Patent Publication Number: US-2021187515-A1

Title: Magnetic separator for an automated single cell sequencing system

Description:
CROSS REFERENCE TO OTHER APPLICATIONS 
     This application claims priority to U.S. Provisional Patent Application No. 62/953,050 entitled AUTOMATED SINGLE CELL SEQUENCING SYSTEM filed Dec. 23, 2019 and U.S. Provisional Patent Application No. 62/980,768 entitled MAGNETIC SEPARATOR FOR AN AUTOMATED SINGLE CELL SEQUENCING SYSTEM filed Feb. 24, 2020, both of which are incorporated herein by reference for all purposes. 
    
    
     BACKGROUND OF THE INVENTION 
     Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA-sequencing (RNA-Seq) uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment. RNA-Seq analyzes the transcriptome of gene expression patterns encoded within the RNA. 
     Traditional RNA-Seq techniques analyze the RNA of an entire population of cells, but only yield a bulk average of the measurement instead of representing each individual cell&#39;s transcriptome. By analyzing the transcriptome of a single cell at a time, the heterogeneity of a sample is captured and resolved to the fundamental unit of living organisms—the cell. Single-cell transcriptomics examines the gene expression level of individual cells in a given population by simultaneously measuring the messenger RNA (mRNA) concentration of hundreds to thousands of genes. 
     Automated single cell sequencing systems have been developed integrating various components to achieve RNA sequencing. One important component is a magnetic separator which interacts with a fluid in a vial. There is a need to improve the interaction in a way that allows fluid to be used efficiently and to provide consistent results. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Various embodiments of the invention are disclosed in the following detailed description and the accompanying drawings. 
         FIG. 1  illustrates a front view of one embodiment of an automated single cell sequencing system  100 . 
         FIG. 2  illustrates another view of one embodiment of an automated single cell sequencing system  200 . 
         FIG. 3  illustrates yet another view of one embodiment of an automated single cell sequencing system  300 . 
         FIG. 4A  illustrates a top view of an embodiment of a magnetic separator plate  402 . 
         FIG. 4B  illustrates a cross sectional view of the magnetic separator plate  402 . 
         FIG. 4C  illustrates another view of the magnetic separator plate  402 . 
         FIG. 5  illustrates a plurality of strip tubes  502  that may be loaded onto the magnetic separator plate  214  where the magnetic bead based cleanup may be performed. 
         FIG. 6  illustrates an exemplary consumable  602  that may be loaded onto the magnetic separator plate  214  where the magnetic bead based cleanup may be performed. 
         FIG. 7A  illustrates a top view of the 96-tube PCR plate  602  being loaded onto the magnetic separator plate  402 . 
         FIG. 7B  illustrates a cross sectional view of the 96-tube PCR plate  602  being loaded onto the magnetic separator plate  402 . 
         FIG. 7C  illustrates a portion of a magnified cross sectional view of the 96-tube PCR plate  602  being loaded onto the magnetic separator plate  402 . 
         FIG. 8A  illustrates a top view of a magnetic separator plate adapter  802 . 
         FIG. 8B  illustrates a cross sectional view of the magnetic separator plate adapter  802 . 
         FIG. 8C  illustrates a bottom view of the magnetic separator plate adapter  802 . 
         FIG. 8D  illustrates another view of the top surface of the magnetic separator plate adapter  802 . 
         FIG. 8E  illustrates another view of the bottom surface of the magnetic separator plate adapter  802 . 
         FIG. 9A  illustrates a top view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 . 
         FIG. 9B  illustrates a cross sectional view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 . 
         FIG. 9C  illustrates another cross sectional view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 . 
         FIG. 9D  illustrates a portion of a magnified cross sectional view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 . 
         FIG. 10A  illustrates a view of the magnetic separator plate adapter  802  about to be loaded onto the magnetic separator plate  402  and the 96-tube PCR plate  602  about to be loaded onto the magnetic separator plate adapter  802 . 
         FIG. 10B  illustrates another view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402  and the 96-tube PCR plate  602  being loaded onto the magnetic separator plate adapter  802 . 
         FIG. 11A  illustrates a top view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 , and the 96-tube PCR plate  602  being loaded onto the magnetic separator plate adapter  802 . 
         FIG. 11B  illustrates a cross sectional view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 , and the 96-tube PCR plate  602  being loaded onto the magnetic separator plate adapter  802 . 
         FIG. 11C  illustrates another cross-sectional view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 , and the 96-tube PCR plate  602  being loaded onto the magnetic separator plate adapter  802 . 
         FIG. 11D  illustrates a portion of a magnified cross sectional view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 , and the 96-tube PCR plate  602  being loaded onto the magnetic separator plate adapter  802 . 
     
    
    
     DETAILED DESCRIPTION 
     The invention can be implemented in numerous ways, including as a process; an apparatus; a system; a composition of matter; a computer program product embodied on a computer readable storage medium; and/or a processor, such as a processor configured to execute instructions stored on and/or provided by a memory coupled to the processor. In this specification, these implementations, or any other form that the invention may take, may be referred to as techniques. In general, the order of the steps of disclosed processes may be altered within the scope of the invention. Unless stated otherwise, a component such as a processor or a memory described as being configured to perform a task may be implemented as a general component that is temporarily configured to perform the task at a given time or a specific component that is manufactured to perform the task. As used herein, the term ‘processor’ refers to one or more devices, circuits, and/or processing cores configured to process data, such as computer program instructions. 
     A detailed description of one or more embodiments of the invention is provided below along with accompanying figures that illustrate the principles of the invention. The invention is described in connection with such embodiments, but the invention is not limited to any embodiment. The scope of the invention is limited only by the claims and the invention encompasses numerous alternatives, modifications and equivalents. Numerous specific details are set forth in the following description in order to provide a thorough understanding of the invention. These details are provided for the purpose of example and the invention may be practiced according to the claims without some or all of these specific details. For the purpose of clarity, technical material that is known in the technical fields related to the invention has not been described in detail so that the invention is not unnecessarily obscured. 
     Preparing consistent single cell gene expression libraries is labor intensive and requires extensive hands-on (i.e., manual) time. It would be beneficial if this could be automated, freeing lab personnel to perform other tasks. 
     Automated techniques for the preparation of single cell gene expression libraries are disclosed in the present application. The techniques provided herein allow for the maximization of consistency in the libraries prepared and productivity of the personnel. The techniques improve quality and performance by 1) decreasing technical variability and generating reproducible results; 2) running pre-validated protocols for single cell assays; and 3) providing a robust workflow and ready-to-use solution. The techniques save time and resources by 1) reducing hands-on time in the lab; 2) eliminating the need for dedicated resources; and 3) requiring no specialized expertise. The techniques are integrated and validated. Single cell partitioning, barcoding, and library preparation are integrated together in one optimized instrument. As a result, less customization and optimization are needed, thereby improving productivity. 
       FIG. 1  illustrates a front view of one embodiment of an automated single cell sequencing system  100 . The system includes an automated controller  102  on deck for single cell partitioning and barcoding. Reagents and consumables may be loaded onto the deck area  104  at the beginning of each run. Operations may be guided through an easy-to-use touchscreen computer  106  with Internet connectivity. System  100  includes a liquid handling gantry  108  that may perform pipetting steps throughout the entire single cell workflow. System  100  further includes one or more barcode scanners that enable lot and reagent tracking for reagents and consumables. 
       FIG. 2  illustrates another view of one embodiment of an automated single cell sequencing system  200 . Automated single cell sequencing system  200  includes five carriers ( 202 ,  204 ,  206 ,  208 , and  210 ) on the deck  201 . Some of the carriers are stationary and some of the carriers may slide in and out for loading and unloading items. Each of the carriers may be loaded with different types of labwares, modules, and consumables, such as a magnetic separator plate, a thermal cycler block, tips, reagent reservoirs, plates (e.g., polymerase chain reaction (PCR) plates and deep well plates), tubes, and the like. The terms labwares and modules may be used interchangeably in the present application. 
       FIG. 3  illustrates yet another view of one embodiment of an automated single cell sequencing system  300 . Automated single cell sequencing system  300  includes five carriers ( 302 ,  304 ,  306 ,  308 , and  310 ) and a disposal bin  336  on a deck  301 . 
     As shown in  FIG. 2 , an automated controller  212  for single cell partitioning and barcoding is located adjacent to the left most carrier  202 . The leftmost carrier  202  includes a magnetic separator plate  214 . An array of magnets  218  is located above magnetic separator plate  214 . Arrays of wells, tips or tubes may be placed above the array of magnets  218 . In some embodiments, a magnetic separator plate adapter  217  may be mounted on top of the magnetic separator plate  214  to keep the array of tips/tubes stable and sitting at the exact locations. The magnetic separator plate adapter  217  may rest above the magnetic separator plate  214  and the array of magnets  218 . The magnetic separator plate adapter  217  may be formed of plastic and include skirts. Magnetic separator plate adapter  217  may include a plurality of calibration posts  216 . Carrier  202  may further receive a cold plate reagent module  220  and other reagent modules  222 . 
     In some embodiments, automated single cell sequencing system  200  may include a barcode reading system. A barcode reader is used to scan reagents and consumables. The barcode reading system enables experiment tracking and prevents reagent mix-ups. A barcode reader (not shown in  FIG. 2 ) may be placed above the five carriers ( 202 ,  204 ,  206 ,  208 , and  210 ) on deck  201 . The barcode reader may be used to read the slots for holding the tips/tubes and the tips/tubes that go into the slots at different locations. The barcode reading system may include software logic to make sure that the right tubes (with reagents) are put at the right slots. The barcode reading system may also detect that the tubes are missing such that the system may inform the user about these errors. The system may check for color matching, lot numbers, and expiration dates. As shown in  FIG. 2 , automated single cell sequencing system  200  may include a plurality of mirrors  223  to allow the barcode reader to read sideways and at more locations. In some embodiments, stickers with barcodes on the slots are covered by the tips/tubes if they are placed there. If the barcode reader reads the barcodes on the slots, then the slots are determined as being empty. If the barcode reader reads the barcodes on the tips/tubes, then the system may match the two barcodes. 
     Carrier  204  (the second carrier from the left) includes an on-deck thermal cycler  224  (ODTC). A thermal cycler may be used to amplify segments of Deoxyribonucleic acid (DNA) via the polymerase chain reaction (PCR). Thermal cyclers may also be used to facilitate other temperature-sensitive reactions. In some embodiments, a thermal cycler has a thermal block with holes where tubes holding reaction mixtures may be inserted. The thermal cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps. Carrier  204  further includes a rack  226  for storing disposable ODTC lids. 
     Carrier  206  (the third carrier from the left) includes carrier spaces for receiving, storing, or loading tube strips, chips, gel beads, core or lifting paddles, ethanol reservoirs, primer, glycerol, and the like. Carrier  208  (the fourth carrier from the left) includes a sample index plate holder  230 . The carrier further includes a unit  232  for formulations and bead cleanups. Carrier  208  and carrier  210  (the fifth carrier from the left) may receive different consumables, such as pipette tips  234 . 
     Automated single cell sequencing system  200  may further include a waste disposal bin  236  that is adjacent to carrier  210 . In some embodiments, a divider may be added to the waste disposal bin for separating the recycled tips and lids. With the added divider, one side of the disposal bin is used for storing the tips and the other side of the disposal bin is used for storing the lids. A gantry  238  may be programmed to drop the tips and the lids on different sides of the disposal bin. This prevents the lids from stacking up and toppling over, causing the system to malfunction. This allows the recycling of the lids while preventing contamination. 
     The liquid handling gantry  238  in automated single cell sequencing system  200  may perform automated pipetting steps throughout the entire single cell workflow. Liquid handling gantry  238  is a movable liquid-handling pipetting device with precision positioning. 
     A traditional manual pipette is a laboratory tool commonly used in chemistry, biology and medicine to transport a measured volume of liquid. A pipette can be used to aspire (or draw up) a liquid into a pipette tip and dispense the liquid. In manual pipetting, a piston is moved by a thumb using an operation knob. Accuracy and precision of pipetting depend on the expertise of the human operator. 
     Automated pipetting has many advantages over manual pipetting. Automated pipetting enhances the throughput and the reproducibility of laboratory experiments. Automated pipetting takes the manual labor out of repeated pipetting, thereby shortening manual hands-on time. Reducing manual hands-on time frees up time and effort for other tasks, thereby greatly improving throughput. Furthermore, automated pipetting significantly reduces errors from manual pipetting, thereby enhancing reproducibility. 
     The liquid handling gantry  238  in automated single cell sequencing system  200  includes a pipetting head, which is the mechanical component for liquid transfer. In some embodiments, the pipetting head is a multi-channel pipetting head for increased throughput. In some embodiments, the pipetting head may be an 8-channel pipetting head coupled to a pump system such that for each channel, a volume of liquid may be aspirated from a source container by creating suction and then dispensed into a destination container (e.g., a tube or a well). A disposable tip may be attached to each of the eight channels of the pipetting head, such that the liquid is not in direct contact with the pipetting head, preventing cross contamination. 
     The liquid handling gantry  238  with the pipetting head may be programmed to move within a working area where liquid aspirating and dispensing take place. The working area may be the deck area  201  including the five carriers ( 202 ,  204 ,  206 ,  208 , and  210 ) that may be loaded with different types of labwares, modules, or consumables, such as reagent reservoirs, plates (e.g., polymerase chain reaction (PCR) plates and deep well plates), tubes, and the like. For example, the pipetting head may be moved to the position of the reagent module  240  to dispense liquid into a row  242  of eight wells of the reagent module  240 . The position of the reagent module  240  and the position of the row of wells may each be specified by a set of offset distances in the x, y, and z axes from one or more reference points within deck area  201 . In some embodiments, the position of a certain module or labware may be recorded by single cell sequencing system  200  as a first set of offset values (in x, y, and z) from a reference point within deck area  201 , and the position of a row of wells within the module or labware may further be recorded by the system as another set of offset values from the position of the module or labware. In some embodiments, different positions within the working area are recorded by single cell sequencing system  200  as different sets of offset values from a single reference point within deck area  201 . 
     In order to place the pipetting head into the appropriate source and destination containers, the liquid handling gantry  238  with the pipetting head may be moved by one or more actuators to different x and y positions in a plane substantially parallel to the floor of deck  201 . In addition, the pipetting head may be moved by one or more actuators in a direction substantially perpendicular to the plane, such that the pipetting head and the tips attached to the pipetting head may be inserted into or withdrawn from the source and destination containers. 
     Magnetic separator plate  214  in  FIG. 2  performs magnetic bead based cleanup. Magnetic beads are used for DNA purification and fragment size selection. Automated single cell sequencing system  200  uses the single-cell RNA-seq technology to analyze transcriptomes on a cell-by-cell basis through the use of microfluidic partitioning to capture single cells and prepare barcoded, next-generation sequencing (NGS) cDNA libraries. Specifically, single cells, reverse transcription (RT) reagents, gel beads containing barcoded oligonucleotides, and oil are combined on a microfluidic chip to form reaction vesicles called Gel Beads in Emulsion, or GEMs. After incubation, GEMs are broken and pooled fractions are recovered. Silane magnetic beads are used to purify the first-strand cDNA from the post GEM-RT reaction mixture, which includes leftover biochemical reagents and primers. In particular, consumables (e.g., test tubes or wells) containing the post GEM-RT reaction mixture and the magnetic beads may be loaded onto the magnetic separator plate  214  where the magnetic bead based cleanup is performed. Barcoded, full-length cDNA is then amplified via PCR to generate sufficient mass for library construction. 
       FIG. 4A  illustrates a top view of an embodiment of a magnetic separator plate  402 .  FIG. 4B  illustrates a cross sectional view of the magnetic separator plate  402 .  FIG. 4C  illustrates another view of the magnetic separator plate  402 . 
     As shown in  FIG. 4A , magnetic separator plate  402  is a magnet holder plate that holds an array of magnets  404 . Magnetic separator plate  402  is a 96-ring magnet plate, and the array of magnets  404  is an 8×12 array of magnets with eight magnets in a row and twelve magnets in a column. In some embodiments, each of the magnets  404  is a ring magnet. As shown in  FIG. 4B , a ring magnet may be a magnet with a shape of a hollow cylinder that is empty from inside and with differing internal and external radii. The hollow space of the cylinder allows a bottom end of a tube to be inserted therein. For example, a tube received by a ring magnet may be a finger-like length of glass or plastic tubing that is open at the top and closed at the bottom. 
       FIG. 5  illustrates a plurality of strip tubes  502  that may be loaded onto the magnetic separator plate  214  or magnetic separator plate  402  where the magnetic bead based cleanup may be performed. As shown in  FIG. 5 , each of the strip tubes  502  includes eight tubes  504  for storing the reaction mixture and the magnetic beads. 
       FIG. 6  illustrates an exemplary consumable  602  that may be loaded onto the magnetic separator plate  214  or magnetic separator plate  402  where the magnetic bead based cleanup may be performed. In this example, consumable  602  is a 96-tube polymerase chain reaction (PCR) tube holder plate with an array of tubes  604  arranged as an 8×12 array of tubes with eight tubes in a row and twelve tubes in a column. 
       FIG. 7A  illustrates a top view of the 96-tube PCR plate  602  being loaded onto the magnetic separator plate  402 .  FIG. 7B  illustrates a cross sectional view of the 96-tube PCR plate  602  being loaded onto the magnetic separator plate  402 .  FIG. 4C  illustrates a portion of a magnified cross-sectional view of the 96-tube PCR plate  602  being loaded onto the magnetic separator plate  402 . 
     As shown in  FIGS. 7B and 7C , the hollow space of a ring magnet (e.g.,  404 A or  404 B) allows the bottom end of a tube (e.g.,  604 A or  604 B) to be inserted therein. However, both the PCR plate  602  and the magnetic separator plate  402  are manufactured parts that have their respective sets of associated tolerances. All dimensions of a manufactured part have their associated tolerance, the amount that the particular dimension is allowed to vary. The tolerance is the difference between the maximum and minimum limits. Therefore, the length  606 A (the length from the center of the ring magnet  404 A to the center of the ring magnet  404 B) and the length  606 B (the length from the center of the ring magnet  404 B to the center of the ring magnet  404 C) may not be the same. Similarly, the length  608 A (the length from the center of the tube  604 A to the center of the tube  604 B) and the length  608 B (the length from the center of the tube  608 B to the center of the tube  604 C) may not be the same. These variations in dimensions may cause misalignments of the tubes and their corresponding ring magnets. As a result, some of the bottom ends of the tubes may no longer be inserted into the hollow spaces and resting within the hollow spaces of the ring magnets at the same depth, causing the PCR plate  602  to be tilted instead of leveled, and causing it to rest on the magnetic separator plate  402  at an angle, thereby degrading the performance of the magnetic bead based cleanup process. 
     In the present application, an improved magnetic separator is disclosed. The magnetic separator comprises an array of magnets configured to interact with an array of tubes, wherein the array of tubes is attached to a plate. The magnetic separator further includes a magnetic separator plate adapter. In some embodiments, the adapter comprises a raised frame extending around a periphery of the array of magnets such that the raised frame is configured to support the plate, such that the array of tubes are suspended above the array of magnets. By suspending the array of tubes above the array of magnets, the bottom ends of the tubes are no longer resting within the hollow spaces of the ring magnets at different depths, thereby keeping the plate with the array of tubes leveled with respect to the array of magnets. The benefit is that the performance of the magnetic bead based cleanup process may be significantly improved. 
       FIG. 8A  illustrates a top view of a magnetic separator plate adapter  802 .  FIG. 8B  illustrates a cross sectional view of the magnetic separator plate adapter  802 .  FIG. 8C  illustrates a bottom view of the magnetic separator plate adapter  802 .  FIG. 8D  illustrates another view of the top surface of the magnetic separator plate adapter  802 .  FIG. 8E  illustrates another view of the bottom surface of the magnetic separator plate adapter  802 . As shown in  FIG. 8A , magnetic separator plate adapter  802  includes four collars  804  at the four corners of the adapter. The collars  804  may be used to fix the location (the x and y location on the deck) of a consumable, such as a 96-tube PCR plate. For example, each of the collars  804  constrains the x location and the y location of the tube holder plate by having a tube inserted into the collar. The magnetic separator plate adapter  802  further includes four cylindrical feet  806  at the four corners of the adapter, such that the magnetic separator plate adapter  802  may be mounted on the magnetic separator plate  402 . In some embodiments, magnetic separator plate adapter  802  may be formed of plastic and includes skirts. Magnetic separator plate adapter  802  may include a plurality of calibration posts  808 . 
       FIG. 9A  illustrates a top view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 .  FIG. 9B  illustrates a cross sectional view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 .  FIG. 9C  illustrates another cross sectional view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 .  FIG. 9D  illustrates a portion of a magnified cross sectional view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 . As shown in  FIGS. 9B, 9C, and 9D , a cylindrical foot  806  of the magnetic separator plate adapter  802  fits into a cylindrical hole on the magnetic separator plate  402 , thereby mounting the magnetic separator plate adapter  802  on the magnetic separator plate  402  and raising the magnetic separator plate adapter  802  above the magnetic separator plate  402 . 
       FIG. 10A  illustrates a view of the magnetic separator plate adapter  802  about to be loaded onto the magnetic separator plate  402  and the 96-tube PCR plate  602  about to be loaded onto the magnetic separator plate adapter  802 .  FIG. 10B  illustrates another view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402  and the 96-tube PCR plate  602  being loaded onto the magnetic separator plate adapter  802 . 
       FIG. 11A  illustrates a top view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 , and the 96-tube PCR plate  602  being loaded onto the magnetic separator plate adapter  802 .  FIG. 11B  illustrates a cross-sectional view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 , and the 96-tube PCR plate  602  being loaded onto the magnetic separator plate adapter  802 .  FIG. 11C  illustrates another cross-sectional view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 , and the 96-tube PCR plate  602  being loaded onto the magnetic separator plate adapter  802 .  FIG. 11D  illustrates a portion of a magnified cross-sectional view of the magnetic separator plate adapter  802  being loaded onto the magnetic separator plate  402 , and the 96-tube PCR plate  602  being loaded onto the magnetic separator plate adapter  802   
     The magnetic separator plate adapter  802  comprises a raised frame extending around the periphery of the magnetic separator plate  402 , such that the raised frame supports the 96-tube PCR plate  602  in such a way that the array of tubes  604  are suspended above the array of magnets  404 . As shown in  FIG. 11D , the array of tubes is suspended above the array of magnets  404  at a height such that the tubes  604  do not come in contact with their corresponding magnets  404 . By suspending the array of tubes  604  above the array of magnets  404 , the bottom ends of the tubes  604  are no longer resting within the hollow spaces of the ring magnets at different depths, thereby keeping the 96-tube PCR plate  602  with the array of tubes  604  leveled with respect to the array of magnets  404 . The benefit is that the performance of the magnetic bead based cleanup process may be significantly improved. 
     Although the foregoing embodiments have been described in some detail for purposes of clarity of understanding, the invention is not limited to the details provided. There are many alternative ways of implementing the invention. The disclosed embodiments are illustrative and not restrictive.