WO2019136153A1

WO2019136153A1 - High-efficiency, array-based, single-cell sequencing prep system that correlates cell phenotype with genotype

Info

Publication number: WO2019136153A1
Application number: PCT/US2019/012189
Authority: WO
Inventors: Harold E. Ayliffe; Donald O'neil
Original assignee: E. I. Spectra, Llc
Priority date: 2018-01-03
Filing date: 2019-01-03
Publication date: 2019-07-11

Abstract

A particle-sorting device includes a housing having an inlet for receiving first and second types of cells, a first outlet through which cells of the first type are expelled from the housing and a second outlet through which cells of the second type are expelled from the housing. The housing further includes an interrogation zone in which cells passing through the interrogation zone are determined by a sensing device to be of the first type or the second type. An electromagnet is in signal communication with a processing device. A shunting device is disposed within the housing, which includes ferromagnetic material and is configured to block flow through the second outlet in response to creation of a magnetic field by the electromagnet. The processing device causes the electromagnet to create the magnetic field in response to the sensing device determining that a cell of the first type has passed through the interrogation zone.

Description

High- Efficiency, Array-Based, Single-Cell Sequencing Prep System that Correlates Cell Phenotype with Genotype

PRIORITY CLAIM

[0001] The present application claims priority from U.S. Provisional Application No. 62/613,348 filed January 3, 2018, U.S. Provisional Application No. 62/615,381 filed January 9, 2018, and U.S. Provisional Application No. 62/619,577 filed January 19, 2018. All of the aforementioned applications are hereby incorporated by reference in their entireties as if fully set forth herein.

BACKGROUND OF THE INVENTION

[0002] In the field of genomics, Single-cell DNA sequencing is quickly gaining traction in both academic and industry cellular research labs. The driving force behind this is the fact that populations of cells are known to have genetic outliers and are believed to contain DNA and mRNA that is different from the mass population. Understanding these outliers is critical to unlocking genetic keys to diseases like cancer. When mRNA and DNA from an entire population of cells is sequenced, individual genetic outliers are lost, as they are relatively rare in numbers. Because of this, tools are being created to enable populations of cells to be prepared for individual downstream DNA sequencing.

[0003] Currently, the industry standard method to prep samples for single-cell sequencing is by randomly encapsulating cell-bead conjugates into emulsion droplets. Prior to encapsulation, each bead is coated with thousands of barcoded primer oligonucleotides. The“barcode” is a short, DNA sequence that is used downstream of sequencing to determine which of the oligos belonged to the same bead... and thus cell.

[0004] The procedure is generally as follows: [0005] 1) Prepare a library of a mRNA capture probe set with an integrated unique oligo barcode.

[0006] 2) Attach the barcoded mRNA capture probes to beads. The barcodes are the same on any individual bead, but unique from bead to bead.

[0007] 3) Bind the beads to the cells, ideally, just one cell per bead.

[0008] 4) Attempt to form emulsion droplets with just one cell-bead conjugate per droplet using an automated droplet generator. This is very dependent on getting the cell- droplet concentration correct.

[0009] 5) As the droplets are being formed, a lyse reagent is added.

[0010] 6) The cell membranes are lysed and the free flowing (formally intracellular) mRNA binds to the capture probes.

[0011] 7) The emulsion droplets are broken up, oligos with cellular mRNA are cleaved from the beads and the entire solution is combined.

[0012] 8) The mixture is prepared for DNA sequencing and sequenced.

[0013] 9) A computer is used to re-combine the mRNA sequences using the barcodes to yield“single-cell” information.

[0014] 10) The data is analyzed.

[0015] The current emulsion droplet method has a number of limitations:

[0016] The theoretical maximum capture efficiency is only 60%, but in practice, capture efficiencies are much lower (20% is not uncommon).

[0017] Droplets often contain no cells, cell debris, or multiple cells. These types of droplets cannot be sequenced correctly and create issues with the integrity of “single-cell” sequencing results.

[0018] The resulting data quality is heavily dependent on obtaining one cell-bead per emulsion droplet and the droplet formation is random. State of the art technologies use statistical models and cell concentrations to drive the efficiency of cell/droplets.

[0019] There is no way to perform any quality control check on the“single-cell” emulsion droplets prior to performing the very expensive sequencing operation. [0020] Any multi-cell droplets or debris leads to data contamination.

[0021] There is currently no tie between the starting cell phenotype, starting cell morphology, starting cell health (i.e. viability, apoptosis assessment, ROS level, mito potential, etc.) and the resulting genotype. Single cell results are literally blind.

[0022] Because of the high failure rate of current droplet single cell sequencing methods, and the cost of the sequencing step, there is a strong market need for a highly efficient single-cell sequencing prep method that has inline quality control checks and the ability to correlate the in-tact single cell information to its genotype.

[0023] Additionally, in biological related sciences, including genomics, there is an ever growing need to be able to“pull out” specific cell sub-populations from a mixed population of cells. Flow cytometry based cell sorting has been around since the l970’s, but the vast majority of systems are still very expensive, difficult to use, and therefore are out of reach for everyday life science research labs.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

[0024] Preferred and alternative examples of the present invention are described in detail below with reference to the following drawing figures:

[0025] FIGS. 1-8 illustrate structure and functionality of a first embodiment of the invention; and

[0026] FIG. 9-12 illustrate structure and functionality of one or more additional embodiments of the invention an embodiment of the invention can be implemented.

DETAILED DESCRIPTION

[0027] This patent application is intended to describe one or more embodiments of the present invention. It is to be understood that the use of absolute terms, such as“must,” “will,” and the like, as well as specific quantities, is to be construed as being applicable to one or more of such embodiments, but not necessarily to all such embodiments. As such, embodiments of the invention may omit, or include a modification of, one or more features or functionalities described in the context of such absolute terms. [0028] An embodiment includes a high-efficiency, array -based single-cell sequencing prep structure and method that enables in-process quality control, in-tact single cell interrogation via imaging and/or fluorescence detection via laser scanning. Because this method can also allow users to track cell viability, the system may only use and analyze data generated by single, viable cells.

[0029] An embodiment includes an array of thousands of micro-wells that are sized appropriately for single cells. A similar library of barcoded, oligonucleotide mRNA target probes can be used. Each well can be printed with a unique barcoded mRNA target molecule (multiple thousands of the same type per well). Once the array is fully printed, cells can be deposited onto the array and allowed to settle, with the target being one cell per well. This can be achieved by having approximately the correct starting cell concentration, although it is not as critical for the experiment’s success as the droplet system. Once the cells have sehled, each well can be automatically imaged or fluorescently scanned (using a focused laser). Fluorescent labeling can provide valuable data on cell count (per well), viability, mitosis, phenotype, morphological information and other cell health data that can be directly tied to the subsequent resulting genetic sequencing at the single cell level. An embodiment includes the first system to directly link in-tact single-cell phenotype with single-cell genotype.

[0030] Once the array is scanned/imaged, a lyse reagent is added and the wells are fluidically isolated. This can be done in a number of ways such as pressing down a silicone membrane, or adding a perfluorocarbon (or fluorocarbon or PFC) solution, etc. The cells are then lysed and the free-floating mRNA binds to the barcoded target molecules that are bound to the base of the micro-well. Following incubation, the wells are fluidically“reconnected” and a solution is added to cleave the barcoded mRNA target probes, thereby releasing them from the array substrate and allowing for recollection. The sample is then prepared for genetic sequencing and the barcoded results can be directly linked to each specific starting cell/well. By utilizing and analyzing only the wells with one, viable cell each, the resulting genetic data is, by default, 100% true single-cell data. [0031] Figure 1 illustrates a top view and section view of an embodiment of a High Efficiency, Array-based, Single Cell Sequencing prep cassette. As shown in the Section View, the cassette is a five-layer laminate with the bottom layer being a clear outer, capping layer with adhesive on the top side. The next layer up from the bottom is a double-sided adhesive layer that has fluid channels (and other features) cut into it. The center layer may contain the microwell vias that may most likely be laser processed. Alternatively, the center layer could be a molded PDMS layer or some other material. The next layer up is a double sided adhesive layer containing fluid channels and other features. Finally, the top layer is a transparent capping layer containing access vias and other features.

[0032] Figure 2 illustrates two section views through the SCS Prep Cassette with the pre-printed microwell vias and the five construction layers. In the top image, the cell- containing sample is aliquoted into the top channel layer of the cassette and flows above the microwells using positive pressure or vacuum assist. The sample is then forced to flow through the vias using either mechanical, downward force or vacuum assist applied to the lower channel layer. Once all the vias have been wetted, the bottom capping layer is pressed upward and the adhesive seals the microwells closed (one the bottom). The cells in solution then slowly settle randomly into the wells. A cell concentration is used to result in a high ratio of wells containing only one cell. The microwells can then be alternately fluidically “sealed” and“unsealed” by pipetting in a fluorocarbon solution. Each well can be scanned to measure fluorescence levels and/or imaged to determine the number of cells per well and the health of the cells. Only data from the cells with one, viable cell per well will be used in the downstream sequencing operations.

[0033] Figure 3 illustrates a work-flow sequence slide for array-based single-cell sequencing system/method. Sample is aliquoted onto the pre-printed micro-array slide. Cells sehle into the micro-wells at a targeted one cell per well concentration. Some wells will not end up with cells in them and these will be actively excluded from the final data results through the in-tact single cell scanning and or imaging step. Each micro-well is scanned/imaged to determine cell count per well and cell viability. Cell lysate is added to the array via the automated system and the wells are“closed” so they are fluidically separate from each other. The cellular mRNA binds to the barcoded target oligonucleotides in each well. A cleaving reagent is added to the array via the automated system and the barcoded mRNA complexes are collected from the cassette.

[0034] Figure 4 illustrates a representation of the barcoded mRNA target molecules, etc. Top left, the target molecules are“printed” into each well individually and recorded in the computer. Top right is a schematic of the barcoded mRNA target molecule. Bottom left image shows the cellular mRNA binding to the printed target molecule. The resulting cDNA complex is synthesized and amplified in preparation for downstream sequencing.

[0035] Referring to Figure 5, shown is a 3D rendering of the array-based, single-cell prep chip is shown in the upper left. 2500 laser drilled holes form the 2.5 x 2.5mm array (upper right). In an embodiment each microwell is approximately 40 micrometers in diameter and 50 micrometers deep.

[0036] Figure 6 illustrates in exploded view of the array-based, single-cell prep chip showing the thin-film construction with one-plastic injection molded piece that forms the top capping layer, input sample reservoir, waste reservoir, and sample re-collection reservoir.

[0037] Figure 7 illustrates an array-based single-cell prep chip shown in position on the modified 96 well plate. To run an experiment, the user pipettes the required reagents and fluorocarbon into pre-identified wells, pipettes the cell sample directly into the prep chip, and inserts the full assembly into the automated system. The system can apply pneumatic pressure (or vacuum) directly to the prep chip to move fluids around the chip, etc. The system can automatically pipette the reagents and fluorocarbon into the chip using a up/down pipetter and an XYZ plate motion robot.

[0038] Figure 8 is a schematic representation of the laser scanning light engine used to scan the micro-array cassette. Laser light is re-imaged onto the cassette at a 5X reduction so that the laser beam diameter is approximately the same size as the diameter of the microwells (lO-lOOum). A PIN diode can be placed under the cassette to be used to orient the laser scanner to the microwells. The Steering Mirror is used to move the focused laser beam across the cassette. This arrangement can be used to either collect total fluorescence of each microwell in PMT(s) or create images of each microwell.

[0039] An embodiment includes a novel method to sort desired sub-population(s) of cells using a very low-cost method. In an embodiment, a one-time (or multi-time) use consumable cartridge (see Figure 9) includes multiple (e.g., 5) laminated layers. The middle, sensor layer incorporates a small“valve” section that is printed with ferromagnetic ink and has a laser (or die cut) opening configured in such a way as to allow a portion of the center film layer to be deflected when an external (of the cassette) magnetic field is supplied (most likely electromagnetic). By rapidly applying a magnetic field at the correct time, desired cells can be sorted immediately following analysis.

[0040] Using generally understood principles of flow cytometry, the cells are analyzed as they flow substantially single file through a laser drilled hole (detection zone) in the middle, sensor layer. In preferred embodiments, as the cells flow through the detection zone, their volume can be precisely determined using the Coulter Principle. Simultaneously, the cells can also be fluorescently interrogated by focusing one or more high intensity wavelengths of light into the detection zone. Any resulting cell-specific fluorescence emission is collected by one or more photo-detectors, in addition to any side-scattered light and forward scatter, or forward extinction (depending on the configuration). These data are used by researchers to specify which populations of cells are to be removed from the mixed population or cells, or debris. A cross-section of one possible electro-magnetic valve configuration is seen in Figures 9 and 10.

[0041] In real-time, a processing device of the system in signal communication with the photo-detectors determines if each individual cell (based on cell volume and fluorescence emission) belongs to the population that is to be separated, as it flows through the detection zone. Based on this determination (pre-set by the researcher), the processing device of the system will either trigger an electro-magnetic transducer to flex the ferromagnetic coated “valve”, thereby forcing the cell into a separate reservoir (i.e., not allowing the cell/particle to flow into the central waste reservoir). Once the entire sample is processed, the researcher can re-collect each of the, now, sub-populations of cells from each of the perspective reservoirs.

[0042] Alternate methods/embodiments include:

[0043] Using other cell detection methods (instead of the Coulter orifice) to analyze the cells to make sorting determinations. An embodiment may combine a more traditional capillary flow detection method with a thin-film, electro-magnetic valve.

[0044] Multiple valves in series can be used to sort multiple populations.

[0045] As shown in Figure 9, cells are analyzed as they transition through the laser drilled detection hole (left side of image). If the cell is of interest, an electromagnetic pulse is applied to move the ferromagnetic valve to force the cell into a separate“Collection Reservoir”, based on pre-set conditions.

[0046] The Coulter Principle Component.

[0047] Referring to Figures 11 and 12, one or more embodiments use conductive saline to drive the constant (Coulter) current across a vertical flow channel. The side channels (with printed electrodes) can be wetted by fabricating relatively large dead spaces beyond each electrode. When a vacuum is applied to draw the fluid through the cassette, the residual vacuum in these dead spaces draws the fluid in. Alternatively, a light active vacuum can be applied individually to the“dead” space to draw the fluid in. In an alternative embodiment, capillary action could also be used to draw the fluid in.

[0048] As cells move between the printed electrodes, the constant current is disturbed and a voltage spike is measured that is proportional to the cell/particle volume (i.e., the volume of conductive media that is displaced is measured).

[0049] Fluorescence Component.

[0050] In an embodiment, an EPI Fluorescence measurement technique can be used to measure any fluorescence as the cell/particle is transitioning the“detection zone” (which is where the Coulter component is measured). The laser(s) are actively aligned to the detection zone after the cassette is inserted into the system. This can be accomplished by forming the fluid channels in an opaque, or semi-opaque material and measuring the total light collected under the cassette using, for example, a PIN diode. The laser beam is then steered around and the location of the channel intersection is determined using feedback from the PIN diode. Alternatively, a black dot can be printed with a hole in it and aligned to the printed feature.

[0051] This new planar structure is preferred, specifically when integrated with the magnetic sorter, because it is easier to keep track of the individual cells from when they are detected (Coulter and/or Fluorescence) to when one needs to actuate the magnetic value (or not).

[0052] One or more embodiments of the invention include:

[0053] 1) The use of the conductive saline to move current from the printed electrodes to the flow channel.

[0054] 2) The combination of this planar structure Coulter sensor with EPI fluorescence.

[0055] 3) The alignment of the laser to the flow channels using a PIN diode under the cassette.

[0056] 4) The planar Coulter sensor (and/or epifluorescence sensor) in combination with the electro-magnetic flapper valve using printed ferromagnetic ink.

[0057] An embodiment employs a five-layer structure for this new cassette, as depicted in Figure 9. The center layer may be printed with conductive inks for the Coulter electrodes and any fluid front detection electrodes that we likely use (for on-cassette volumetric counts), as well as with the printed ferromagnetic ink for the sorting valve. The center layer could also have laser drilled features to form the sorting valve and any vias needed. On the top and bottom of the center layer may be double-sided pressure sensitive adhesive (or possibly thermal, heat-stake films) to bond the center layer to the clear outer/capping layers. One may laser cut the double-sided adhesive layers to form the fluid channels, vias, etc.

[0058] While the preferred embodiment of the invention has been illustrated and described, as noted above, many changes can be made without departing from the spirit and scope of the invention. Accordingly, the scope of the invention is not limited by the disclosure of the preferred embodiment. Instead, the invention should be determined entirely by reference to the claims that follow.

Claims

CLAIMS The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of enabling single-cell DNA sequencing, the method comprising the steps of:

introducing a suspension containing a set of cells into a chamber in fluid communication with an array of micro-wells, each said micro-well being sized to receive at least one cell of the set, each said micro-well being printed with a mRNA target molecule barcoded to uniquely correspond to a respective micro-well;

allowing the cells of the set to settle into the micro-wells;

acquiring an image of each micro-well;

determining from each image whether a corresponding micro-well contains a cell of the set;

adding a lyse reagent to each of the micro-wells such that free-floating mRNA binds to the barcoded target molecules;

adding a cleaving solution to each of the micro-wells operable to create barcoded mRNA target probes associated with each micro-well containing at least one cell of the set; collecting the barcoded mRNA target probes; and

determining from the barcoded mRNA target probes which of the micro-wells contained a single cell of the set.

2. A particle-sorting device, comprising:

a housing comprising at least one inlet for receiving first and second types of cells, a first outlet through which cells of the first type are expelled from the housing and a second outlet through which cells of the second type are expelled from the housing, the housing further including an interrogation zone in which cells passing through the interrogation zone are determined by a sensing device to be of the first type or the second type;

an electromagnet in signal communication with a processing device; and a shunting device disposed within the housing, the shunting device comprising ferromagnetic material and being configured to block flow through the second outlet in response to creation of a magnetic field by the electromagnet, wherein the processing device causes the electromagnet to create the magnetic field in response to the processing device determining that a cell of the first type has passed through the interrogation zone.