving a plurality of tubes, respectively, wherein said tube holder is movable between a first position in which each of said apertures is oriented in a vertical direction relative to the base and a second position in which each of said apertures is oriented in a horizontal direction relative to the base.
Each of the plurality of apertures is shaped and/or sized to retain a respective tube therein and configured to subject any tubes within to vortex forces from a vortex mixer. The apertures may be shaped and/or sized to receive and retain a tube comprising of volume of between 0.1 mL and 5 mL. When the tube holder is in the first position, each of the apertures retains respective tubes received therein in a vertical direction. In such a configuration, a longitudinal axis of each tube is orthogonal relative to a surface upon which a vortex mixer is placed. For example, in such a position, tubes received and retained within respective apertures may be subjected to vertical vortexing upon receipt of vortex forces from the vortex mixer. When the tube holder is in the second position, each of the apertures retains respective tubes received therein in a horizontal direction. In such a configuration, a longitudinal axis of each tube is parallel relative to a surface upon which a vortex mixer is placed. For example, in such a position, tubes received and retained within respective apertures are subjected to horizontal vortexing upon receipt of vortex forces from the vortex mixer.
In some embodiments, the tube holder comprises at least a first set of apertures arranged in a row. For example, the tube holder may include a single set of apertures arranged in a row and provided on one side of the tube holder. In some embodiments, the tube holder comprises two sets of apertures, each set arranged in a separate room and provided on opposing side of the tube holder.
The tube holder may further include one or more counterweights provided on an opposing side of the tube holder for balancing inertial forces upon receipt of vortex forces from the vortex mixer.
The vortex adapter may further include a connection member provided at the distal portion of the base and selectively moveable between an engaged position and a disengaged position. When in an engaged position, the connection member maintains the tube holder in one of the first and second positions and, when in a disengaged position, the tube holder is moveable between the first and second positions. The distal portion of the base may include a channel defined between two knuckle members. The connection member may include an adjustable bolt assembly extending between the knuckle members and configured to draw the knuckle members together upon movement of the bolt assembly to an engaged position. The tube holder may include a body portion sized to fit within the channel in either of the first and second positions such that, when in an engaged position, the connection member causes the knuckle members to apply a retention force upon the body portion of the tube holder and prevent movement thereof within the channel.
Another aspect of the present invention includes a magnetic separator device. The magnetic separator device includes: a platform comprising a plurality of apertures for receiving and retaining a plurality of tubes therein; and a magnetic assembly comprising a guide member and a plurality of magnets provided on a surface thereof, wherein the magnetic assembly provided beneath the plurality of apertures and is moveable in both vertical and horizontal directions relative to the apertures.
The magnetic assembly is movable between an upper-most position, in which the surface of the guide member is substantially parallel with a longitudinal axis of each aperture and the magnets are a closer distance to the plurality of apertures, and a lower-most position, in which the surface of the guide member is orthogonal relative to the longitudinal axis of each aperture and the magnets a farther distance to the plurality of apertures.
The plurality of apertures may generally be arranged in a row on the platform and the plurality of magnets are arranged in a corresponding row on the guide member. For example, each of the plurality of magnets may correspond to a separate one of the plurality of apertures, such that each of the plurality of magnets is substantially aligned with the corresponding aperture. Accordingly, when a tube is received and retained within an aperture, a corresponding magnet is positioned adjacent to a bottom of the tube when the magnetic assembly is in the lower-most position and positioned adjacent to a side portion of the tube when the magnetic assembly is in the upper-most position. Each of the apertures is shaped and/or sized to receive and retain a tube comprising of volume of between about 0.1 mL and about 50 mL. As such, each of the apertures is configured to retain a corresponding tube in a vertical direction.
When the magnetic assembly is in the upper-most position, magnetic particles within a tube are attracted to a sidewall of the tube due to magnetic attractive forces and when the magnetic assembly is in the lower-most position, magnetic particles within a tube are attracted to a bottom of the tube due to magnetic attractive forces. As such, when the magnetic assembly transitions from the upper-most position to the lower-most position, magnetic particles within a tube correspondingly move from a sidewall of the tube toward a bottom of the tube due to magnetic attractive forces.
The platform of the magnetic separator device may include opposing sidewalls, wherein each sidewall comprising a corresponding slot for guiding the magnetic assembly between the upper-most and lower-most positions. For example, the guide member may generally be positioned between the opposing sidewalls and comprises support members extending through the corresponding slots on the opposing sidewalls. The slots may be L-shaped.
The platform of the magnetic separator device may include a wall member that comprises one or more magnets provided thereon. The magnetic assembly may be releasably maintained in the upper-most position due to magnetic attractive forces between the one or more magnets of the wall member in the platform and one or more magnets of the magnetic assembly.
The magnets used in the magnetic separator device may be permanent magnets, such as rare earth magnets. In some embodiments, the magnets used in the magnetic separator device may be electromagnets. The electromagnets may be operably coupled to one or more timers such that one or more magnetic fields are produced in an automated fashion based, at least in part, on said one or more timers.
Another aspect of the present invention includes a centrifuge adapter for use with a microcentrifuge. The centrifuge adapter is generally shaped and/or sized to fit within a microwell plate holder of a microcentrifuge and support individual tubes. The adapter generally comprises a base portion including one or more recesses shaped and/or sized to receive and retain portions of tube holders thereto. For example, the base portion may include at least a first recess defined on a first end and a second recess defined on a second, opposing end. The centrifuge adapter further includes a first tube holder and a second tube holder configured to be releasably coupled to the base portion via engagement between respective portions of the first and second tube holders and corresponding first and second recesses. Each tube holder may include a frame including a plurality of apertures for receiving individual tubes within (e.g., tubes comprising a volume of between 0.1 mL to 5.0 mL).
A portion of the frame of each tube holder may generally be shaped and/or sized to be received within at least one of the first and second recesses. Furthermore, the frame of each tube holder may further include one or more gradation lines or other visual indicia adjacent to the apertures for providing a technician with a visual indication of fill volumes.
When assembled, the base portion of the adapter comprises the first and second tube holders releasably coupled thereto and the apertures on the frame are oriented in a substantially horizontal direction. Accordingly, tubes received within the respective apertures may be positioned in a substantially horizontal direction once the base portion is loaded into a microwell plate holder of a microcentrifuge, wherein the bottom of a tube is facing in an outward direction and the top of a tube is facing an inward direction.
In some embodiments, each tube holder may be releasably attachable to each other via a corresponding connection assembly. For example, the frame of each tube holder may include one or more magnets provided on both sides of the frame. As such, two tube holders may be releasably attachable to each other via attraction forces between magnets on respective sides of the tube holders.
By way of overview, the present invention provides devices for improving assay protocol compliance, reproducibility, sensitivity, and specificity. As described in detail herein, devices of the present invention are particularly useful when performing certain single-cell sequencing assays, including single-cell RNA sequencing assays that utilize pre-templated instant partitions (PIPs). However, it is noted that devices according to the invention are useful when performing most assays, particularly when preparing biological samples for processing (including isolation of specific target cells of interest from a biological sample), and during most fluid handling and sample washing steps.
For example, performing a single-cell RNA sequencing (ScRNA-Seq) assay utilizing PIPs templates requires careful sample handling and manipulation of multiphase samples, which can be challenging for standard hand-held liquid handling pipettors. In such sequencing assays, PIPs templates are used to simultaneously segregate complex cell mixtures into partitions with barcoded template particles that can be easily processed for single cell applications, such as ScRNA-Seq.
Devices of the present invention generally improve the manner with which a medical technologist (or other lab personnel) carries out the various sample preparation steps involved in ScRNA-Seq using PIPs templates. In particular, devices of the present invention improve the reliability of liquid handling, which ultimately results in improved protocol compliance, improved assay performance, and improved assay reproducibility.
ScRNA-Seq assays utilize PIPs templates to form monodisperse droplets for segregating single cells and preparing a library preparation thereof to profile expression of the single cells. The PIPs template particles are used to template the formation of monodisperse droplets to generally capture a single target cell in an encapsulation, derive a plurality of distinct RNA from the single target cell, and prepare a library of nucleic acids that can be traced to the cell from which they were derived, and quantify distinct RNA to generate an expression profile of the single target cell. Such assays can be used to prepare libraries for single cell analysis of, for example, at least 100 cells, at least 1000 cells, at least 1,000,000 cells, at least 2,000,000 cells, or more, from a single reaction tube.
The typical workflow begins with sample preparation, in which a single cell suspension of interest is mixed with PIPs. The PIPs partition the mixture and isolate single cells inside compartments for conducting individual, parallel processes. The PIPs include template particles, which are generally hydrogel particles that function as templates, causing water-in-oil emulsion droplets to form when mixed inside a mixture of aqueous solution with oil and vortexed or sheared. The template particles may be provided in the aqueous solution (e.g., saline, nutrient broth, water) inside a tube or dried to be rehydrated at time of use. A sample comprising cells may be added into a tube (e.g., directly upon sample collection from a cell culture dish, or after some minimal sample prep step such as centrifuging the cells and re-suspending the cells in a buffered saline solution). Preferably an oil is added to the tube (which will typically initially overlay the aqueous mixture).
For example, an aqueous mixture can be prepared in a reaction tube that includes template particles and cells in aqueous media (e.g., water, saline, buffer, nutrient broth, etc.). The cells can be any cell type that contains RNA. The cells can be obtained from cellular tissue taken from a subject. For example, the cells may be cells taken from a subject by a blood draw. The subject may be suspected of carrying a contagious pathogen. Alternatively, the cells may be tissue culture cells. The cells can be nonadherent or adherent cells, e.g., HeLa cells. The cells can be primary cells, stem cells, epithelial cells, endothelial cells, fibroblast cells, or neurons.
After combining the cells with template particles inside a tube, an oil is added to the tube, and the tube is agitated (e.g., on a vortexer, also referred to as a vortex mixer). The particles act as templates in the formation of monodisperse droplets that each contain one particle in an aqueous droplet, surrounded by the oil. The pre-templated instant partitions are useful to segregate large numbers of cells into single cell compartments quickly, and without any expensive instrumentation (e.g., microfluidic devices). As such, samples for single cell RNA sequencing can be initially prepared at almost any location, such as in the field or at a remote laboratory. The partitions are formed around hydrogels and provide stable reaction chambers that can be transported by courier and/or where RNA is prepared for sequencing.
As noted, the partitioning step involves vortexing, which is preferred for its ability to reliably generate partitions of a uniform size distribution. However, the PIPs templates have a tendency to settle and clump at the bottom of sample tubes. It has been found that standard vertical vortexing is inefficient and unreliable at disaggregating clumped hydrogel particles, and may ultimately result in non-uniform vortexing conditions required for reproducible PIPs partitioning.
Recognizing the limitations of vertical vortexing, the present invention provides a vortex adapter configured to mount to a standard benchtop vortex unit and retain sample tubes in either a horizontal position or a vertical position, thereby allowing for horizontal or vertical vortexing as desired.
For example,
As shown, the tube holder 104 is releasably mounted to a distal portion of the base 102, wherein said tube holder 104 is movable between a first position in which each of said apertures is oriented in a vertical direction relative to the base 102 and a second position in which each of said apertures is oriented in a horizontal direction relative to the base 102. For example, the tube holder may be mounted to the distal end of the base in a vertical position (i.e., the cap of each tube is facing upwards and away from the vortex mixer), as shown in
By providing a vortex adapter that allows for a selectable position (i.e., vertical or horizontal position), a technician can perform both horizontal and vertical vortexing as needed and realize the benefits of each. In particular, horizontal vortexing generates more chaotic mixing and is effective at disaggregating settled PIPs templates while vertical vortexing is useful for providing uniform particle templated emulsification.
As illustrated in at least
The vortex adapter further includes a connection member 106 provided at the distal portion of the base and is selectively moveable between an engaged position and a disengaged position. When in an engaged position, the connection member 106 maintains the tube holder 104, 204 in one of the first and second positions. When in a disengaged position, the tube holder 104, 204 is moveable between the first and second positions.
For example, as illustrated, the distal portion of the base may include a channel defined between two knuckle members. The connection member 106 may include an adjustable bolt assembly extending between the knuckle members and configured to draw the knuckle members together upon movement of the bolt assembly to an engaged position.
The tube holder 104, 204 may generally comprise a body portion that is sized to fit within the channel in either of the first and second positions. Accordingly, when in an engaged position, the connection member 106 causes the knuckle members to apply a retention force upon the body portion of the tube holder 104, 204 and prevent movement thereof within the channel. In order to transition between vertical and horizontal positions, a technician need only disengage the tension (i.e., unscrew the bolt), adjust the body portion of the tube holder (i.e., rotate the tube holder to either a vertical or horizontal position) and then engage the connection assembly (i.e., screw the bolt to apply tension).
In some assays, magnetic nanoparticles may be used in partitioning and isolating target cells (via an associated binding element). Accordingly, when exposed to a sample, the binding elements bind with their partners (i.e., target analyte) and the emulsion forms partitions (e.g., droplets) that sequester the analyte. The partitions may then be manipulated using a magnetic field applied to the vessel containing the emulsion. However, magnetic particle-loaded PIPs templates are weakly magnetic, and, as a result, are unable to be efficiently collected via standard magnetic separators.
Accordingly, the present invention provides a magnetic separator device that enables at least a two-stage magnetic separation by providing at least two orientations of a magnetic field relative to sample tubes.
For example,
As shown, a plurality of apertures 504 are arranged in a row on the platform 502 and the plurality of magnets 510 are arranged in a corresponding row on the guide member 508, wherein each of the plurality of magnets corresponds to a separate one of the plurality of apertures. Furthermore, each of the plurality of magnets is substantially aligned with the corresponding aperture. Accordingly, when a tube is received and retained within an aperture, a corresponding magnet is positioned adjacent to a bottom of the tube when the magnetic assembly is in the lower-most position and positioned adjacent to a side portion of the tube when the magnetic assembly is in the upper-most position. As such, when the magnetic assembly is in the upper-most position, magnetic particles within a tube are attracted to a sidewall of the tube due to magnetic attractive forces, and when the magnetic assembly is in the lower-most position, magnetic particles within a tube are attracted to a bottom of the tube due to magnetic attractive forces. As shown in
As previously described, the magnetic assembly 506 is configured to move both vertically and horizontally relative to the platform, and specifically relative to the tubes. As shown in
The platform 502 further includes a wall member that comprises one or more magnets 516 provided thereon. The magnetic assembly is releasably maintained in the upper-most position due to magnetic attractive forces between the one or more magnets 516 of the wall member in the platform and one or more magnets 510 of the magnetic assembly 506.
In some embodiments, the magnets are permanent magnets. For example, the magnets may be rare earth magnets. In other embodiments, the magnets are electromagnets. The electromagnets may be operably coupled to one or more timers such that one or more magnetic fields are produced in an automated fashion based, at least in part, on said one or more timers.
After agitation of the sample with the PIPs templates (e.g., vortexing), a plurality (e.g., thousands, tens of thousands, hundreds of thousands, one million, two million, ten million, or more) of aqueous partitions is formed simultaneously inside the tube. Vortexing causes the fluids to partition into a plurality of monodisperse droplets. A substantial portion of droplets will contain a single template particle and a single cell. Droplets containing more than one or none of a template particle or target cell can be removed, destroyed, or otherwise ignored.
The next steps in the workflow involves lysing the single cells and capturing released mRNA inside the partitions. First-strand cDNA is then generated via reverse transcription (RT) and amplified to create a cDNA library for each individual cell. These are then processed into sequencing libraries using standard library preparation methods and subsequently sequenced and analyzed. Such processes generally involve, and require, careful sample handling and manipulation of the multiphase samples, including washing and fluid handling. Recognizing the difficulties technicians face with standard hand-held liquid handling pipettors, the present invention provides a sample tube holder, generally in the form of a guide rack, that incorporates certain visual aids to enable precise removal of excess volume in washing and fluid handling steps. In particular, the guide rack includes a plurality of slots or apertures configured to receive and releasably retain sample tubes therein, and further includes a visual guide rod positioned relative to each slot or aperture. The visual guide rod may be at a fixed position that corresponds to a particular volume at which excess fluid can be removed from a given tube. Accordingly, the guide rack improves the ease with which a technician can work with the sample tubes, particularly during washing and fluid handling steps.
It should be noted that certain steps (including washing steps) may further require the use of a centrifuge. For example, centrifuging a sample tube prior to performing PCR ensures that all reactants are in the bottom of the tube for proper concentrations and improved yields. A common issue encountered with a standard benchtop centrifuge is that sample tubes sit at a fixed and angled position, which results in the formation of a slanted pellet within the tube, which is not optimal. Some centrifuges have been developed that are configured to receive microwell plates (i.e., typically 96-, 384-, or 1536-well plates) and swing the wells into a horizontal position during operation, thereby concentrating the resulting pellet into the well bottoms. However, such centrifuges (also referred to as microplate microcentrifuges) are limited to receiving microwell plates and are unable to accept individual sample tubes.
The present invention provides a centrifuge adapter allowing for standard benchtop microwell plate centrifuges to accommodate individual sample tubes and/or strip tube configurations.
As shown, the adapter 600 is shaped and/or sized to fit within a microwell plate holder of a microcentrifuge and support individual tubes, such as sample tubes (i.e., 0.5 mL, 1.5 mL, 2 mL, etc., tubes) held via tube holders 700(1) and 700(2) and sets of strip tubes (e.g., 0.2 mL tubes in a strip) held via holders 800(1) and 800(2). In particular, the adapter 600 includes a base portion 602 that has an exterior profile matching that of a microwell plate, thereby allowing for the base portion to be placed within a microwell plate holder of a microcentrifuge. The base portion 602 is configured to receive and releasably retain sample tube holders thereto. In particular, the base portion 602 includes multiple recesses shaped and/or sized to receive and retain portions of sample tube holders thereto. For example, in one embodiment, the base portion 602 may include a recess 604, 606 defined on each end thereof in which portions of respective sample tube holders 700(1), 700(2) may be received and retained. For example, a first tube holder 700(1) and a second tube holder 700(2) may be configured to be releasably coupled to the base portion 602 via engagement between respective portions of the first and second tube holders and corresponding first and second recesses 604, 606.
In some embodiments, each sample tube holder may be configured to releasably attach to another sample tube holder via magnets 708 or other connecting means that are integrated into sides of the sample tube holder, to thereby arrange the sample tubes in a row. This is specifically shown in
When assembled, the base portion 602 comprises the first and second tube holders 700(1), 700(2) releasably coupled thereto and the apertures 704 on the frame 702 are oriented in a substantially horizontal direction. For example,
Accordingly, the devices of the present invention improve upon protocol compliance, reproducibility, and sensitivity, particularly in single-cell sequencing assays. Such devices are tailored to single-cell RNA sequencing assays that utilize PIPs and will ultimately improve the manner with which a medical technologist (or other lab personnel) can carry out the various sample preparation steps involved in ScRNA-Seq using PIPs templates, thereby reducing overall time required in performing a given assay and further reducing the risk of inaccurate diagnoses.
References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.
Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.
This application claims priority to, and the benefit of, U.S. Provisional Application No. 63/275,152, filed Nov. 3, 2021, the content of which is incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63275152 | Nov 2021 | US |
