Systems and methods for preparing one or more samples on a flow cell device

Abstract

The present disclosure provides flow cell devices, systems, and methods for facilitating and performing DNA sequencing analysis with reduced system complexity and cost, COGS saving and reduced contamination level.

Description

SYSTEMS AND METHODS FOR PERFORMING DNA SEQUENCING CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 63 / 706,241, filed Oct. 11, 2024, U.S. Provisional Patent Application Ser. No. 63 / 707,675, filed Oct. 15, 2024, and U.S. Provisional Patent Application Ser. No. 63 / 811,004, filed May 23, 2025. The entirety of the above-mentioned patent applications are hereby incorporated by reference in their entireties. TECHNICAL FIELD

[0002] This disclosure relates generally to flow cell devices, fluidic dispensing and control devices for performing DNA sequencing. BACKGROUND

[0003] Flow cell devices are used in chemistry and biotechnology applications. In next- generation sequencing (NGS) systems, flow cell devices are used to immobilize template nucleic acid molecules derived from biological samples and then introduce a repetitive flow of sequencing reagents, which can be used to bind labeled nucleotides or labeled multivalent molecules to specific positions in the nucleic acid template molecules. A series of label signals are detected and decoded to reveal the nucleotide sequences of the nucleic acid template molecules, e.g., immobilized, or amplified, or combinations thereof, nucleic acid template molecules attached to a surface of the flow cell.

[0004] Existing NGS flow cells are multi-layered structures fabricated from planar surface substrates and other flow cell components, which are then bonded to form fluid flow channels. Such flow cell devices may require costly, multi-step precision fabrication techniques to achieve the required design specifications. On the other hand, inexpensive and off-the-shelf, single channel capillaries are available in a variety of sizes and shapes, but are generally not suited for ease of handling and compatibility with the repetitive switching between reagents required for applications such as NGS. There thus exists a need for additional flow cell devices, and methods of using the same in applications such as NGS. SUMMARY

[0005] Described herein are flow cell devices and systems for sequencing nucleic acids. The flow cell devices, systems, and methods described herein can advantageously achieveefficient delivery and usage of reagents to significantly lower consumable costs (“cost of goods,” or “COGS”) and reduce delivery time of reagents in sequencing analysis. The devices, systems, and methods described herein can advantageously achieve more efficient and effective cleaning and alleviate contamination caused by residual reagents, thereby increasing accuracy and reliability of sequencing analysis. The devices, systems, and methods herein can advantageously allow or cause delivery or purge of an air gap (e.g., an amount of air, a bolus of air, quantum of air, or a flow of air or otherwise gaseous flow) between administration of two liquid reagents without impairing chemical functioning of the flow cell device (also referred to herein as “flow cells”) and its coating(s), which may be infeasible with existing flow cells and their coatings. Such air gaps may greatly facilitate cleaning, and thus reduces contamination by left-over or residual reagents in subsequent reactions on the flow cell. The air gap may improve homogeneity of the reagents across a channel of the flow cell device, thereby reducing concentration gradients of the reagent and improving accuracy of methods carried out on the device, such as sequencing.

[0006] The devices, systems, and methods herein may further eliminate series of tubing, e.g., a common line for different reagents, for reagent administration so that the flow cell devices can be more robust against fluidics errors and adaptable to different fluidic control and administration. As such, the flow cells disclosed herein are more flexible for various sequencing applications. For example, the flow cell devices herein do not require locked-in tubing, so that errors caused by malfunction of the tubing, e.g., a clogged tube, can be easily resolved in comparison to existing sequencing systems. As another example, the flow cell devices herein can be conveniently adapted for the addition / removal of nozzles or dispensing tips for a new sequencing application. Furthermore, the flow cell devices and systems herein can utilize an open landing area in combination with the air gap, which advantageously achieves a more homogenous distribution of reagents on the flow cell and / or less consumption of reagents in comparison to existing devices. The flow cell devices and systems described herein are suitable for rapid DNA sequencing and can help realize more efficient use of expensive reagents and reduce the amount of time required for sample pre- treatment and replication compared to other DNA sequencing techniques. Therefore, flow cell devices and systems described herein can result in a faster and more cost-effective sequencing method than other systems known in the art.

[0007] The disclosure provides a system for preparing one or more samples on a flow cell device, comprising: a plate with one or more wells extending from a top surface of the plate to a bottom surface of the plate; a flow cell substrate configured to receive the one or moresamples through the one or more wells and to immobilize the one or more samples on the flow cell substrate; a seal that seals a gap between the plate and the flow cell substrate; and a cover that protects the flow cell substrate from contamination.

[0008] In some embodiments of the system, the bottom surface of the plate is coupled to the seal, with the plate being positioned above the flow cell substrate. In some embodiments, the seal is preassembled to the bottom surface of the plate. In some embodiments, the seal comprises a layout identical to a layout of the bottom surface of the plate. In some embodiments, the seal protects a top surface of the flow cell substrate from mechanical impact from the plate. In some embodiments, the seal comprises one or more adhesive layers for attaching to the plate, the flow cell substrate, or both.

[0009] In some embodiments of the system, the flow cell substrate is a bottom substrate of the flow cell device. In some embodiments, the flow cell substrate is a top substrate of the flow cell device. In some embodiments, the flow cell substrate is configured to be fixedly coupled to a top substrate, a middle substrate, or both to form one or more microfluidic channels. In some embodiments, the flow cell substrate is configured to be fixedly coupled to a top substrate or a bottom substrate to form the one or more microfluidic channels. In some embodiments, the one or more samples are received and immobilized on a top surface of the flow cell substrate, a bottom surface of the flow cell substrate, or both. In some embodiments, the flow cell substrate is configured to be fixedly coupled to another flow cell substrate to form the flow cell device. In some embodiments, the flow cell substrate comprises glass or plastic.

[0010] In some embodiments of the system, the flow cell device comprises an adhesive layer configured to fixedly attach the top substrate or the bottom substrate to the flow cell substrate. In some embodiments, the adhesive layer comprises an area that does not overlap with the area of the one or more microfluidic channels along a x-y plane that is orthogonal to a z axis along which the plate, the seal, and the flow cell substrate are coupled.

[0011] In some embodiments of the system, the seal comprises a gasket. In some embodiments, the seal comprises a shape that surrounds at least part of each of the one or more microfluidic channels.

[0012] In some embodiments of the system, the adhesive layer comprises a shape that surrounds at least part of each of the one or more microfluidic channels. In some embodiments, the adhesive layer comprises a shape that surrounds the gasket.

[0013] In some embodiments of the system, the one or more microfluidic channels provide fluidic communication between a landing area and the samples immobilized on theflow cell device. In some embodiments, the one or more microfluidic channels provide fluidic communication between a landing area and the samples immobilized on a surface of the one or more microfluidic channels.

[0014] In some embodiments of the system, the cover is coupled to a top surface of the plate.

[0015] In some embodiments of the system, the system further comprises a removable clamp configured to hold the plate, the seal, and the flow cell substrate in a stack for sample preparation, sample examination, or both. In some embodiments, the plate comprises a groove or a tongue configured to reversibly couple the plate to the removable clamp. In some embodiments, the plate comprises a height along a z axis in a range from 0.5 mm to 5 cm, from 0.5 mm to 3 cm, or from 1 mm to 3 cm.

[0016] In some embodiments of the system, each of the one or more wells comprises a corresponding cross-sectional area that matches a size and a shape of the cross-sectional area of the corresponding microfluidic channel of the flow cell device. In some embodiments, each of the one or more wells comprises a cross-sectional area that is less than ±5%, ±10%, ±15%, or ±20% different from a corresponding cross-sectional area of a microfluidic channel of the flow cell device. In some embodiments, each of the one or more wells has a uniform cross-sectional area along the z axis. In some embodiments, at least one of the one or more wells has a non-uniform cross-sectional area along the z axis. In some embodiments, the one or more wells comprise a single well.

[0017] In some embodiments of the system, the one or more samples comprise at least 1 x 106, 2 x 106, 3 x 106, 4 x 106, 5 x 106, 6 x 106, 8 x 106, or 1 x 107cells received through the single well and immobilized on the flow cell substrate.

[0018] In some embodiments of the system, the one or more wells comprise two, three, or four wells. In some embodiments, the one or more wells comprise 12 wells.

[0019] In some embodiments of the system, the one or more samples comprise cells, tissue, or organoids. In some embodiments, the one or more samples comprise at least 5 x 103, 1 x 104, 5 x 104, or 1 x 105cells received through each of the one or more wells and immobilized on the flow cell substrate.

[0020] In some embodiments of the system, the one or more wells comprise a first number of wells that matches a second number of microfluidic channels of the flow cell device. In some embodiments, the one or more wells comprise multiple wells arranged in a predetermined spatial pattern. In some embodiments, the one or more wells comprise at least two wells comprising a different cross-sectional area or cross-sectional shape at the bottomsurface of the plate. In some embodiments, the one or more wells comprise at least two wells comprising an identical cross-sectional area or cross-sectional shape at the bottom surface of the plate.

[0021] In some embodiments of the system, the flow cell device is configured to be inserted into a flow cell cartridge or a manifold that is configured to hold the flow cell device and enable fluidic communication between the flow cell device and a fluidic dispensing device, a fluidic reservoir, a waste container, or a combination thereof. In some embodiments, flow cell device, the manifold, or both enables open fluidic communication to the one or more microfluidic channels. In some embodiments, the flow cell device, the manifold, or both enables open dispensing of a reagent to the landing area of the flow cell device. In some embodiments, flow cell device, the manifold, or both enables closed fluidic communication of a reagent between the fluidic reservoir and the one or more microfluidic channels. In some embodiments, the flow cell device, the manifold, the flow cell cartridge, or a combination thereof enables closed fluidic communication of a reagent between the fluidic reservoir and the one or more microfluidic channels. In some embodiments, the closed fluidic communication is through one or more fluidic pathways connecting the manifold or flow cell cartridge to the fluidic reservoir.

[0022] In some embodiments of the system, each of the one or more wells comprises a cross-sectional area that is less than ±5%, ±10%, ±15%, or ±20% different from a corresponding cross-sectional area of a microfluidic channel of the flow cell device at the bottom surface of the plate.

[0023] In some embodiments of the system, the adhesive layer comprises one or more selected from the group comprising: a pressure sensitive adhesive, a double-sided tape, a photo-crosslinkable adhesive, thermo-crosslinkable adhesive, a chemically-cured adhesive, an adhesive made from a monomer, and an adhesive gel.

[0024] The disclosure provides a method for preparing one or more samples on a flow cell device, comprising: coupling a plate with one or more wells to a flow cell substrate, wherein the flow cell substrate is configured to hold the one or more samples immobilized on the flow cell substrate, and wherein the one or more wells extend from a top surface of the plate to a bottom surface of the plate; delivering one or more samples through the one or more wells to the flow cell substrate, wherein the one or more samples are three-dimensional (3D); coupling a cover to the top surface of the plate to protect the flow cell substrate from contamination from the top surface of the plate; incubating or imaging the one or more samples immobilized on the flow cell substrate; analyzing image data of the one or moresamples to determine characteristics describing the one or more samples; determining whether a predetermined criterion has been met by comparing the characteristics against the predetermined criterion; in response to determining that the predetermined criterion has been met, removing the flow cell substrate from the plate and the cover; and forming a flow cell device comprising one or more microfluidic channels by coupling the flow cell substrate to a top substrate or a bottom substrate, wherein the one or more microfluidic channels enables fluidic communication from a dispensing device to the one or more samples.

[0025] The disclosure provides a method for preparing one or more samples on a flow cell device, comprising: providing: a plate with one or more wells extending from a top surface of the plate to a bottom surface of the plate; a flow cell substrate configured to immobilize the one or more samples; a seal that seals a gap between the plate and the flow cell substrate; and a cover that protects the flow cell substrate from contamination; coupling the plate to the flow cell substrate with the seal disposed between the plate and the flow cell substrate; openly delivering the one or more samples through the one or more wells to the flow cell substrate, wherein the one or more samples are three-dimensional (3D); removing the flow cell substrate from the plate, in response to determining that a predetermined criterion has been met; and forming a flow cell device comprising one or more microfluidic channels by coupling the flow cell substrate to a top substrate or a bottom substrate, wherein the one or more fluidic channels provides fluidic communication from a dispensing device to the one or more samples.

[0026] In some embodiments of the methods, coupling the flow cell substrate to the top substrate or the bottom substrate comprises providing a pressure, a vacuum, a temperature change, or a photonic energy to fixedly couple the flow cell substrate to the top substrate or the bottom substrate.

[0027] The disclosure provides a method for detecting leakage of a flow cell device, comprising: positioning a flow cell device on a sequencing system for a sequencing run, comprising: connecting an outlet of the flow cell device to a pump in a first sealed connection; connecting an inlet of the flow cell device to a valve in a second sealed connection, wherein the valve is in a closed position; pumping, by the pump, gas via the outlet to the flow device for a first predetermined period of time at a predetermined pressure level; sensing, by a pressure sensor, a gas pressure in a microfluidic channel of the flow cell device during the predetermined period of time; in response to detecting that the gas pressure is over a predetermined threshold pressure over a threshold duration, switching the valve to an open position; introducing liquid via the valve or the inlet to the flow cell device for asecond predetermined period of time; acquiring, by an image sensor of the sequencing system, a first image of the flow cell device at a first z-level and at a first time point and a second image of the flow cell device at the first z-level and at a second time point while introducing gas via the valve or the outlet to the flow cell device; in response to determining that the first image and the second image are different, determining that the microfluidic channel has leakage; and in response to determining that the first image and the second image are not different, determining that the microfluidic channel lacks leakage.

[0028] The disclosure provides a method for detecting leakage of a flow cell device, comprising: positioning a flow cell device on a sequencing system for a sequencing run, comprising: connecting an outlet of the flow cell device to a pump in a first sealed connection; connecting an inlet of the flow cell device to a valve in a second sealed connection, wherein the valve is in a closed position; pumping, by the pump, gas via the outlet to the flow cell device for a first predetermined period of time at a predetermined pressure level; sensing, by a pressure sensor, gas pressure in a microfluidic channel of the flow cell device during the predetermined period of time; and in response to detecting that the gas pressure is below a predetermined threshold pressure over a threshold duration, determining, by a processor of the sequencing system, that the microfluidic channel has leakage.

[0029] The disclosure provides a method for detecting leakage of a flow cell device, comprising: positioning a flow cell device on a sequencing system for a sequencing run, comprising: connecting an outlet of the flow cell device to a pump in a first sealed connection; connecting an inlet of the flow cell device to a valve in a second sealed connection, wherein the valve is in an open position; acquiring, by an image sensor of the sequencing system, a first image of the flow cell device at a first z-level and at a first time point and a second image of the flow cell device at the first z-level and at a second time point while introducing liquid via the valve or the outlet to the flow cell device for a predetermined period of time; in response to determining that the first image and the second image are different, determining that the microfluidic channel has leakage; and in response to determining that the first image and the second image are not different, determining, by a processor of the sequencing system, that the microfluidic channel lacks leakage.

[0030] In some embodiments of the methods disclosed herein, the microfluidic channel lacks any fluid therewithin. In some embodiments, the microfluidic channel comprises a surface on which the one or more samples are immobilized.

[0031] In some embodiments of the methods, the flow cell device comprises a flow cell substrate on which the one or more samples are immobilized.

[0032] In some embodiments of the methods, each of the first and the second sealed connection is liquid-sealed. In some embodiments, at least one of the first and the second sealed connection is not gas-sealed.

[0033] In some embodiments of the methods, connecting the outlet of the flow cell device to the pump in the first sealed connection comprises: sealing a landing area of the flow cell device with a sealing element external to the sequencing system. In some embodiments, connecting the outlet of the flow cell device to the pump in the first sealed connection comprises: connecting the outlet of the flow cell device to the pump via one or more first closed fluidic pathways. In some embodiments, the one or more first closed fluidic pathways comprise tubing and a common line. In some embodiments, connecting the inlet of the flow cell device to the valve in the second sealed connection comprises: connecting the inlet to the valve via one or more second closed fluidic pathways.

[0034] In some embodiments of the methods, the pressure sensor is located in the one or more first closed fluidic pathways. In some embodiments, the pressure sensor is located in the one or more second closed fluidic pathways.

[0035] In some embodiments of the methods, the gas comprises air.

[0036] In some embodiments of the methods, the first image of the flow cell device is at the first time point when the microfluidic channel lacks any gas bubbles at the first z-level and the second image of the flow cell device at the second time point when the microfluidic channel has gas bubbles at the first z-level. In some embodiments, the first image or the second image comprises light intensities of a light source reflected by the flow cell device.

[0037] In some embodiments of the methods, the valve is in fluidic connection with a buffer reservoir.

[0038] In some embodiments of the methods, the pump is a syringe pump in fluidic connection with a reagent reservoir or a buffer reservoir. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The novel features of the inventive concepts are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present inventive concepts will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the inventive concepts are utilized, and the accompanying drawings of which:

[0040] FIG. 1 illustrates a block diagram of a computer-implemented system for performing operations in DNA sequencing and sequencing analysis, according to some embodiments.

[0041] FIG. 2 is a schematic showing a flow cell system, according to some embodiments.

[0042] FIG. 3 is a schematic showing a flow cell device , according to some embodiments.

[0043] FIG. 4 is a schematic showing a flow cell device, according to some embodiments.

[0044] FIGS. 5A-5F show flow cell devices, according to some embodiments. FIG. 5A is a perspective view of the substrates, according to some embodiments. FIG. 5B is a top view of the flow cell device in FIG. 5A. FIG. 5C is a cross-sectional view of the flow cell device at D-D’ in FIG. 5B. FIG. 5D is a perspective view of substrates of a flow cell device, according to some embodiments. FIG. 5E is a perspective view of substrates of a flow cell device, according to some embodiments. FIG. 5F shows a perspective view and a top view of a flow cell device, according to some embodiments.

[0045] FIGS. 6A-6C show fluidic control devices of the flow cell systems for delivery of reagents to a flow cell device, according to some embodiments. FIG. 6A shows a fluidic control device including a dispenser (vertical line, 680) and a continuous track (left most arrow, 691). FIG. 6B shows a fluidic control device including a dispensing plate (692) with an electrowetting surface. FIG. 6C shows a fluidic control device comprising a reagent reservoir (694) and a sipper (693).

[0046] FIG. 7A shows a graph illustrating contamination levels achieved by flow cell systems disclosed herein in comparison to existing flow cell systems.

[0047] FIG. 7B shows a table illustrating reduction of reagent consumption during a same sequencing application achieved by a flow cell system disclosed herein in comparison to an existing flow cell system. COGS: cost of goods sold.

[0048] FIG. 8 illustrates a block diagram of a computer system for fluidic control and for performing sequencing and sequencing analysis, according to some embodiments.

[0049] FIG. 9 is a schematic showing an exemplary linear single stranded library molecule (900) which comprises: a surface pinning primer binding site (920); an optional left unique identification sequence (980); a left index sequence (960); a forward sequencing primer binding site (940); an insert region having a sequence of interest (910); a reversesequencing primer binding site (950); a right index sequence (970); and a surface capture primer binding site (930).

[0050] FIG. 10 is a schematic showing an exemplary linear single stranded library molecule (900) which comprises: a surface pinning primer binding site (920); a left index sequence (960); a forward sequencing primer binding site (940); an insert region having a sequence of interest (910); a reverse sequencing primer binding site (950); a right index sequence (970); an optional right unique identification sequence (990); and a surface capture primer binding site (930).

[0051] FIG. 11 is a schematic of various configurations of multivalent molecules. Left (Class I): schematics of multivalent molecules having a “starburst” or “helter-skelter” configuration. Center (Class II): a schematic of a multivalent molecule having a dendrimer configuration. Right (Class III): a schematic of multiple multivalent molecules formed by reacting streptavidin with 4-arm or 8-arm PEG-NHS with biotin and dNTPs. Nucleotide units are designated ‘N’, biotin is designated ‘B’, and streptavidin is designated ‘SA’.

[0052] FIG. 12 is a schematic of a multivalent molecule comprising a generic core attached to a plurality of nucleotide-arms.

[0053] FIG. 13 is a schematic of a multivalent molecule comprising a dendrimer core attached to a plurality of nucleotide-arms.

[0054] FIG. 14 shows a schematic of a multivalent molecule comprising a core attached to a plurality of nucleotide-arms, where the nucleotide arms comprise biotin, spacer, linker and a nucleotide unit.

[0055] FIG. 15 is a schematic of a nucleotide-arm comprising a core attachment moiety, spacer, linker and nucleotide unit.

[0056] FIG. 16 shows the chemical structure of a spacer (top), and the chemical structures of various linkers, including an 11-atom Linker, a 16-atom Linker, a 23-atom Linker and an N3 Linker (bottom).

[0057] FIG. 17 shows the chemical structures of various linkers, including Linkers 1-9.

[0058] FIG. 18 shows the chemical structures of various linkers joined / attached to nucleotide units.

[0059] FIG. 19 shows the chemical structures of various linkers joined / attached to nucleotide units.

[0060] FIG. 20 shows the chemical structures of various linkers joined / attached to nucleotide units.

[0061] FIG. 21 shows the chemical structures of various linkers joined / attached to nucleotide units.

[0062] FIG. 22 shows the chemical structure of a biotinylated nucleotide-arm. In this example, the nucleotide unit is connected to the linker via a propargyl amine attachment at the 5 position of a pyrimidine base or the 7 position of a purine base.

[0063] FIG. 23 shows a schematic illustration of one embodiment of the flow cell devices in which the support comprises a glass substrate and alternating layers of hydrophilic coatings which are covalently or non-covalently adhered to the glass, and which further comprises chemically-reactive functional groups that serve as attachment sites for oligonucleotide primers.

[0064] FIGS. 24A-24E show an embodiment of the flow cell device depicted in FIGS. 5A-5D. FIG. 24A is a perspective view of the flow cell device. FIG. 24B is a perspective view of the flow cell device showing the top, middle and bottom substrates. FIG. 24C is a top view of the top substrate of the flow cell device. FIG. 24D is a top view of the middle substrate of the flow cell device. FIG. 24E is a top view of the bottom substrate of the flow cell device.

[0065] FIGS. 25A-25E show an embodiment of the flow cell device disclosed herein. FIG. 25A is a perspective view of the flow cell device. FIG. 25B is a perspective view of the flow cell device showing the top, middle and bottom substrates. FIG. 25C is a top view of the top substrate of the flow cell device. FIG. 25D is a top view of the middle substrate of the flow device. FIG. 25E is a top view of the bottom substrate of the flow cell device.

[0066] FIGS. 26A-26C show embodiments of the flow cell device disclosed herein. FIG. 26A is a top view of an embodiment of the flow cell device. FIG. 26B is a top view of another embodiment of the flow cell device. The flow device in FIG. 26A comprises a differently sized open landing area and inlet as compared to the flow cell device in FIG. 26B or FIGS. 24A-24E. The tapered transition portion from the cleaning outlet to the open landing area of the flow cell device in FIG. 26A is also altered from embodiments in FIG. 26B or FIGS. 24A-24E. FIG. 26C is a top view of yet another embodiment of the flow cell device. The flow device in FIG. 26C comprises a differently sized open landing area and inlet as compared to the flow cell device in FIGS. 25A-25E.

[0067] FIGS. 27A-27G show an embodiment of the flow cell device disclosed herein. FIG. 27A is a side view of the flow cell device. FIG. 27B shows a cross-sectional view at A- A in FIG. 27A. FIG. 27C is a top view of the flow cell device. FIG. 27D is a cross-sectional view at B-B in FIG. 27B. FIG. 27E shows an expanded view of area A in FIG. 27B. FIG. 27Fshows an expanded view of area C in FIG. 27C. FIG. 27G shows an expanded view of area B in FIG. 27D.

[0068] FIGS. 28A-28C show an embodiment of the flow cell device in FIG. 5E in a top view (FIG. 28A), a perspective view (FIG. 28B), and a top view of the bottom, middle, and top substrates (FIG. 28C).

[0069] FIG. 29 shows a graph illustrating contamination levels of individual tiles and average contamination level across multiple tiles of the flow cell device achieved by flow cell systems disclosed herein.

[0070] FIGS. 30A-30B show schematics of two embodiments of the fluid dispensing device herein.

[0071] FIGS. 31A-31B show schematics of an embodiment of the fluid dispensing device herein. The fluid dispensing device is coupled to an actuator configured to actuate the pump(s) of the fluid dispensing device. The fluid dispensing device, in this particular embodiment, includes a microfluidic chip (FIG. 31B).

[0072] FIG. 31C shows a schematic of the fluid dispensing device in FIGS. 31A-31B coupled to an actuator configured to move relative to the microfluidic chip and actuate the pump(s) of the fluid dispensing device.

[0073] FIGS. 32A-32B shows a schematic of the cross section of the fluid dispensing device in FIGS. 31A-31B with the movable pin configured to deliver fluid from the microfluidic chip to the dispensing tips. The movable pin is mechanically coupled to the actuator (FIG. 32A) or the microfluidic chip (FIG. 32B).

[0074] FIGS. 33A-33B shows a schematic of an embodiment of the fluid dispensing device herein with barrels and plungers. Each of the barrel and plunger pairs connects a different compartment of the fluid dispensing device to a microfluidic pathway of the microfluidic chip, and is configured to deliver fluid from the cartridge to the microfluidic chip, and then to the dispensing tips.

[0075] FIGS. 34A-34C show schematics of an embodiment of the fluid dispensing device herein with barrels and plungers. The barrel and plunger pairs connect to individual reservoirs and then to corresponding compartments of the fluid dispensing device through individual valves. The barrel and plunger pair is configured to deliver fluid from the cartridge to the microfluidic chip, and then to the dispensing tips through the corresponding valve.

[0076] FIGS. 35A -35C show schematics of an embodiment of the fluid dispensing device with the reagent cartridge, the transportation valve, and the microfluidic chip.

[0077] FIGS. 36A-36B show schematics of a cross-sectional view of the fluidic dispensing device in FIGS. 35A-35C.

[0078] FIGS. 37A-37D show schematics of a bottom view of the fluidic dispensing device (FIG. 37A) in relation to the flow cell device, and a bottom view of the microfluidic chip in relation to the transportation valve (FIGS. 37B-37D).

[0079] FIGS. 38A-38B show schematics of an embodiment of the fluid dispensing device herein with the transportation valve and the microfluidic chip.

[0080] FIGS. 39A-39C show the at least two opening positions for reagent aspiration and dispensing (FIG. 39B, FIG. 39C) and the closed position (FIG. 39A) of the transportation valve in relation to the microfluidic chip.

[0081] FIG. 40 shows a schematic of an embodiment of the fluid dispensing device herein with the transportation valve and the microfluidic chip.

[0082] FIG. 41 shows a schematic of an embodiment of the system for preparing samples on the flow cell device disclosed herein.

[0083] FIGS. 42A-42D show schematics of embodiments of the plate of the sample preparation system with one or more wells for delivering one or more samples to the flow cell substrate.

[0084] FIG. 43A shows an exemplary embodiment in which the flow cell substrate is the bottom substrate of the flow cell device.

[0085] FIG. 43B shows an exemplary embodiment in which the flow cell substrate is coupled to a top substrate or bottom substrate with a seal and an optional adhesive layer.

[0086] FIG. 44 shows an exemplary embodiment of a cartridge configured to contain the flow cell device therein and enable fluidic communication of the flow cell device to the fluidic reservoir, the dispensing device, and / or a waste container.

[0087] FIG. 45 shows an exemplary embodiment of the sample preparation system that can prepare multiple flow cell substrates in parallel.

[0088] FIGS. 46A-46B show exemplary embodiments of the flow cell substrate, after sample preparation, which can be coupled with another flow cell substrate to form a flow cell device.

[0089] FIGS. 46C-46E show exemplary embodiments of the flow cell substrate, after sample preparation, which can have one or more sample distribution areas and be coupled with another flow cell substrate to form a flow cell device with one or more open landing areas.

[0090] FIG. 47A shows an exemplary embodiment of detecting leakage of the microfluidic channel(s) of the flow cell device using pressure changes therewithin.

[0091] FIG. 47B shows exemplary pressure changes over time in flow cell devices with different levels of leakage.

[0092] FIG. 48 shows an exemplary embodiment of detecting leakage of the microfluidic channel(s) of the flow cell device using an auto-focus (AF) laser and AF sensor of the sequencing system. DETAILED DESCRIPTION

[0093] Described herein are systems and devices to analyze different nucleic acid sequences, e.g. from amplified nucleic acid arrays in flow cells, from an array of immobilized nucleic acids, or from a sample disposed within a flow cell. The systems and devices described herein can also be useful in, e.g., sequencing for comparative genomics, tracking gene expression, microRNA sequence analysis, epigenomics, and aptamer and phage display library characterization, and other sequencing applications. The systems and devices herein comprise various combinations of optical, mechanical, fluidic, thermal, electrical, and computing devices / aspects. The systems and devices described herein can also be useful for imaging applications that use sequencing systems to image target analytes such as proteins or nucleic acid in cells or tissues disposed within flow cell devices. See, for example, WO2021 / 061841, WO2023 / 205707, WO2024040068, WO2025147667 and WO2021061841, the contents of which are incorporated by reference in their entireties herein.

[0094] The advantages of the disclosed flow cell devices, fluidic control devices, and systems include, but are not limited to: significantly lower consumable costs (e.g., as compared to those for currently available nucleic acid sequencing systems); efficient and effective cleaning of flow cell devices, thereby reducing contamination of sequencing processes by residual reagent(s); reduced delivery time of reagents, reduced washing time, and increased homogeneity of reagents on the flow cells; reduced device and system manufacturing / maintenance complexity and cost; flexible system throughput and flexible adaptation of the systems to different sequencing applications.

[0095] Capillary flow cell devices, cartridges, and systems may include, but are not limited to: an open dispensing tip in the fluidic control device and an open landing area on the flow cell device to allow open delivery of reagents without the complexity and cost of existing tubing and to enable flexibility in the systems to adapt to different sequencing applications; a slippery coating that facilitates fluidic transfer and residual cleaning from theopening landing area; a cleaning outlet in fluidic connection to the open landing area to facilitate cleaning of liquid meniscus that cannot be effectively cleaned using washing reagents alone; a channel coating that allows purging of an air gap between two fluidic reagents without damaging subsequent sequencing reactions; and compatibility with a wide variety of detection methods such as fluorescence imaging.

[0096] Although the disclosed flow cell devices and systems are described primarily in the context of their use for nucleic acid sequencing applications, various aspects of the disclosed devices and systems may be applied not only to nucleic acid sequencing but also to any other type of chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis applications. It shall be understood that different aspects of the disclosed devices and systems can be appreciated individually, collectively, or in combination with each other. Sequencing Systems

[0097] Disclosed herein, in some embodiments, are flow cell devices and systems that can be employed for performing or facilitating DNA sequencing analysis using sequencing systems. The sequencing systems may utilize various sequencing techniques including but not limited to the sequencing techniques disclosed herein.

[0098] FIG. 1 illustrates a block diagram of a computer-implemented system 100 for performing sequencing and sequencing analysis, according to one or more embodiments disclosed herein. The system 100 has a sequencing system 110 that includes a flow cell device 112, a sequencer 114, an imager 116, a data storage device 122, and a user interface 124. The sequencing system 110 may optionally be connected to a cloud 130 (e.g., coupled to a server, compute device, database, etc.). The sequencing system 110 may include one or more of dedicated processors 118, an integrated circuit (e.g., Field-Programmable Gate Array(s) (FPGAs)) 120, and a computer system 126.

[0099] In some embodiments, the flow cell device 112 is configured to capture DNA fragments and form DNA sequences for base-calling on the flow cell device. The flow cell device 112 can include a support as disclosed herein. The support can be a solid support. The support can include a surface coating thereon as disclosed herein. The surface coating can be a polymer coating as disclosed herein. The surface coating can be on a surface of the one or more channels of the flow cell device. A different or identical surface coating can be placed on a surface of the inlet of the flow cell device.

[0100] In some embodiments, the flow cell device 112 can include a plurality of tiles (e.g., portions, locations, areas, sections, etc.) thereon configured to be imaged by the imager 116, and each tile may be separated into a plurality of subtiles. In some embodiments, the subtiles may be organized in a grid. Each subtile can include a plurality of clusters or polonies (e.g., a collection of DNA molecules such as the concatemer template molecules disclosed herein) thereon. In some embodiments, the flow cell device 112 may include a number of tiles in a range of about 1 tile to about 2000 tiles, for example about 100 tiles to about 1500 tiles, or about 200 tiles to about 500 tiles, inclusive of all ranges and subranges therebetween. In some embodiments, each tile may be divided into a number of subtiles in a range of about 2 subtiles to about 200 subtiles, about 10 subtiles to about 100 subtiles, or about 20 subtiles to about 50 subtiles, inclusive of all ranges and subranges therebetween. In some embodiments, the subtiles may be organized in a grid that may have M by N subtiles. As a nonlimiting example, a flow cell can have 424 tiles, and each tile can be divided into a 6 x 9 grid, and therefore contains 54 subtiles. In some embodiments, the imager 116 may be configured to obtain one or more images (hereinafter, “flow cell image(s)” of the plurality of tiles, a subset of the plurality of tiles, and / or a subset of the plurality of subtiles. The flow cell image(s) as disclosed herein can include an image including signals (e.g., fluorescence levels) of the plurality of clusters or polonies. The flow cell image can include one or more tiles of signals or one or more subtiles of signals. In some embodiments, a flow cell image can be an image that includes all the tiles and approximately all signals thereon. The flow cell image can be acquired from a channel during (i) an imaging cycle or (ii) a sequencing cycle using the imager 116. In some embodiments, each tile may include millions of polonies or clusters. As a nonlimiting example, a tile can include about 1 to 10 million clusters or polonies. Each polony can be a collection of many copies of DNA molecules. Imaging systems and methods of using same to image flow cells are described, for example in US. Patent No. 11,408,032, WO2025024672, WO2025196650, WO2020118255, WO2021 / 146597, WO2023004014, WO2024 / 118641, WO 2024 / 158927, WO2024173403 and WO2024243548, the contents of which are incorporated by reference in their entireties herein.

[0101] More details of the flow cell device 112 and its functional and structural elements are disclosed herein in relation to figures, e.g., FIGS. 2-4, 5A-5F, 6A-6C, 7A-7B, 24A-24E, 25A-25E, 26A-26C, 27A-27G and 28A-28C.

[0102] The sequencer 114 may be configured to flow mixtures of reagents onto the flow cell. Such mixtures of reagents include nucleotide mixtures, polymerases, reagents to add or cleave chain terminating moieties from the nucleotides in between nucleotide addition steps,and perform other steps for the formation of the DNA molecules suitable for sequencing applications on the flow cell 112. Methods of sequencing, and of preparing nucleic acid libraries for sequencing, are described, for example in WO2022266470, US Publication No. 2023-0279382, U.S. Publication No. US 2023-0279483, WO2024159166, U.S. Publication No. US 2024-0084380, U.S. Publication No. US 2024-0011022, U.S. Publication No. 2024- 0191225, WO2025120579, WO2025024465, WO2020102594 and WO2020243017, the contents of which are incorporated by reference in their entireties herein. The nucleotides may have fluorescent elements (also referred to as “labels” or “moieties”) attached that emit light or energy at a wavelength that indicates the type of nucleotide. Suitable fluorescent elements, also referred to as dyes and fluorophores, are described, for example, in U.S. Publication No. 2024-0240249 the contents of which are incorporated by reference in their entirety herein. Each type of fluorescent element may correspond to a particular nucleotide base (e.g., A, G, C, T). The fluorescent elements may emit light in visible wavelengths, and / or in non-visible wavelengths. In some embodiments, the sequencer 114 and the flow cell device 112 may be configured to perform various sequencing methods disclosed herein or known in the art, for example, sequencing-by-avidite, sequencing by binding, or sequencing by synthesis.

[0103] For example, each nucleotide base may be assigned a color. Different types of nucleotides can be assigned different colors, for example by conjugation to different fluorescent elements (sometimes referred to herein as “labels” or “dyes”). Adenine (A) may be red, cytosine (C) may be blue, guanine (G) may be green, and thymine (T) may be yellow, for example. The color or wavelength of the fluorescent element for each nucleotide may be selected so that the nucleotides are distinguishable from one another based on the wavelengths of light emitted by the fluorescent elements.

[0104] The imager 116 may be configured to capture images of the flow cell device 112 after each flowing step. In an embodiment, the imager 116 is a camera configured to capture digital images, such as a CMOS or a CCD camera. The camera may be configured to capture images at the wavelengths of the fluorescent elements bound to the nucleotides. The images can be called flow cell images.

[0105] In some embodiments, the imager 116 can include one or more optical systems disclosed herein. Exemplary optical systems are described, for example, in WO2023004014, WO2024158927 and WO2024173403, the contents of which are incorporated by reference in their entireties herein. The optical system(s) can be configured to capture optical signals fromthe flow cell and generate corresponding digital images thereof. The digital images can then be used for base calling.

[0106] In some embodiments, the images of the flow cell may be captured in groups, where each image in the group is taken at a wavelength or in a spectrum that matches or includes one of the fluorescent elements. In other embodiments, the images may be captured as single images that captures all of the wavelengths of the fluorescent elements.

[0107] The resolution of the imager 116 controls the level of detail in the flow cell images, including pixel size. In existing systems, this resolution controls the accuracy with which a spot-finding algorithm identifies the polony centers. In some embodiments, the image resolution of flow cell images disclosed herein can be about 10 nanometers (nm) to 900 nm, inclusive of all ranges or subranges therebetween. In some embodiments, the image resolution of the flow cell images can be between about 10 nm to about 900 nm, about 10 nm to about 500 nm, about 10 nm to about 200 nm, about 20 nm to about 500 nm, about 20 nm to about 200 nm, or any range or subrange therebetween. One way to increase the accuracy of spot finding is to improve the resolution of the imager 116, or improve the processing performed on images taken by imager 116. Detecting polony centers in pixels other than those detected by a spot-finding algorithm can be performed. Suitable spot-finding algorithms will be known to persons of ordinary skill in the art. These methods can allow for improved accuracy in detection of polony centers without increasing the resolution of the imager 116. The resolution of the imager 116 may even be less than existing systems with comparable performance, which may reduce the cost of the sequencing system 110.

[0108] The image quality of the flow cell images can control the base calling quality / accuracy. The imager 116 disclosed herein can increase the accuracy of base calling Alternatively, the processing performed on images taken by imager 116 can result in a better image quality.

[0109] After base calling is performed, a processor (e.g., dedicated processors 118, FPGA(s) 120, computer system 126, or a combination thereof) may optionally perform additional processing and / or analysis of base calling results. In some embodiments, after base calling is performed, the sequencing read(s) (processed and / or raw) can be outputted from the system to an external device (e.g., the cloud 130 and / or to a computer system 126). The sequencing read(s) herein can include a forward read (R1), a reverse read (R2), or both. The sequencing reads herein can be any orderly sequence of bases of A, T, C, and G.

[0110] In some embodiments, the sequencing read(s) can be communicated (e.g., directly or indirectly) to the computer system 126 for subsequent analysis such as adaptor trimming, index sequence identification, or phasing, for example.

[0111] These sequencing analysis methods, including primary analysis, secondary analysis, or combinations thereof, can be advantageously performed in parallel in the computer system 126, without interference with or delay of the existing sequencing workflow of the computer-implemented system 100. The results of sequencing analysis can be made available for generating sequencing results for users. Some or all operations of the sequencing process can be advantageously performed by the FPGA(s) and data can be communicated between the CPU(s) and FPGA(s) to reduce the total operational time from methods operating without the FPGA(s).

[0112] The operations or actions disclosed herein may be performed by the dedicated processors 118, the FPGA(s) 120, the computer system 126, or a combination thereof. One or more operations or actions in methods disclosed herein may be performed by the dedicated processors 118, the FPGA(s) 120, the computer system 126, or a combination thereof. In some embodiments, which operations or actions are to be performed by the dedicated processors 118, the FPGA(s) 120, the computer system 126, or their combinations can be determined based on one or more of: a computation time for the specific operation(s), the complexity of computation in the specific operation(s), the need for data transmission between the hardware devices, or combinations thereof.

[0113] The computer system 126 can include one or more general purpose computers that provide interfaces to run a variety of programs in an operating system, such as Windows™ or Linux™. Such an operating system may provide great flexibility to a user.

[0114] In some embodiments, the dedicated processors 118 may not be general-purpose processors, but instead custom processors with specific hardware or instructions for performing method steps. Dedicated processors directly run specific software without an operating system. The lack of an operating system reduces overhead, at the cost of the flexibility in what the processor may perform. A dedicated processor may make use of a custom programming language, which may be designed to operate more efficiently than the software run on general-purpose computers. This may increase the speed at which the steps are performed and allow for real time processing.

[0115] In some embodiments, the FPGA(s) 120 may be configured to perform operations of the sequencing analysis methods described herein. An FPGA is programmed as hardware that may perform a specific task. A special programming language may be used to transformsoftware steps into hardware componentry. Once an FPGA is programmed, the hardware directly processes digital data that is provided to it without running software. The FPGA instead uses logic gates and registers to process the digital data. Because there is no overhead required for an operating system, an FPGA may process data faster than a general-purpose computer. Similar to dedicated processors, this is at the cost of flexibility.

[0116] The lack of software overhead may also allow an FPGA to operate faster than a dedicated processor, although this will depend on the exact processing to be performed and the specific FPGA and dedicated processor.

[0117] A group of FPGA(s) 120 may be configured to perform the steps in parallel. For example, a number of FPGA(s) 120 may be configured to perform a processing step for an image, a set of images, a subtile, or a select region in one or more images. Each FPGA(s) 120 may perform its own part of the processing step at the same time, reducing the time needed to process data. This may allow the processing steps to be completed in real time. Further discussion of the use of FPGAs is provided below.

[0118] Performing the processing steps in real time may allow the system to use less memory, as the data may be processed as it is received. This improves over existing systems, which may need to store the data before it may be processed, and which may require more memory or accessing of a computer system located in the cloud 130.

[0119] In some embodiments, the data storage device 122 is used to store information used in or obtained from sequencing analysis. For example, the DNA sequences determined after adaptor trimming may be stored in the data storage device 122. Compressed, or uncompressed, or combinations thereof, sequencing data may be stored in the data storage device 122. The FASTQ file may also be stored in the data storage device 122.

[0120] The user interface 124 may be used by a user to operate the sequencing system or access data stored in the data storage device 122 or the computer system 126. The computer system 126 may control the general operation of the sequencing system and may be coupled to the user interface 124. In some embodiments, the computer system 126 may perform one or more steps in sequencing analysis, such as base calling, adaptor trimming, demultiplexing, phasing etc. In some embodiments, the computer system 126 may be structurally and / or functionally similar to computer system 800, as described in more detail in FIG. 8. The computer system 126 may include a memory configured to store information regarding the operation of the sequencing system 110, such as, for example, configuration information, instructions for operating the sequencing system 110, or user information. The computer system 126 may be configured to pass information between the sequencing system 110 andthe cloud 130. For example, the computer system 126 may be configured to receive base calling results from the dedicated processors 118 and / or FPGA(s) and send the base calling results to the cloud 130 for storage and / or further analysis.

[0121] As discussed above, the sequencing system 110 may have dedicated processors 118, FPGA(s) 120, or the computer system 126. The sequencing system may use one, two, or all of these elements to accomplish the processing described above. In some embodiments, when these elements are present together, the processing tasks are split between them. For example, the FPGA(s) 120 may be used to perform some portion or all of sequencing analysis operations, while the computer system 126 may perform other processing functions for the sequencing system 110. Various combinations of these elements may allow various system embodiments that balance efficiency and speed of processing with cost of processing elements.

[0122] The cloud 130 may be a network, remote storage, or some other remote computing system separate from the sequencing system 110. The connection to cloud 130 may allow access to data stored externally to the sequencing system 110 or allow for updating of software in the sequencing system 110. Flow Cell Devices

[0123] Disclosed herein are flow cell devices and systems that can be employed for performing or facilitating DNA sequencing analysis. Flow cell devices herein can be used to immobilize biological samples, or template nucleic acid molecules derived from biological samples, and introduce a repetitive flow of sequencing reagents (e.g., sequencing-by-binding, sequencing-by-synthesis, or sequencing-by-avidite, or combinations thereof) to attach labeled nucleotides or labeled multivalent molecules to specific positions in the template sequences. A series of labeled signals are detected and decoded to reveal the nucleotide sequences of the template molecules, e.g., immobilized, or amplified, or combinations thereof, nucleic acid template molecules attached to a surface of the flow cell.

[0124] In some embodiments, a flow cell device disclosed herein can comprise a support having one or more substrates, a number of channels, an inlet, and an outlet. FIGS. 2-4, and 5A-5F show embodiments of flow cell devices.

[0125] In some embodiments, the flow cell device 200, 300, 400, 500 disclosed herein can include a support 210, 510. The support 210, 510 can be solid. At least part of the support 210, 510 can be transparent so that light transmitting from a light source of the imager (116 inFIG. 1) can travel through the transparent portion of the support and reach the samples located on the flow cell device 200, 300, 400, 500.

[0126] The support 210, 510 can comprise one or more substrates 320, 322, 330, 420, 422, 430, 520, 522, 530. As shown in FIGS. 3-4, 5A and 5C, the one or more substrates can include a top substrate 320, 420, 520 and a bottom substrate 330, 430, 530. When the flow cell device 112 is placed in the sequencing system 110 for imaging, the top substrate 320, 420, 520 can be closer to the camera of the imager 116, along the z direction, than the bottom substrate 330, 430. The bottom substrate 330, 430, 530 can be closer to a translation stage of the sequencing system 110 for holding and supporting the flow cell device 112 during sequencing than the top substrate 320, 420, 520.

[0127] In some embodiments, the flow cell device 200, 300, 400, 500 can further include a middle substrate 322, 422, 522 in between the top 320, 420, 520 and the bottom substrate 330, 430, 530 as shown in FIGS. 3-4, 5A and 5C.

[0128] Each substrate can have a predetermined thickness, and different substrates can have different thickness. In some embodiments, each substrate can have a uniform thickness along the z direction. In some embodiments, each substrate can have a uniform thickness along the z direction in at least a portion of the substrate. For example, the portion with uniform thickness can encompass the channel(s) or the imaging areas of the flow cell device 112.

[0129] In some embodiments, the top or bottom substrate can have a thickness of about 0.2 mm to about 5 mm. In some embodiments, the top or bottom substrate can have a thickness of about 0.6 mm to about 3 mm. In some embodiments, the top or bottom substrate can have a thickness of about 0.8 mm to about 2 mm. In some embodiments, the top or bottom substrate can have a thickness of about 0.8 mm to about 1.5 mm. In some embodiments, the top or bottom substrate can have a thickness of about 0.8 mm to about 1.2 mm. In some embodiments, the top or bottom substrate can have a thickness of about 0.9 mm to about 1.1 mm.

[0130] In some embodiments, the top or bottom substrate can have a thickness of 0.2 mm to 5 mm. In some embodiments, the top or bottom substrate can have a thickness of 0.6 mm to 3 mm. In some embodiments, the top or bottom substrate can have a thickness of 0.8 mm to 2 mm. In some embodiments, the top or bottom substrate can have a thickness of 0.8 mm to 1.5 mm. In some embodiments, the top or bottom substrate can have a thickness of 0.8 mm to about 1.2 mm. In some embodiments, the top or bottom substrate can have a thickness of0.9 mm to 1.1 mm. In some embodiments, the top or bottom substrate can have a thickness of 0.95 mm to 1.05 mm.

[0131] In some embodiments, the middle substrate can have a thickness of about 40 to 200 . In some embodiments, the middle substrate can have a thickness of about 40 to 150 . In some embodiments, the middle substrate can have a thickness of about 40 to 70 . In some embodiments, the middle substrate can have a thickness of about 80 to 120 . In some embodiments, the middle substrate can have a thickness of about 60 to 90 .

[0132] In some embodiments, the middle substrate can have a thickness of 40 to 200 . In some embodiments, the middle substrate can have a thickness of 40 to 150 . In some embodiments, the middle substrate can have a thickness of 40 to 70 . In some embodiments, the middle substrate can have a thickness of 80 to 120 . In some embodiments, the middle substrate can have a thickness of 60 to 90 .

[0133] In some embodiments, the substrate(s) can have an elongate shape extending along the y axis. In some embodiments, the substrate(s) can have various shapes such as rectangular, square, etc.

[0134] In some embodiments, the one or more substrates can have one or more surfaces that are planar. In some embodiments, the one or more substrates contain no curvature perceivable to naked eyes, e.g., as shown in FIGS. 2-4, so that the one or more substrates can have planar surfaces. In some embodiments, the flatness of the surface(s) of the substrates can be measured as the height from its peak to valley in an direction orthogonal to the surface(s). The height can be less than about 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, or 0.09 mm, e.g., along a direction orthogonal to the surface. In other words, the flat surface(s) of the substrates may fit between two parallel planar 2D planes that less than 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, or 0.09 mm apart from each other. In some embodiments, the flatness of the surface(s) of the substrates can include a height from its peak to valley that is less than 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, or 0.09 mm. However, the substrates do not have to be planar in certain embodiments. Alternatively, a part or the entirety of one or more substrates can be curved.

[0135] In some embodiments, the support or the one or more substrates can comprise glass, plastic, or polymers. In some embodiments, the support or one or more substrates are all-glass or all-plastic. In some embodiments, the support or the one or more substrates can comprise a tape such as a pressure sensitive adhesive (PSA) tape. For example, the middlesubstrate as shown in FIG. 3 can be made from PSA tape and can conveniently tape the top and bottom substrates to it fixedly.

[0136] The substrate(s) 320, 322, 330, 420, 422, 430, 520, 522, 530 can define one or more channels 250, 350, 550 of the flow cell devices 200, 300, 400, 500. The channels 250, 350, 550 can allow reagents, e.g. a fluid such as a liquid, or a gas, to flow therethrough.

[0137] The gas herein can comprise one type of gas or a combination of different types of gases. In some embodiments, the gas comprises air. The gas can comprise dry air. In some embodiments, the gas comprises one or more inert gases. In some embodiments, the gas comprises one or more active gases.

[0138] The reagents herein can comprise liquid. In some embodiments, the liquids are deprived of air bubbles that are greater than a predetermined size, e.g., with a diameter of greater than 0.01mm, 0.05 mm, 0.1mm, 0.2 mm, 0.5 mm, or 1mm. In some embodiments, the first reagent is configured to wet the first coating of the surface of the one or more channels. The surface of the one or more channels is equivalent to the surface of the flow cell substrate on which the samples are located. In some embodiments, the second reagent is configured to rewet the surface of the one or more channels after the surface of the channel(s) have at least partly dried as a result of the air gap (the bolus of air) or gas flow flowing through the channel(s).

[0139] In some embodiments, the first and / or second reagents may be introduced to the microfluidic channel(s) and the one or more sample(s) via the inlet(s). In some embodiments, the sequencing system and the fluidic dispensing device herein may comprise an actuator configured to actuate and cause flow and delivery of the first and / or second reagent to the microfluidic channel(s). In some embodiments, the actuator may actuate the flow of the first reagent to enter the microfluidic channel via the inlet(s) in a first direction at a predetermined flow rate with a predetermined duration of time which results in a first unit volume of first reagent to be delivered to the flow cell device. Such flow of the first reagent may be repeated for a number of times, e.g., 1- 20 times, to increase spatially homogenous delivery of reagent concentration to the samples across the microfluidic channel in comparison to a single unit flow of the first reagent. In some embodiments, the actuator or a second actuator may actuate a flow of the first reagent to enter the microfluidic channel via the outlet(s) in a second direction at a second predetermined flow rate with a second predetermined duration of time which results in a second unit volume of reagent to be delivered to the flow cell device. Such flow of the first reagent may be repeated for a number of times, e.g., 1- 20 times, to increase spatially homogenous delivery of reagent concentration to the samples across themicrofluidic channel in comparison to a single flow of the first reagent. The flow of the first reagent in the second direction may re-utilize some or all of the volume of the first reagent that has flowed through the sample and exited the flow cell via the outlet(s), thereby resulting in saving of reagent consumption. The predetermined flow rates and the predetermined duration of time for the flow may be customized based on various factors including but not limited to: the reagent type, the size of the microfluidic channels, the type of sample(s), and the sequencing chemistry. In some embodiments, the first or second unit volume may be within a range from 1 μL to 1000 μL. In some embodiments, the first or second unit volume may be within a range from 5 μL to 500 μL. In some embodiments, the first or second unit volume may be within a range from 10 μL to 150 μL. In some embodiments, the first or second predetermined flow rate may be in a range from 1 μL / second (s) to 1000 μL / s. In some embodiments, the first or second predetermined flow rate may be in a range from 1 μL / s to 500 μL / s. In some embodiments, the first or second predetermined flow rate may be in a range from 2 μL / s to 100 μL / s. As a nonlimiting example, the first reagent may be delivered at a first unit volume of 20 μL with 5 repetitions in the first direction from the inlet(s), and then the first reagent may be delivered at a second unit volume of 20 μL with 1 repetition in the second direction from the outlet(s). The order of the reagent delivery in the first and second directions may be mixed. For example, each repetition of delivery in the first direction may be followed by 0, 1, 2, or more repetitions of delivery in the second direction before the repetition of the delivery in the first direction.

[0140] In some embodiments, the channel(s) 250, 350, 450, 550 can be defined by a top interior surface 521 and a bottom interior surface 521 of the substrates. In specific embodiments, the channels 250, 550 can each include a lumen 551 defined by a top interior surface 521 and a bottom interior surface 521 of the substrates surrounding the lumen 551, and a groove in either the top, bottom, or both surfaces, without a middle substrate.

[0141] In some embodiments, the channels 350, 450, 550 can be defined by the top and bottom substrates with an addition of a middle substrate 322, 422, 522. The middle substrate can include a void, e.g., an elongated void, extending along a longitudinal axis, or y axis, of the middle substrate. The void’s width can define the width of the channel 350, 450, 550, along the x axis, and the void’s length, along the y direction, can define the length of the channel. FIGS. 3-4 and 5A show flow cell devices with channels 350, 450, 550 defined by the top, middle, and bottom substrates.

[0142] In some embodiments, the channels are microfluidic channels. In some embodiments, a gap or height between the top interior surface and the bottom interior surfaceof the substrates that defines the channels, along the z direction, is about 150 , 130 , 120 , 110 , 100 , 90 , 80 , 70 , 60 , 50 , or 40 . In some embodiments, the gap or height between the top interior surface and the bottom interior surface of the substrates is between about 40 and about 150 , about 50 and about 130 , about 60 and about 120 , or about 70 and about 110 , or any range therebetween. In some embodiments, the gap or height of the channel is no more than about 100 . In some embodiments, the gap or height of the channel is no more than about 80 , 70 , 60 , 50 , or 40 , inclusive of all range and subranges therebetween.

[0143] In some embodiments, a gap or height between the top interior surface and the bottom interior surface of the substrates that defines the channels, along the z direction, is 150 , 130 , 120 , 110 , 100 , 90 , 80 , 70 , 60 , 50 , or 40 . In some embodiments, the gap or height of the channel is no more than 100 . In some embodiments, the gap or height of the channel is no more than 80 , 70 , 60 , 50 , or 40 , inclusive of all range and subranges therebetween.

[0144] In some embodiments, a length of the channel, along the y direction, is about 120 mm, 100 mm, 90 mm, 80 mm, 70 mm, 60 mm, 50 mm, 40 mm, or 30 mm. In some embodiments, the length of the channel, along the y direction, is about between about 120 mm and about 30 mm, between about 100 mm and about 40 mm, between about 90 mm and about 50 mm, between about 80 mm and about 60 mm, or any range therebetween. In some embodiments, the length of the channel is no more than about 100 mm. In some embodiments, the length of the channel is no more than about 80 mm, 75 mm, 70 mm, 65 mm, 60 mm, 55 mm, 50 mm, 45mm, or 40 mm.

[0145] In some embodiments, a length of the channel, along the y direction, is 120 mm, 100 mm, 90 mm, 80 mm, 70 mm, 60 mm, 50 mm, 40 mm, or 30 mm. In some embodiments, the length of the channel is no more than 100 mm. In some embodiments, the length of the channel is no more than 80 mm, 75 mm, 70 mm, 65 mm, 60 mm, 55 mm, 50 mm, 45mm, or 40 mm, inclusive of all range and subranges therebetween.

[0146] In some embodiments, a width of the channel, along the x direction, is about 50 mm, 40 mm, 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, 8 mm, or 5 mm. In some embodiments, the width of the channel, along the x direction, is between about 50 mm and about 5 mm, between about 40 mm and about 8 mm, between about 30 mm and about 10 mm, between about 25 mm and about 15 mm, or any range therebetween. In some embodiments, the length of the channel is no more than about 10 mm or about 7 mm. In someembodiments, the width of the channel is no more than about 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, or 5 mm, inclusive of all range and subranges therebetween.

[0147] In some embodiments, a width of the channel, along the x direction, is 50 mm, 40 mm, 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, 8 mm, or 5 mm. In some embodiments, the width of the channel is no more than 10 mm or 7 mm. In some embodiments, the width of the channel is no more than 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, or 5 mm, inclusive of all range and subranges therebetween.

[0148] In some embodiments, the distance between two adjacent channels or the distance from an edge of the channel to the edge of the flow cell device, along the x axis, is about 0.5 mm to about 15 mm. In some embodiments, the distance between two adjacent channels or the distance from an edge of the channel to the edge of the flow cell device, along the x axis, is about 1 mm to about 8 mm. In some embodiments, the distance between two adjacent channels or the distance from an edge of the channel to the edge of the flow cell device, along the x axis, is about 2 mm to 6 mm.

[0149] In some embodiments, the distance between two adjacent channels or the distance from an edge of the channel to the edge of the flow cell device, along the x axis, is 0.5 mm to 15 mm. In some embodiments, the distance between two adjacent channels or the distance from an edge of the channel to the edge of the flow cell device, along the x axis, is 1 mm to 8 mm. In some embodiments, the distance between two adjacent channels or the distance from an edge of the channel to the edge of the flow cell device, along the x axis, is 2 mm to 6 mm.

[0150] In some embodiments, the flow cell devices can have more than one channel, and all the channels can have a uniform size and shape. FIGS. 5A, 5E, and 5F show embodiments of flow cell devices with two channels of the identical size and shape. In some embodiments, the flow cell devices can have channels of different sizes, or shapes, or combinations thereof. FIGS. 3-4 show embodiments of flow cell devices with similar channel length but different channel widths.

[0151] In some embodiments, the channels may include a tapered portion that connects the open landing area to the body of the channel (e.g., FIGS. 2-4). The tapered area and its taper angle can be determined by the size of the open landing area that it is connecting to, and also the width of the channel body. The size of the tapered area and its taper angle can be adjusted to facilitate efficient fluid transfer from the open landing area to the body of the channel. FIG. 4 shows an embodiment of the flow cell device with a tapered transition portion 451 connecting the open landing area 441 to the body of the channel 452. A second tapered area 453 can be used to connect the body of the channel 452 to the outlet 460.

[0152] The size and shape of the tapered transition portion 451 may be varied depending on the applications of the flow cell device.

[0153] FIGS. 27A-27G show an embodiment with the size and dimensions of the tapered transition portion connecting the body of the channel to the outlet.

[0154] In some embodiments, the tapered transition portion from the outlet to the body of the channel can be about 3 mm to about 15 mm along the y axis. In some embodiments, the tapered transition portion from the outlet to the body of the channel can be about 5 mm to about 12 mm along the y axis. In some embodiments, the tapered transition portion from the outlet to the body of the channel can be about 6 mm to about 9 mm along the y axis. In some embodiments, the tapered transition portion from the outlet to the body of the channel can be 3 mm to 15 mm along the y axis. In some embodiments, the tapered transition portion from the outlet to the body of the channel can be 5 mm to 12 mm along the y axis. In some embodiments, the tapered transition portion from the outlet to the body of the channel can be 6 mm to 9 mm along the y axis.

[0155] In some embodiments, the tapered angle, e.g., the acute angle between an edge of the flow cell device and an edge of the tapered area is 25.1 degrees as shown in FIG. 27B. In some embodiments, the tapered angle can be in the range of about 15 degrees to about 40 degrees. In some embodiments, the tapered angle can be in the range of about 20 degrees to about 30 degrees. In some embodiments, the tapered angle can be in the range of 15 degrees to about 40 degrees. In some embodiments, the tapered angle can be in the range of 20 degrees to about 30 degrees.

[0156] In some embodiments, each channel has its own corresponding open landing area, or inlet, or combinations thereof, e.g., in FIGS. 4, 5A, 5F and 25A-25F. In some embodiments, two or more channels share a single open landing area, or inlet, or combinations thereof, e.g., in FIGS. 5E and 28A-28C.

[0157] In some embodiments, the open landing area is directly connected to the body of the channel. In some embodiments, the open landing area is connected to the body of the channel without a tapered transition portion in between. FIGS. 25A-25E show an embodiment of the flow cell device disclosed herein. The flow cell device 700 includes a circular open landing area 741 that is directly connected to the body of the channel 752 without a tapered transition portion. In some embodiments, the channel 750 starts where the open landing area ends, and the channel width is substantially identical or exactly identical to the diameter of the opening landing area. As shown in FIG.26C, the size of the open landing area can be different from the embodiment in FIGS. 25A-25E either in one channel or in bothchannels, so that the diameter of the open landing area is smaller than the width of the channel along the x axis. When the diameter of the open landing area is smaller, there can be a tapered transition region 751 between the open landing area and the body of the channel.

[0158] The flow cell device 200, 300, 400, 500 can include one or more inlets 540 and one or more outlets 560. Referring to FIG. 5C, a channel 550 can run from its corresponding inlet 540 to its corresponding outlet 560, thereby allowing fluidic communication from the inlet to the outlet. Sequencing reagents can be introduced to the flow cell device 200, 300, 400, 500 via the inlet 540, flow through the channels 550 and interact with samples located therein, and exit from the outlet 560.

[0159] The size and shape of the inlet and outlet can be customized to suit various sequencing applications. For example, the size and shape can be determined based on the specific sequencing application(s), such as, a minimal flush volume, a contamination threshold, the parameters of the flow cell, e.g., the size of the flow cell channels, or the parameters of the dispenser, e.g., the size of the dispensing tip. As a nonlimiting example, the inlet can be cylindrical as shown in FIG. 5C with walls extending along the z direction and orthogonal to the substrates. As shown in FIG. 4, at the bottom of the cylindrical void / hole, the inlet 440 can be connected to a cleaning outlet 470, 570. The inlet can be shaped differently. For example, the inlet can have an inverted cone shape with wider openings at the top and narrows down toward the channel to reduce the residuals of reagents that can remain in the inlet. FIG. 5D shows an embodiment with an inlet as a cylindrical shape. Without the cleaning outlet, the inlet has no connection to the cleaning outlet. In another embodiment, as in FIG. 5E, the inlet 540 can be part or all of the open landing pad. In yet other embodiments of the flow cell device, as in FIG. 5F, the inlet may be a groove of various sizes or shapes in the middle substrate, or in the middle and the bottom substrates, that is in fluidic connection to the channels.

[0160] The diameter of the inlet, e.g., the widest dimension in the x-y plane, can be in the range of about 3 mm to about 11 mm. As another example, the height of the inlet, along the z direction, can be the total height of the top substrate and the middle substrate, and it can be in the range of about 1 mm to 12 mm.

[0161] The diameter of the outlet or the cleaning outlet, in the x-y plane, can be in the range of about 0.3 mm to about 4 mm. In some embodiments, the diameter of the outlet can be in the range of about 0.4 mm to about 2 mm, and the outlet can be a cylindrical shape.

[0162] The diameter of the inlet, e.g., the widest dimension in the x-y plane, can be in the range of 3 mm to 11 mm. As another example, the height of the inlet, along the z direction,can be the total height of the top substrate and the middle substrate, and it can be in the range of 1 mm to 12 mm.

[0163] The diameter of the outlet or the cleaning outlet, in the x-y plane, can be in the range of 0.3 mm to 4 mm. In some embodiments, the diameter of the outlet can be in the range of 0.4 mm to 2 mm, and the outlet can be a cylindrical shape.

[0164] The size and shape of the inlet and outlet can be customized to suit various sequencing applications. For example, the size and shape can be determined based on the specific sequencing application(s), such as, a minimal flush volume, a contamination threshold, the parameters of the flow cell, e.g., the size of the flow cell channels, or the parameters of the dispenser, e.g., the size of the dispensing tip. As a nonlimiting example, the inlet can be cylindrical as shown in FIG. 5C with walls extending along the z direction and orthogonal to the substrates. At the bottom of the cylindrical void / hole, the inlet 440, 540 can be connected to a cleaning outlet 470, 570. The inlet can be shaped differently. For example, the inlet can have an inverted cone shape with wider openings at the top and narrows down toward the channel to reduce the residuals of reagents that can remain in the inlet. FIG. 5D shows an embodiment with an inlet as a cylindrical shape. Without the cleaning outlet, the inlet has no connection to the cleaning outlet. In another embodiment, as in FIG. 5E, the inlet 540 can comprise the open landing pad or a portion thereof but no other structural elements in the flow cell device. FIGS. 28A-28C shows the embodiment in FIG. 5E from different views. FIG. 28A is a top view of the flow cell device. FIG. 28B shows three different substrates in a perspective view, and FIG. 28C shows the bottom, middle, and top substrates. In yet another embodiment of the flow cell device, as in FIG. 5F, the inlet may be a groove of various sizes or shape in the middle substrate, or in the middle and the bottom substrates that are in fluidic connection to the channels.

[0165] FIGS. 2- 4, and 5A-5F show flow cell devices with two to three substrates forming one or two channels, and each channel having a corresponding inlet and outlet. However, the number of substrates, channels, inlets, and outlets can vary in different embodiments. In some embodiments, the number of substrates, channels, inlets and outlets can be any integer number that is greater than 0. In some embodiments, the flow cell devices herein have 2, 4, 6, 8, 10, or even more channels. Channel Coatings and Air Gaps

[0166] As shown in FIG. 5C, one or more of the interior surfaces 521 can be coated with a first coating 522.

[0167] In some embodiments, the channels are configured to allow fluids, e.g., liquid reagents, and an air gap (e.g., a flow or bolus of air between the flow of fluids) to flow therethrough. In some embodiments, the air gap can comprise a bolus of gas. The air gap can be introduced similarly to the liquid reagents, e.g., via the inlet to the channels to exit from the outlet. Alternatively, the air gap can be introduced from other openings such as the outlet or the cleaning outlet of the flow cell device. The air gap can be driven mechanically by one or more structural elements of the fluidic control device herein. As an example, the air gap can be sucked into the channels via the inlet by a mechanical force applied at the outlet, e.g., by a pump or a vacuum. As another example, the air gap may be purged by a pump or the like via the inlet.

[0168] The volume of the air gap can vary depending on the geometry, or size, or combinations thereof, of the flow cell devices and channels. For example, the volume of air gap can be selected to fill up about 30%, 40%, 50%, 60%, or 70% of the entire volume of each channel. As another example, the volume of the air gap can be adjusted based on the subsequent reagent to be administered, e.g., air gap can be increased if higher cleaning or reduction in contamination is desired.

[0169] The air gap that flows through the one or more channels can be configured to push existing reagents in the channel(s) toward the outlet and / or to exit from outlet. Subsequent delivery of reagent can achieve high homogeneity in the flow cells. In other words, the air gap can effectively eliminate the first reagent from the channel, e.g., 550, to prepare for a delivery a second reagent with minimum contamination from residual of the first reagent. Known flow cells rely solely on washing buffer(s) between delivery of sequencing reagents, which may result in mixing of the sequencing reagents with washing liquid(s) that can cause a concentration gradient of the sequencing reagent with higher concentration at one end closer to the landing area, and lower concentration at the opposite end closer to the outlet. Such gradient or inhomogeneity can be gradually reduced by repeated washing but remains difficult completely eliminate. The gradient of concentration or inhomogeneity in concentration of reagents may cause sequencing analysis of tiles toward the opposite end of the flow cell from the open landing area or inlet to be less accurate and unreliable at least partly due to inhomogeneous reaction, or attachment, or combinations thereof, of compounds in the reagent to the polonies. In addition, known flow cell devices, sequencing systems, and sequencing methods can cause an introduction of air bubbles into the channels between reagents, which can damage the channel coating, the polonies tethered thereon and being imaged, or combinations thereof, thereby impairing the sequencing process. The flow celldevices herein can utilize the air gap to minimize or eliminate concentration gradient or inhomogeneity in the flow cells (e.g., the channels of the flow cells), along the y axis (axis shown in FIG. 2) without damage to the reagents reactions at the flow cell device and the sequencing process.

[0170] In some embodiments, the concentration variation of the first reagent or second reagent across the sample(s) located on the flow cell substrate is less than ±2%, 5%, 10%, 15% or 20%. In some embodiments, the cell density or polony density variation across the flow cell substrate is less than ±2%, 5%, 10%, 15% or 20%.

[0171] In some embodiments, the air gap and washing liquid(s) can be combined to achieve optimal cleaning of the channel(s). In some embodiments, the air gap can be used alone to achieve optimal cleaning of the channels. In some embodiments, the washing scheme, using an air gap, washing liquid(s), or both can be determined based on the contamination level of the reagent to be delivered. In some embodiments, the washing scheme, using an air gap, washing liquid(s), or both can be determined based on the cost of the reagent, along or in combination with other factors such as contamination levels. In some embodiments, the order of using an air gap and washing liquid(s) can vary when the two are combined in the washing scheme. The air gap can be applied after or before any number of flushing with washing liquid(s). In some embodiments, the air gap can be purged in between any selected flushing of washing liquids.

[0172] The air gap that flows through the one or more channels may dry the coating of the one or more channels, but the functionality of the coating can remain unaltered after one or more air gaps flows therethrough. In some embodiments, the air gap that flows through the one or more channels may dry the polonies tethered thereon the channel coating. However, the air gap (e.g., one or more parameters of the air gap) can be configured to prevent damage to the polonies and ensure proper sequencing of the polonies when a subsequent liquid reagent is flushed through the channel(s). As such, the flow cell devices with such channels can be cleaned by delivering (e.g., purging, flowing, expelling, moving) air gaps into the channels, alone or in combination with washing with reagents and / or washing buffers. Usage of air gaps for cleaning can increase the efficiency and effectiveness of cleaning the channels while simultaneously reducing the costs of reagents that is required for washing and performing sequencing analysis, while satisfying a predetermined contamination requirement (e.g., an amount of reagent present in the channel(s) from a previous step that has been predetermined to be sufficiently low that it does not negatively impact subsequent steps in a workflow). The predetermined contamination requirement may be customized to be atvarious levels. For example, the predetermined contamination level may be based on the sequencing application and the reagent(s) being applied. As a nonlimiting example, the contamination level may be below 0.1%, 0.01%, 0.005%, or 0.001%.

[0173] In some embodiments, cleaning of the microfluidic channel after delivery of the first or second reagent includes one or more cleaning operations: delivering a bolus of air of a first unit volume to the microfluidic channel(s); delivering a washing buffer of a second unit volume to the microfluidic channel(s); and delivering a third unit volume of a next reagent to be used to the microfluidic channel(s). The one or more cleaning operations may be in a first direction from the inlet(s) toward the outlet(s) or in a second direction from the outlet(s) toward the inlet(s). Each of the one or more cleaning operations may be performed in 0 to 50 repetitions, for example 1-50, 2-40, 10-45, 15-20, 1-25, 2-5, 3-15 or any range therebetween. Each of the one or more cleaning operations may be performed in 0 to 10 repetitions. Each of the one or more cleaning operations may be performed in 1 to 10 repetitions. The order of performing the one or more cleaning operations may be customized by a user depending on various factors including but not limited to: the type of the reagent, the type of the flow cells, the distribution of the samples on the flow cell substrate, the spatial density of the sample(s), and the sequencing chemistry. In some embodiments, the one or more cleaning operations may be performed with a flow rate within a range from 1 μL / s to 1000 μL / s, such as 10 μL / s to 500 μL / s, 20 μL / s to 700 μL / s, 50 μL / s to 500 μL / s, 100 μL / s to 500 μL / s, 20 μL / s to 300 μL / s or any range therebetween. In some embodiments, the one or more cleaning operations may be performed with a flow rate within a range from 1 μL / s to 100 μL / s. In some embodiments, the one or more cleaning operations may be performed with a flow rate within a range from 1 μL / s to 50 μL / s. In some embodiments, the one or more cleaning operations may be performed with a flow rate within a range from 1 μL / s to 1000 μL / s. In some embodiments, the one or more cleaning operations may be performed with a flow rate within a range from 1 μL / s to 100 μL / s. In some embodiments, the one or more cleaning operations may be performed with a predetermined time duration in a range from 0.1 seconds to 1000 seconds, such as from 0.1 seconds to 500 seconds, 0.5 seconds to 100 seconds, 0.1 second to 10 seconds, 0.5 seconds to 15 seconds, 1 second to 30 seconds, 10 seconds to 60 seconds, or any range therebetween. In some embodiments, the one or more cleaning operations may be performed with a predetermined time duration in a range from 0.2 seconds to 500 seconds. In some embodiments, the one or more cleaning operations may be performed with a predetermined time duration in a range from 0.5 seconds to 200 seconds.

[0174] In some embodiments, the surface can be passivated for the first coating 522. In some embodiments, the surface is passivated with the first coating 522 that immobilizes surface capture primers, nucleic acid template molecules, or both for capturing polynucleotides thereon. In some embodiments, during sequencing, the surface can comprise polynucleotides captured thereon. In some embodiments, the polynucleotides captured thereon are configured to be imaged in a sequencing cycle.

[0175] In some embodiments, the first coating 522 of the surface comprises one or more hydrophilic polymer coating layers. The first coating can comprise a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating layer. The hydrophilic polymer coating layer(s) can comprise polyethylene glycol (PEG). The hydrophilic polymer layer(s) can comprise a branched hydrophilic polymer and the branched hydrophilic polymer can comprise at least 8 branches. In some embodiments, the hydrophilic polymer coating layer(s) has a water contact angle of no more than about 50 degrees.

[0176] In some embodiments, the surface of the one or more channel(s) (which is equivalent to the surface of the flow cell substrate on which the samples are located) comprises at least one discrete region that comprises a plurality of clonally-amplified sample nucleic acid molecules that have been annealed to (e.g., hybridized to, or covalently attached to) a plurality of attached oligonucleotide molecules (e.g., attached surface capture primers). In some embodiments, at least one of the plurality of the clonally-amplified sample nucleic acid molecules comprises a concatemer molecule annealed to at least one of the plurality of attached oligonucleotides.

[0177] The samples disclosed herein on the flow cell device may be two dimensional (2D) or three dimensional (3D) samples. The sample(s) may include samples for in situ sequencing methods, such as cells or tissue (“in situ samples”).

[0178] In some embodiments, the sample(s) may be immobilized on the support of the flow cell device, e.g., on the flow cell substrate. In some embodiments, the sequencing system, including the optical system, advantageously enables sequencing and imaging of target analyte(s) or features while they remain intact inside the cell or tissue. In some embodiments, the cell or tissue and the targets (e.g., target analytes, structure elements, organelles, etc.) therewithin remain intact during sequencing and / or imaging. In some embodiments, the one or more samples being imaged using the optical systems herein can be 2D or 3D samples. The 2D sample(s) may include nucleotide acid molecules extracted from various sources. The 3D samples can include various samples in which polonies within the sample do not fit into a single z-level while keeping the polonies in focus during imaging ofthe flow cell. The 3D samples may include in situ samples such as cells and / or tissues. In some embodiments, the cell or tissue samples are immobilized on the flow cell device or an interior surface thereof for sequencing and / or imaging without modifying the spatial locations of targets within the cells or tissue. In some embodiments, the cell or tissue samples are immobilized on the flow cell device or an interior surface thereof for sequencing or imaging without modifying the spatial relationships of targets or target analytes within the cells or tissue. In some embodiments, the cells and / or tissue are immobilized with the morphological features, RNA, mRNA, and protein targets of the samples intact inside the cell(s) or tissue during sequencing and / or imaging. In some embodiments, the spatial locations or relationships of the target analytes or targets remain intact during sequencing and / or imaging. In some embodiments, the spatial locations or relationships of the target analytes or targets during sequencing and / or imaging are not manually reconstructed using artificially added structure or features in the sample. For example, the nucleus, cell membrane, mitochondria, and extracellular matrix, as well as specific proteins, lipid membranes and RNAs can retain their relative spatial relationship relative to each other in the sample(s) during imaging and / or sequencing.

[0179] In some embodiments, the one or more samples herein may include a cell or cells. In some embodiments, the cells may be cultured on a solid support disclosed herein. The support can be the flow cell device or a flow cell substrate that is prepared by the sample preparation system herein and transferred to the flow cell device. In some embodiments, the cell may be an adherent cell. In some embodiments, the cell may be a non-adherent cell, for example a suspended cell. In some embodiments, the cell may be a confluent cell. In some embodiments, a suspended cell may be adhered to the surface, e.g. a surface of the flow cell, by a specific capture mechanism such as an antigen-antibody interaction, or a receptor-ligand interaction, including an interaction of a known surface receptor with a known ligand; an unknown surface receptor with a known ligand, a known ligand with an unknown ligand, or an unknown receptor with an unknown ligand. In some embodiments, a suspended cell may be adhered to a surface by a specific carbohydrate binding interaction, a specific protein or peptide binding interaction, or a specific lipid-lipid interaction, lipid-peptide interaction, or lipid-carbohydrate interaction. In some embodiments, a suspended cell may be adhered to a surface by a nonspecific interaction with said surface, such as by use of a charged surface (e.g., a polylysine, poly argininine, polyglutamic acid, polyaspartic acid surface or the like, or a charged polymer surface, such as a polyethylenimine surface; or a plasma-treated or ion- treated glass or polystyrene surface, or the like). It will be understood by one of ordinary skillin the art that in addition to the surfaces disclosed herein, any surface useful for, or known to be used for, cell culture, will be useful for capture of adherent cells. In particular embodiments, a surface useful for capture of adherent cells will comprise at least one of polyethylene oxide, streptavidin, protein A, or any combination thereof. In some embodiments, a suspended cell may be introduced to a flow cell by flowing the suspended cells in solution (e.g., cell culture medium or a suitable buffer such as a saline buffer) through the flow cell, by direct pipetting or liquid transfer onto a surface of the flow cell, by gravitational precipitation, by centrifugation, or by any method known in the art for bringing cells into contact with a surface.

[0180] In some embodiments, the one or more samples include biological analytes (e.g., target analyte(s)). In some embodiments, the target analytes(s) are located inside the sample(s) or on the membrane of the sample(s). In some embodiments, the one or more samples include target analyte(s) that are on the exterior or interior surface of the cell. In some embodiments, the one or more samples include target analyte(s) that are on the exterior or interior surface of a cellular membrane. In some embodiments, the one or more samples include target analyte(s) that are part of the extracellular matrix. In some embodiments, the one or more samples include target analyte(s) that are part of and / or located on or in one or more organelles within the cell or tissue (nucleus, mitochondria, chloroplast, Golgi apparatus, lysosome, endoplasmic reticulum, actin or tubulin cytoskeleton and the like). In some embodiments, the one or more samples include target analytes that are on or in the glycocalyx or belong to part of the glycocalyx.

[0181] In some embodiments, the biological analyte(s) or target analyte(s) comprise at least one polypeptide, lipid, nucleic acid or polysaccharide. In some embodiments, the target analyte(s) comprise at least one polypeptide, enzyme or lipid located anywhere in the sample(s) including the cytoplasm and nucleus. In some embodiments, the target analyte(s) comprise at least one polypeptide, enzyme or lipid located in or on a cellular structure including without limits any cellular membrane, nucleus, nucleolus, mitochondria, chloroplast, Golgi apparatus, ribosome, endoplasmic reticulum, microtubules, peroxisome and lysosome.

[0182] In some embodiments, the one or more samples herein include analytes (e.g., nucleic acids, DNA, RNA, mRNA, polysaccharides, lipids and / or proteins) obtained from cell or tissue with preserved spatial information to undergo sequencing and / or imaging outside the cell or tissue. In some embodiments, the one or more samples herein include analytes removed from the cell or tissue so that the analytes are not inside the cell or tissueanymore when they undergo sequencing and / or imaging outside, while keeping the rest of the cell or tissue, e.g., the structure of the cell or tissue, intact while the analytes are outside. In some embodiments, the one or more samples include analytes transferred to the outside of the cell or tissue with artificially reconstructed spatial information to undergo sequencing and / or imaging outside the cell or tissue.

[0183] In some embodiments, the one or more sample(s) comprises a cell, a plurality of cells, a section of a cell, an intact tissue, an organ, a tissue section, an intact tumor, or a tumor section. In some embodiments, the sample(s) comprises a fresh cellular sample, a freshly- frozen cellular sample, a sectioned cellular sample, or an formalin-fixed paraffin embedded (FFPE) cellular sample. In some embodiments, the sample(s) comprises one or more living cells or non-living cells. In some embodiments, the sample(s) can be obtained from a virus, fungus, prokaryote or eukaryote. In some embodiments, the sample(s) can be obtained from an animal, fungus, plant, yeast, or bacterium. In some embodiments, the animal is a mammal or an insect. In some embodiments, the sample(s) comprises one or more virally-infected cells. In some embodiments, the sample(s) comprises transfected cells, displaced cells or genetically engineered cells. In some embodiments, the sample(s) comprises mammalian transfected or displaced cells. In some embodiments, the sample(s) comprises a biofilm, i.e. a consortium of microorganisms that adhere together. In some embodiments, the sample(s) can be obtained from any organism including a human, simian, ape, canine, feline, bovine, equine, murine, porcine, caprine, lupine, ranine, piscine, plant, insect, or bacterium. In some embodiments, the sample(s) can be obtained from any organ including head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testicles, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin, heart, larynx, or other organs.

[0184] The methods, devices, and systems disclosed herein may allow preparation and sequencing and analysis of various samples and sources. The samples may include nucleic acids extracted from any of a variety of biological samples, e.g., blood samples, saliva samples, urine samples, cell samples, tissue samples, and the like. In some embodiments, the samples here may include a variety of different cell, tissue, or sample types known to those of ordinary skill in the art. For example, the sample(s) may be from eukaryotes (such as animals, plants, fungi, protista), archaebacteria, or eubacteria. In some embodiments, the sample(s) may include prokaryotic or eukaryotic cells, such as adherent or non-adherent eukaryotic cells. In some embodiments, the sample(s) may be from, for example, primary or immortalized rodent, porcine, feline, canine, bovine, equine, primate, or human cell lines. Insome embodiments, the sample(s) may include a variety of different cell, organ, or tissue types (e.g., white blood cells, red blood cells, platelets, epithelial cells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts, skeletal muscle cells, smooth muscle cells, gametes, or cells from the heart, lungs, brain, liver, kidney, spleen, pancreas, thymus, bladder, stomach, colon, or small intestine). In some embodiments, the sample(s) may include normal or healthy cells. Alternately or in combination, the sample(s) may include diseased cells, such as cancerous cells, cells infected with a disease (such as a viral or bacterial disease) or from pathogenic cells that are infecting a host. In some embodiments, the sample(s) may include a distinct subset of cell types, e.g., immune cells (such as T cells, cytotoxic (killer) T cells, helper T cells, alpha beta T cells, gamma delta T cells, T cell progenitors, B cells, B-cell progenitors, lymphoid stem cells, myeloid progenitor cells, lymphocytes, granulocytes, Natural Killer cells, plasma cells, memory cells, neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells, and / or macrophages, or any combination thereof), undifferentiated human stem cells, human stem cells that have been induced to differentiate, rare cells (e.g., circulating tumor cells (CTCs), circulating epithelial cells, circulating endothelial cells, circulating endometrial cells, bone marrow cells, progenitor cells, foam cells, mesenchymal cells, or trophoblasts). Other cells are contemplated and consistent with the disclosure herein.

[0185] In some embodiments, the sample(s) harbors or contains a plurality of biological analytes or target analytes including polypeptides, lipids, nucleic acids and polysaccharides, or a mixture thereof. In some embodiments, the sample(s) harbors 2-10,000 different biological or target analytes. In some embodiments, the target analytes comprise a plurality of target polypeptides. In some embodiments, the plurality of target polypeptides have different amino acid sequences. In some embodiments, the sample(s) harbors 1-10,000 different target polypeptides, the sample(s) harbors 1-1,000 different target polypeptides, the sample(s) harbors 1-500 different target polypeptides, the sample(s) harbors 1-100 different target polypeptides, the sample(s) harbors 1-25 different target polypeptides, or harbors 25-50 different target polypeptides, or harbors 50-75 different target polypeptides, or harbors 75- 100 different target polypeptides, or harbors any range therebetween of different target polypeptides. In some embodiments, the sample(s) harbors more than 100 different target polypeptides, or more than 250 different target polypeptides, or more than 500 different target polypeptides, or more than 1000 different target polypeptides. In some embodiments, the sample(s) harbors more than 10,000 different target polypeptides.

[0186] In some embodiments, the sample(s) can be deposited (e.g., seeded) onto a support, e.g., a flow cell substate, which is passivated with a coating that promotes cell adhesion. In some embodiments, the sample(s) can be deposited on a support that lacks immobilized capture primers which can bind target polynucleotide analytes from the sample(s). In some embodiments, the support can be coated with one or more compounds that generate a charged coated surface. In some embodiments, the support is coated with a lysine compound, poly-lysine compound, arginine compound, poly-arginine compound, or an amino-terminated compound (e.g., including amino-terminated PEG). The support can be coated with an unbranched compound, a branched compound, or a mixture of unbranched and branched compounds. In some embodiments, the support can be coated with modified peptides, including, for example and without limitation, cationic anti-microbial peptides or dual surface anti-microbial peptides. In some embodiments, the support can be coated with polycyclic peptide antibiotics comprising thioether amino acids lanthionine or methyllanthionine and / or unsaturated amino acids dehydroalaine and 2-aminoisobutryic acid. In some embodiments, the support can be coated with at least one small peptide such as melittin. In some embodiments, the support can be coated with a compound that promotes integrin-mediated cell adhesion. For example, and without limitation, the support can be coated with tripeptide arginyl-glycyl-aspartic acid (Arg-Gly-Asp; also known as RGD). In some embodiments, the support can be coated with amines or polymers having -NH2 groups which promote cell adhesion, including for example polyethyleneimine (PEI) or polydopamine (PDA).

[0187] Depending on the sample(s) immobilized on the support (e.g., a flow cell substrate), the flow cell images may include single or multiple z-levels along an z axis orthogonal to the image plane of the flow cell images. In particular, for three dimensional samples, e.g., cells, tissues, or other in situ samples, the flow cell images herein can include multiple z-levels (i.e., axial locations) in order to cover the whole sample(s) in 3D. The z axis can extend from the objective lens of the optical system disclosed herein to the support, e.g., flow cell. The z axis can be orthogonal to the image plane of the flow cell images. Each z- level of flow cell images may be separated from the adjacent z-level(s) for a predetermined distance, for example, for about 0.1 μm to about 15 μms. Each z-level of flow cell images may be separated from the adjacent level(s) for 0.5 μm to 10 μms. Each z-level of flow cell images may be separated from the adjacent level(s) for 0.2 μm to 2 μms. At each z-level, flow cell images can be acquired from one or more sequencing cycles and / or one or more channels. Each flow cell image may include in its field of view at least part of one or moretiles or subtiles of the flow cell. The image plane is defined by the x and y axis. And the axial axis (i.e., z axis) is orthogonal to the x-y plane. Preparation of the 3D sample(s) and immobilization on flow cells for sequencing reactions and imaging using the optical systems herein are disclosed in U.S. Patent Publication No. US 2023-0326065 , and is herein incorporated by reference in its entirety.

[0188] In some embodiments, the sample comprises a plurality of nucleic acid molecules. In some embodiments, at least one of the plurality of sample nucleic acid molecules comprises a single-stranded multimeric nucleic acid molecule comprising repeats of a regularly occurring monomer unit (a “concatemer molecule”). The single-stranded multimeric nucleic acid molecules can be at least 10 kilobases in length. In some embodiments, the at least one of the plurality of sample nucleic acid molecules further comprises a double-stranded monomeric copy of the regularly occurring monomer unit. The plurality of nucleic acid molecules can be present at about a uniform surface density across the surface. The plurality of nucleic acid molecules can be present at a local surface density of at least about2at a first position on the surface, and at a second local surface density at a second position on the surface. In some embodiments, the plurality of oligonucleotide molecules is present at a surface density of at least about 1,000 molecules / m2.

[0189] In some embodiments, the first coating can comprise multiple hydrophilic polymer coating layers. The first coating can include a first layer comprising a monolayer of polymer molecules tethered to the surface of the substrate. The first coating can further include a second layer comprising a second monolayer of polymer molecules tethered to the polymer molecules of the first layer; and a third layer comprising a third monolayer of polymer molecules tethered to the polymer molecules of the second layer, wherein at least one of the first layer, the second layer, or the third layer comprises branched polymer molecules.

[0190] In some embodiments, the third layer can comprise oligonucleotides (for example, capture primers) tethered to the polymer molecules of the third layer. The oligonucleotides tethered to the polymer molecules of the third layer can be distributed at a plurality of depths throughout the third layer.

[0191] In some embodiments, the first coating can comprise a fourth layer comprising branched polymer molecules tethered to the polymer molecules of the third layer, and a fifth layer comprising polymer molecules tethered to the branched polymer molecules of the fourth layer. In some embodiments, the polymer molecules of the fifth layer further compriseoligonucleotides (for example, capture primers) tethered to the polymer molecules of the fifth layer. The oligonucleotides tethered to the polymer molecules of the fifth layer are distributed at a plurality of depths throughout the fifth layer.

[0192] In some embodiments, the hydrophilic polymer coating layer of the first coating can comprise a molecule selected from the group consisting of: polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly- glucoside, streptavidin, and dextran.

[0193] In some embodiments, the plurality of nucleic acid molecules from the sample are clonally amplified. In some embodiments, when the clonally-amplified nucleic acid molecules or complementary sequences thereof are labeled with Cyanine dye-3, an image of the surface exhibits a ratio of fluorescence intensities for the clonally-amplified Cyanine dye- 3-labeled nucleic acid molecules, or complementary sequences thereof, and nonspecific Cyanine dye-3 dye adsorption background (Binter) of at least 3:1.

[0194] In some embodiments when the image of the surface exhibits a ratio of fluorescence intensities for clonally amplified Cyanine dye-3-labeled nucleic acid molecules, or complementary sequences thereof, and a combination of nonspecific Cyanine dye-3 dye adsorption background and nonspecific amplification background (Binter+Bintra) of at least 3:1.

[0195] In some embodiments, when the clonally-amplified nucleic acid molecules or complementary sequences thereof are labeled with Cyanine dye-3, the image of the surface exhibits a ratio of fluorescence intensities for clonally-amplified Cyanine dye-3-labeled nucleic acid molecules, or complementary sequences thereof, and nonspecific dye adsorption background (Binter) of at least 5:1.

[0196] In some embodiments, when the image of the surface exhibits a ratio of fluorescence intensities for clonally-amplified, Cyanine dye-3-labeled nucleic acid molecules, or complementary sequences thereof, and a combination of nonspecific Cyanine dye-3 dye adsorption background and nonspecific amplification background (Binter+Bintra) of at least 5:1.

[0197] In some embodiments, when the clonally-amplified nucleic acid molecules or complementary sequences thereof are labeled with Cyanine dye-3, the fluorescence image of the surface exhibits a contrast-to-noise ratio (CNR) of at least 20 when the fluorescenceimage is acquired using an inverted microscope equipped with a 20× objective, NA=0.75, dichroic mirror optimized for 532 nm light, a bandpass filter optimized for Cyanine dye-3 emission, and a camera under non-signal saturating conditions, while the surface is immersed in a buffer.

[0198] In some embodiments, one or more of the interior surfaces 521 of the flow cell can be coated, in combination with the first coating 522, a third coating of fluorescent beads (not shown). Exemplary flow cells coating in beads are described, for example, in WO2025062341, the contents of which are incorporated by reference in their entirety herein.

[0199] The fluorescent beads can be chemically immobilized to the surface. The fluorescent beads can be covalently immobilized to the surface. The fluorescent beads can be immobilized or fixedly attached to the surface by forming a coating thereon, e.g., a third coating, so that the fluorescent beads remain fixed or immobilized relative to the surface 521. The coating can be applied directly to and in contact with the surface 521. Alternatively, the third coating can be applied indirectly to or not in direct contact with the surface 521. In some embodiments, the third coating can be applied in between the surface 521 and the first coating 522.

[0200] In some embodiments, the fluorescent beads are chemically immobilized to the surface. In some embodiments, the fluorescent beads are covalently immobilized to the surface. In some embodiments, the fluorescent beads are pre-activated to enable chemical attachment to the surface. In some embodiments, the fluorescent beads are pre-activated to enable covalent attachment to the surface. In some embodiments, the clusters or polonies of polynucleotides captured thereon and the fluorescent beads are imaged simultaneously in one or more sequencing cycles using the sequencing system 110. Landing Areas

[0201] The flow cell devices, fluidic control devices, and systems herein can include a landing area. The landing area may be comprised in each inlet of the microfluidic channels of the flow cell devices. The landing area may be the area that the fluids travels to after being dispensed from the dispensing tips and before it enters the microfluidic channels.

[0202] In some embodiments, the landing area is an open landing area and it is openly exposed to air at least when the dispensing tip is not dispensing fluids to the landing area. FIGS. 2-4, and 5A-5F shows flow cell devices with an open landing area for one or more channels.

[0203] The open landing area 341, 441, 541 can be part of the inlet 340, 440, 540. The open landing area 341, 441, 541 can be on a bottom substrate 330, 430, 530. The open landing area 341, 441, 541 can be in fluidic connection with its corresponding channel(s) 350, 550.

[0204] The inlet 340, 440, 540 can comprise a void or hole in the top substrate 320, 420, 520 that is located above at least part of the open landing area 341, 441, 541. The air gap, or liquid reagents, or combinations thereof, can be introduced via the void or hole of the inlet 340, 440, 540 to reach the open landing area 341, 441, 541, and then transfer from the open landing area 341, 441, 541 into the corresponding channel(s) 350, 450, 550. In some embodiments, the void or hole can have a cross-sectional area in the x-y plane that is substantially identical (e.g., within about 2%, about 5%, about 10% of) or identical to the area of the open landing area, e.g., as in FIGS. 3-4, 5A and 5F. In some embodiments, the void or hole can have a cross-section area in the x-y plane that is greater than the area of the open landing area, e.g., in FIG. 5E. The void or hole in FIG. 5E can be considered to have a rectangular shaped cross section in the x-y plane which is as wide as the flow cell device along the x axis.

[0205] The inlet 340, 440, 540 and the open landing area 341, 441, 541, 741 can advantageously enable open administration of liquids or gas to the flow cell devices. The open administration enabled by the flow cell devices herein can remove series of closed tubing or locked-in tubing thereby greatly reducing system complexity and cost and allowing more flexible adaptation of the systems and devices for various sequencing applications.

[0206] The size and shape of the hole or void, and the size and shape of the open landing area can vary in different embodiments. The sizes and shapes may be determined based on parameters in the specific sequencing application(s), such as, a flush volume, a contamination threshold, the dimensions of the flow cell, e.g., the width of the flow cell channels, or the parameters of the dispenser, e.g., the size of the dispensing tip. As a nonlimiting example, the hole or void is cylindrical as shown in FIG. 5C with walls extending along the z direction and orthogonal to the substrates. However, the hole or void can be shaped, or sized, or combinations thereof, differently. For example, the hole or void can have an inverted cone shape with wider openings at the top and narrows down toward the channel to reduce the residuals of reagents that can remain in the inlet. In some embodiments, a larger open landing area may better facilitate reagent transfer into the channels and keeping the open landing area’s size to the width of the channels in a predetermined ratio range may also better facilitate reagent transfer into the channels. As a nonlimiting example, the diameter of theopening area, e.g., the widest dimension in the x-y plane, can be in the range of about 3 mm to about 40 mm. In some embodiments, the diameter of the opening area is about identical to the width of the corresponding channel. In some embodiments, the diameter of the opening area is about 10%, 20%, 30%, 40% or 50% less than the width of the corresponding channel. As a nonlimiting example, the diameter of the hole or void, e.g., the widest dimension in the x-y plane, can be in the range of about 3 mm to about 40 mm. In some embodiments, the diameter of the hole or void is about identical to the width of the corresponding channel. In some embodiments, the diameter of the hole or void is about 10%, 20%, 30%, 40% or 50% less than the width of the corresponding channel.

[0207] To work with the open landing area, the flow cell system may include a fluidic operation device which may comprise a dispenser 280, 580 that is configured to openly dispense one or more reagents to the inlet 540. The dispenser can openly dispense from a tip, via the void or hole of the inlet, to the open landing area 341, 541. In some embodiments, there is no tubing connecting the dispenser and the inlet. In some embodiments, the dispenser directly contacts part of the inlet, e.g., the landing area, or a wall of the void, to openly dispense the reagents. In some embodiments, the dispenser does not directly contact any physical part of the inlet, but its tip may extend into the void or hole of the inlet. In some embodiments, at least part of the tip of the dispenser is in contact with the open landing area. In some embodiments, the tip of the dispenser is not in direct physical contact with the open landing area.

[0208] The dispenser may include more than one dispensing tips, e.g., pipette tips, so that each different reagent can have its own dispensing tip without mixing of reagents occurring in the dispenser or the dispensing tips. In a sense, the dispenser disclosed herein removed the common line in existing flow cell devices and reduces the dead volume in the common line in existing flow cell systems so that the required consumption of reagents for identical sequencing process can be significantly reduced. Further, removal of common line and usage of separate dispensing tips reduces mixing of reagents and the resulting contamination of reagents dispensed to the flow cell devices.

[0209] In some embodiments, the dispenser and its tip(s) may be manually operated for moving, or dispensing, or combinations thereof. In some embodiments, the dispenser and its tip(s) may be automatically operated for moving, or dispensing, or combinations thereof. For example, the dispenser may include an array of dispensing tips, each in fluidic communication with a reagent reservoir in a cartridge, and a robotic arm moves the array to position a corresponding tip above the landing area and then controls the dispensing. When anext reagent needs to be delivered, the robotic arm can withdraw the previous dispensing tip and locate the next reagent tip in the array for dispensing.

[0210] In some embodiments, the flow cell devices 400, 500, 700 further comprises a cleaning outlet 470, 570, 770. The cleaning outlet 470, 570, 770 can be located in the one or more substrates, for example, in the bottom substrate 430, 530. In some embodiments, the cleaning outlet 470, 570, 770 may be located on a top substrate or in a middle substrate as a side port (not shown). The cleaning outlet 470, 570, 770 can be in fluidic connection with the inlet 440, 540. In some embodiments, the cleaning outlet 470, 570, 770 is configured to be coupled with a fluid driving device, e.g., a pump or vacuum 471 of the fluidic control device. The vacuum or pump 471 may be in addition to the vacuum or pump 472 coupled to the outlet 460. In some embodiments, a same fluid driving device, e.g., pump, can be coupled to the outlet 460, 560 and the cleaning outlet 470.

[0211] The distance from the cleaning outlet 470, 570, 770 can be shorter to the inlet 440, 540 than to the outlet 460, 560. The distance can be within the x-y plane. The shorter distance from the cleaning outlet to the inlet is designed to facilitate transfer of liquid or gas from the open landing area to the cleaning outlet.

[0212] In some embodiments, the relative position of the cleaning outlet 470, 570, 770 to the inlet or open landing area 441, 541, 741 can be different. In some embodiments, the cleaning outlet 770 can be directly underneath the open landing area 741, e.g., in FIGS. 25A- 25E and 26C. In such embodiments, the cleaning outlet 770 is directly connected to the open landing area.

[0213] In some embodiments, the cleaning outlet 470, 570 may not be directly beneath the open landing area but of a distance to the open landing area, e.g., in FIGS. 4, 5A, 24A-E, and 26A-B. In such embodiments, the cleaning outlet 470, 570 is not directly connected to the corresponding open landing area, but instead connected via a tapered transition portion 454, 554 therebetween.

[0214] The distance from the cleaning outlet to the closest edge or the center of the open landing area can be 0 mm or about 0 mm. The distance from the cleaning outlet to the closest edge or the center of the open landing area, when the cleaning outlet is not directly underneath the open landing area, can be from about 0 mm to about 20 mm. The distance from the cleaning outlet to the closest edge or the center of the open landing area, when the cleaning outlet is not directly underneath the open landing area, can be from about 0 mm to about 15 mm. The distance from the cleaning outlet to the closest edge or the center of the open landing area, when the cleaning outlet is not directly underneath the open landing area,can be from about 0 mm to about 10 mm. The distance from the cleaning outlet to the closest edge or the center of the open landing area, when the cleaning outlet is not directly underneath the open landing area, can be from about 3 mm to about 10 mm.

[0215] The distance from the cleaning outlet to the closest edge or the center of the open landing area, when the cleaning outlet is not directly underneath the open landing area, can be from 0 mm to 15 mm. The distance from the cleaning outlet to the closest edge or the center of the open landing area, when the cleaning outlet is not directly underneath the open landing area, can be from 0 mm to 10 mm. The distance from the cleaning outlet to the closest edge or the center of the open landing area, when the cleaning outlet is not directly underneath the open landing area, can be from 3 mm to 10 mm.

[0216] In some embodiments, there can be residuals of reagents, such as meniscus, as shown in the bottom panel of FIG. 4, that remains on the landing pad, or on the wall(s) of the hole of the inlet, or combinations thereof. Such residuals, if not removed, may cause unintended mixing when a subsequent reagent is delivered to the open landing area and consequently contaminate sequencing reactions in the channels. Washing with liquid(s) alone may not be effective in removing such residual reagents as meniscus, so it may take multiple flushing of washing liquids to completely remove the residual in existing flow cell systems, with increased washing time and washing costs. The cleaning outlet 470, 570 in fluidic connection can advantageously facilitate time- and cost- effective removal of such residuals. In some embodiments, a mechanical driving force can be applied, e.g., by a pump or an inlet vacuum, via the cleaning outlet, to completely remove such residual of reagents on the landing pad. As such, the required time and washing volume to remove the residuals to achieve a predetermined contamination level (e.g., less than ) can be effectively improved from existing flow cell devices. As a nonlimiting example, the contamination level may be below 0.1%, 0.01%, 0.005%, or 0.001%.

[0217] The size and shape of the cleaning outlet may be customized to suit different sequencing applications. Although the cleaning outlet is shown as a cylinder in FIG. 5C, it can be made in different shapes, such as a cone, an inverted cone, etc. In some embodiments, the size and shape of the cleaning outlet can be identical to that of the outlet. In some embodiments, the size of the cleaning outlet can be no more than about 10%, 20%, or 30% different from that of the outlet. In some embodiments, the diameter of the cleaning outlet in the x-y plane is about 0.3 mm to about 10 mm. In some embodiments, the height of the cleaning outlet in the z direction is the same as the height of the bottom substrate. In some embodiments, the height of the cleaning outlet is about 0.3 mm to about 3 mm. In someembodiments, the height of the cleaning outlet is about 0.5 mm to about 1 mm. In some embodiments, the diameter of the cleaning outlet in the x-y plane is 0.3 mm to 10 mm. In some embodiments, the height of the cleaning outlet in the z direction is the same as the height of the bottom substrate. In some embodiments, the height of the cleaning outlet is 0.3 mm to 3 mm. In some embodiments, the height of the cleaning outlet is 0.5 mm to 1 mm. Coatings

[0218] In some embodiments, the flow cell device, e.g., 4900, 5000, and / or the one or more dispensing tips may be at least partly coated with a coating disclosed herein, e.g., the second coating. The coating may be a hydrophobic coating. The coating may be a slippery coating. The coating may facilitate transfer of fluids on the coating comparing with the surfaces / structures without the coating. For example, the coating may be applied at least partly on a top surface of the top substrate, e.g., coating 4949 in FIG. 49C and 5049 in FIG. 50B.

[0219] In some embodiments, the flow cell device, e.g., 300, 400, 500, 700, and / or the one or more dispensing tips may lack any coating disclosed herein. In some embodiments, at least some surfaces of the substrate, and / or the one or more dispensing tips may lack any coating disclosed herein. For example, the landing area of flow cell device may lack any hydrophobic or slipper coating in order to reduce residual or dead volume build up.

[0220] In some embodiments, the coating can include one or more layers of liquid- repelling coating. In some embodiments, the coating, can include an omniphobic coating. In some embodiments, the coating comprises a slippery omniphobic covalently attached liquid (SOCAL) coating. In some embodiments, the coating comprises a liquid-like polymer brush surface that is covalently attached to the one or more substrates. In some embodiments, the coating is formed by acid-catalyzed graft polycondensation of one or more saline monomers. The one or more saline monomers can comprise dimethyldimethoxysilane (PDMS). In some embodiments, the one or more saline monomers can have a low surface energy that is below about 10, 15, 20, 25, or 20 mJ / m2.

[0221] The coating can be formed using various methods. For example, the coating can be formed by impregnating lubricants in one or more porous surfaces. In some embodiments, the coating comprises a slippery liquid-infused porous surface (SLIPS). In some embodiments, the lubricants comprise a liquid with a low surface energy, where the low surface energy is below a predetermined threshold. The predetermined threshold can be about 20 milliJoule per square meter (mJ / m2). In some embodiments, the predetermined thresholdcan be about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 mJ / m2, inclusive of all ranges and subranges therebetween. In some embodiments, the lubricants comprise a silicone oil. In some embodiments, the coating comprises low surface energy that is below about 10, 15, 20, 25, or 20 mJ / m2, inclusive of all ranges and subranges therebetween.

[0222] In some embodiments, at least part of the substrate, e.g., surface(s) other than the interior surface of the channels, can be covered with a second coating, e.g., a slippery coating, to facilitate transfer of fluids on the coating, either alone or in combination with the first coating disclosed herein. In some embodiments, at least part of the flow cell device and / or the one or more dispensing tips may be at least partly coated with the second coating. The coating may be a hydrophobic coating. The coating may be a super-hydrophobic coating. The coating may be a slippery coating. The coating may facilitate transfer of fluids on the coating comparing fluids transfer with the surfaces / structures without the coating, thereby reducing or eliminating residuals and / or dead volumes in the fluidic system, e.g., at the open landing area of the flow cell device, which may cause contamination or unnecessary waste of reagents.

[0223] The second coating can be different from the first coating of the channels. The second coating can be applied directly to the substrate(s) without application of the first coating. The second coating can be applied to the substrate(s) on top of the application of the first coating.

[0224] The thickness of the coating along the z axis may be customized so that it does not interfere or reduce fluidic communication speed, or other fluidic parameter(s), or combinations thereof, to the channels in comparison to flow cell devices without the coating. The thickness of the coating along the z axis may be customized so that it increases or facilitates fluidic communication speed, or other fluidic parameter(s), or combinations thereof, to the channels in comparison to flow cell devices without the coating.

[0225] In some embodiments, the second coating can include any liquid-repelling coating. In some embodiments, the second coating can include an omniphobic coating. In some embodiments, the second coating comprises a slippery omniphobic covalently attached liquid (SOCAL) coating. In some embodiments, the second coating comprises a liquid-like polymer brush surface that is covalently attached to the one or more substrates. In some embodiments, the second coating is formed by acid-catalyzed graft polycondensation of one or more saline monomers. The one or more saline monomers can comprise dimethyldimethoxysilane (PDMS). In some embodiments, the one or more saline monomers can have a low surface energy that is below about 10, 15, 20, 25, or 20 mJ / m2.

[0226] The second coating can be formed using various methods. For example, the coating can be formed by impregnating lubricants in one or more porous surfaces. In some embodiments, the second coating comprises a slippery liquid-infused porous surface (SLIPS). In some embodiments, the lubricants comprise a liquid with a low surface energy, where the low surface energy is below a predetermined threshold. The predetermined threshold can be about 20 milliJoule per square meter (mJ / m2). In some embodiments, the predetermined threshold can be about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 mJ / m2, inclusive of all ranges and subranges therebetween. In some embodiments, the lubricants comprise a silicone oil. In some embodiments, the coating comprises low surface energy that is below about 10, 15, 20, 25, or 20 mJ / m2, inclusive of all ranges and subranges therebetween.

[0227] In some embodiments, the second coating comprises a superhydrophobic coating made from various superhydrophobic or ultrahydrophobic materials. The superhydrophobic coating can cause a thin layer of air to form on top of a surface thereby repel liquid droplets from the coating. In some embodiments, the superhydrophobic coating may comprise composite materials including material(s) that provides the roughness and material(s) provides low surface energy. In some embodiments, the second coating comprises one or more of: Manganese oxide polystyrene (MnO2 / PS) nano-composite; Zinc oxide polystyrene (ZnO / PS) nano-composite; Precipitated calcium carbonate; Carbon nano-tube structures; Silica nano-coating; Fluorinated silanes; fluoropolymer coatings; and polydimethylsiloxane (PDMS) and silica particles.

[0228] In some embodiments, superhydrophobic coating may comprise materials with intrinsically low surface energy due to non-polar chemistries (i.e., CH2 / CH3or CF2 / CF3) and close packed, stable atomic structures, resulting in high contact angles (up to 120°) of the surface even without material roughening. In some embodiments, the superhydrophobic materials are either coated onto an already rough material, or are directly processed to induce roughness. Examples of creating the super hydrophobic coating include chemical deposition, assemblies of colloids, layer-by-layer methods, electrospraying, and electrospinning. Chemical deposition, for example, would coat an already rough substrate and impart superhydrophobicity.

[0229] In some embodiments, instead of a coating, e.g., the second coating, the substrate surface itself may be made without the use of any coating through the altering of their surface microscopic contours to achieve superhydrophic characteristics equivalent to the application of the second coating.

[0230] In some embodiments, the open landing area 341 is covered with the second coating 342. FIG. 3 shows an embodiment of the second coating 342 on the open landing area 341, the rest of the landing pad 343, and the part of the top substrate that is above the landing pad 343. The right panel of FIG. 3 shows a schematic drawing of the second coating 342 with a liquid droplet of a reagent thereon. In some embodiments, the second coating 342 can be applied to at least part of the open landing area 341. In some embodiments, the second coating 342 can be applied to any combination of surfaces of the substrates except the interior surfaces defining the lumen of the channels. The second coating 342 can effectively facilitate liquid transfer from the open landing area 341 to the channels 350, 550 or to a cleaning outlet 570 to exit the flow cell device. For example, the second coating 342 may help reduce the volume of residual reagents on the landing pad when the reagent(s) is transferred into the channels. As another example, the second coating 342 may facilitate complete removal of the residual reagents on the landing pad, when an inlet vacuuming force is applied via the cleaning outlet.

[0231] In some embodiments, the second coating 342 can be any liquid-repelling coating. In some embodiments, the second coating can be an omniphobic coating. In some embodiments, the second coating comprises a slippery omniphobic covalently attached liquid (SOCAL) coating. In some embodiments, the second coating comprises a liquid-like polymer brush surface that is covalently attached to the one or more substrates. In some embodiments, the second coating is formed by acid-catalyzed graft polycondensation of one or more saline monomers. The one or more saline monomers can comprise dimethyldimethoxysilane (PDMS). In some embodiments, the one or more saline monomers can have a low surface energy that is below about 10, 15, 20, 25, or 20 mJ / m2.

[0232] The second coating can be formed using various methods. For example, it can be formed by impregnating lubricants in one or more porous surfaces. In some embodiments, the coating comprises a slippery liquid-infused porous surface (SLIPS). In some embodiments, the lubricants comprise a liquid with a low surface energy, where the low surface energy is below a predetermined threshold. The predetermined threshold can be about 20 milliJoule per square meter (mJ / m2). In some embodiments, the predetermined threshold can be about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 mJ / m2. In some embodiments, the lubricants comprise a silicone oil. In some embodiments, the coating comprises low surface energy that is below about 10, 15, 20, 25, or 20 mJ / m2.Contamination Levels and COGS Savings

[0233] In some embodiments, one or more of the open landing area with open dispensing, the channel coating, the slippery coating of the landing pad, and the cleaning outlet and vacuuming can be used alone or in different combinations to achieve cleaning of the flow cell devices. FIGS. 2-4, 5A-5F show nonlimiting embodiments of the combination of one or more of them in the flow cell devices.

[0234] FIG. 7A shows the contamination level of flow cell devices disclosed herein, in comparison to existing flow cell devices. Images of the flow cell channels are acquired perflush volume. The flushing in this embodiment is about 60 microliter ( L), determined basedon at least the channel size and geometry. The contamination of flow cell channels, on average, is about 1% for all three flow cell devices and two existing flow cells. The contamination level starts to decrease as the number of flush volume increases. When the flush factor reaches 5, the total volume of flushed reagents reaches about 300 L. At this flushing factor, the contamination level of three flow cell devices disclosed herein, with SLIPS coating on the open landing read, with inlet vacuum from the cleaning outlet, or their combinations, exhibit a contamination level of lower than 0.01%, while the contamination level of existing flow cells are significantly higher, at above 0.1%. It takes more than 10 flush factors or over 600 L of washing reagents for the existing flow cell devices to achieve similar contamination level as in the three flow cell devices disclosed herein. In this particular embodiment, flushing with more than 300 L does not further decrease the contamination level of the three flow cell devices disclosed herein to a significant level. The contamination level of about 0.001% can be satisfactory for all of the reagents used in the NGS sequencing application(s). The three flow cell devices advantageously achieved contamination levels for accurate and reliable sequencing processes with a significant reduction in Cost of Goods Sold (COGS) than existing methods.

[0235] FIG. 29 shows residual level or contamination level that is averaged among different tiles of a flow cell device disclosed herein. Tile contamination variation across the flow cell device can be caused by the spatial location of the tile on the flow cell and its relative position to the inlet, or the outlet, or combinations thereof. Average contamination levels of different tiles of the flow cell devices disclosed herein are effectively reduced to be less than 1% by the first cycle of flush volume. By the third flush volume or cycle, the residual or contamination level of different tiles are all reduced to below 0.001%. The average tile contamination is below the level of 0.001% by the third flush volume. Theindividual tile contaminations across the flow cell are below the level of 0.001% by the third or fourth flush volume. The flush volume is about 60 L so that the contamination level for individual tiles regardless of its spatial location on the flow cell is reduced to be below 0.001% with a total flush volume of reagents or washing buffer of 240 L.

[0236] In an embodiment, the required volume of sequencing-by-avidite reagents for multiple steps in the sequencing reaction (e.g., stepping, cleaving, and imaging) are all significantly reduced by using the flow cell devices disclosed herein. The stepping reagent requires a volume of about 430 L, and it was reduced to about 90 L with active volume reduction (AVR) to recycle a certain portion of the reagents. The AVR can be used in both existing flow cells systems and the flow cell systems disclosed herein. The AVR can be about 40%, 50%, 60%, 70%, 80%, or 90% of the total volume that is required with respect to a sequencing application. The total volume can be a volume without AVR. The reagents saving with AVR is about 5x in comparison to existing flow cell devices. Without AVR, the reduction can still be about 2.5x in comparison to existing flow cell devices. In some embodiments, the cleaving, trapping, and imaging reagents are reduced from about 300 L to about 60 L, with AVR. Table 1 in FIG. 7B shows the volume of sequencing reagents required in using an existing flow cell system and COGS saving or reduced volume of reagents required using a flow cell device disclosed herein. Fluidic Control Devices

[0237] Disclosed herein are fluidic control devices that can be coupled to the flow cell devices and actively apply mechanical forces for dispensing or collecting liquids, or gas, or combinations thereof, from the flow cell devices.

[0238] In some embodiments, the fluidic control devices comprise a pump, a vacuum, or any other device that can actively apply a mechanical force to the lumen of the channels, or the open landing area, or combinations thereof, via the outlet or cleaning outlet. FIG. 4 shows a fluidic control device with a vacuum or pump 472 that is coupled to all outlets 460 of the flow cell device 400. FIG. 4 shows another vacuum or pump 471 that is coupled to the cleaning outlet 470 of the flow cell device 400. The vacuums 471 and 472 can be the same vacuum or pump.

[0239] In some embodiments, the fluidic control devices can comprise a dispenser 280, 580 with one or more dispensing tips. The dispenser 280, 580 can dispense preset amounts of reagents within a certain time window to the inlet.

[0240] In some embodiments, the fluidic control devices can comprise a robotic arm that controls movement of the dispenser. In some embodiments, the robotic arm can move the dispenser in 3D space so that the dispensing tip can reach a specific location before it starts dispensing. In some embodiments, the robotic arm can retrieve a dispensing tip after one dispense and move a second dispending tip to a location for a subsequent one.

[0241] In some embodiments, the fluidic control devices can comprise a dispensing roller configured to dispense the reagents as shown in FIG. 6A. The reagents can be dispensed by the dispenser 680 to a continuous track 691 rolled on one or more wheels, and the wheels of the roller can roll the track 691 and the reagents to an open landing area of the flow cell. In particular embodiments, the inlet can be a side-port at an edge of the substrate(s). There can be an active force applied at the outlet to facilitate delivery of the reagents from the track to the inlet.

[0242] In some embodiments, the fluidic control devices can comprise a dispensing plate 692 with an electrowetting surface. As shown in FIG. 6B, the dispensing plate 692 can be translated, thereby translating the reagents dispensed thereon to the inlet, which in this embodiment, is a side-port at an edge of the substrates.

[0243] In some embodiments, the fluidic control devices can comprise a reagent reservoir and a sipper as shown in FIG. 6C. In particular embodiments, one end of the sipper 693 can be inserted in a reagent reservoir 694, and the other end of the sipper can point to or be in contact with the inlet. The reagent can be sucked out in a controlled fashion to the open landing area of the flow cell. The open landing area, in this embodiment, is facing downward, and the hole or void of the inlet is in the bottom substrate. Various mechanisms can be used to control the sipping action. For example, an active mechanical force can be applied from the outlet to sip a predetermined amount of reagent from the reservoir. A different sipper can be used for a different reagent to avoid unintended mixing of reagents in the sipper 693. Fluid Dispensing Devices

[0244] In some embodiments, the sequencing system herein may work together with a fluid dispensing device disclosed herein for various NGS sequencing applications. In some embodiments, the fluidic control devices herein may include the fluid dispensing device. In some embodiments, the fluid dispensing device is comprised in the flow cell system disclosed herein.

[0245] The fluid dispensing devices can include a dispensing module. The dispensing module may be disposable. The dispensing module may be removable from the sequencingsystem or flow cell device. The dispensing module may be physically separated and moved relative to the sequencing system or flow cell device.

[0246] In some embodiments, the dispensing module 3100 is reversibly coupled to the one or more actuators 3500 (see FIGS. 30A-30B). In some embodiments, the user may manually couple the dispensing module 3100 to the actuator 3500 or physically removed it from the actuator(s) when needed without damaging the functionality of either one of them.

[0247] The dispensing module may be removably coupled to a flow cell device for sequencing analysis of a sample immobilized on the flow cell device. The dispensing module may be removably coupled to a sequencing system (e.g., NGS system) for sequencing analysis of the sample immobilized on the flow cell device. For example, the dispensing module may be removed and disposed of by a user after a first sequencing application of a first number of samples is completed, and a new dispensing module may be coupled to the sequencing system for a next sequencing application. The sample(s) may be nucleotide acid template molecules immobilized on the flow cell device and can be sequenced after contacting them with predetermined sequencing reagents and / or buffers by introducing the fluid(s) via the dispensing tips to the flow cell device, and allowing the fluid(s) to travel to and contact the sample(s).

[0248] FIGS. 30A and 30B show two exemplary embodiments of the fluid dispensing device herein. The fluid dispensing device 3000 may comprise a dispensing module 3100. The dispensing module 3100 can comprise: a reagent cartridge 3200 with one or more compartments 3210; a microfluidic chip 3300 in fluidic communication with each of the one or more compartments; one or more dispensing tips 3400; or their combinations. The one or more compartments 3210 may hold fluids therein, e.g., reagents.

[0249] In some embodiments, the cartridge 3200 may include a single housing for containing the compartment(s) 3210 as a single unit. In some embodiments, the one or more compartments 3210 can be of various 3D geometrical shapes and sizes. In some embodiments, the one or more compartment 3210 may include various sizes to include predetermined volumes of different reagent. In some embodiments, at least two compartments are of different sizes. In some embodiments, all the compartments may include a dimension, e.g., height, which is identical, as shown in FIGS. 30A-30B. As a non-limiting example, the single housing in FIG. 30A may be 8 inch x 4 cycle x 3.5 inch. In some embodiments, the compartments 3210 are not in fluidic communication with each other within the cartridge. In some embodiments, one or more compartments can be used to hold a same type of liquid or reagent, e.g., with identical concentration and / or mixture of biologicalor chemical compounds therewithin. In some embodiments, two or more compartments can be used to hold different type of liquids or reagent, e.g., a washer buffer and a sequencing reaction reagent, or a clean washing buffer and a contaminated washing buffer, etc.

[0250] In some embodiments, at least one compartment 3210 may be in fluidic communication with only one dispensing tip to avoid cross-contamination of different fluids at the dispensing tip, thereby avoiding consequent cross-contamination at the flow cell device 300, 400, 500, 700. In some embodiments, each different fluidic reagent or each mixture of reagents is configured to travel from its corresponding compartment to its corresponding dispensing tip via its corresponding pathway to avoid cross contamination with other fluidic reagent or mixture in other compartments.

[0251] In some embodiments, at least one compartment 3210 may be in fluidic communication with two or more dispensing tips to allow simultaneously dispensing of different or identical reagent to different locations at the flow cell device 300, 400, 500, 700. For example, two compartments 3210 may be holding same washing buffer therewithin for dispensing washing buffer simultaneously to two inlets which eventually leads different microfluidic channels of the flow cell device 300, 400, 500, 700.

[0252] In some embodiment, each compartment may include various sizes that can fit into the single housing. In some embodiment, each dimension (i.e., length, width, or height) may be within 0.1 inches to 8 inches.

[0253] In some embodiments, each of the one or more compartments may comprise a corresponding compartment outlet 3220 that allows the fluids to travel from a corresponding compartment to the microfluidic chip 3300, and then to the dispending tip(s) 3400. The corresponding compartment outlet may be fluidically connected with a corresponding microfluidic pathway in a fluidically sealed fashion to prevent leakage from the connection, e.g., 3230 in FIG. 32B. In some embodiments, the compartment outlet 3220 and the corresponding inlet 3321 of the microfluidic pathway 3320 can be permanently and non- reversibly connected in a fluidically sealed fashion to avoid leakage from the connection 3230. In embodiments with the dispensing module being disposable, the need for a user to disconnect the cartridge and the microfluidic chip and reconnect a new cartridge to the microfluidic chip is eliminated. It advantageously eliminates possible leakage that can occur at the connection 3230 between the cartridge 3200 and the other part of the dispensing system, e.g., the microfluidic pathways, after a manual connection by the user.

[0254] The fluidic dispensing device may include the microfluidic chip 3300. The microfluidic chip 3300 may be fixedly or permanently coupled to the reagent cartridge 3200.The microfluidic chip may be positioned underneath the reagent cartridge as shown in FIGS. 30A and 33A.

[0255] The microfluidic chip 3300 may include one or more microfluidic pathways (3320 in FIG. 31B) therewithin. The microfluidic chip 3300 may have various sizes and / or geometrical shapes. For example, as shown in FIG.31B, the chip has a rectangular shaped cross-section, e.g., in the x-y plane, and a height that is orthogonal to the cross section, which makes the microfluidic chip substantially a cuboid in 3D. At least part of the pathways can be hollow regions contained between a top surface 3311 and a bottom surface 3312 of the microfluidic chip. At least some portion of the top and / or bottom surface may be flat. For example, the top surface may be flat except the curved portion(s) of the pathways.

[0256] In some embodiments, the top and / or bottom surface may be planar surfaces, and the flatness of the surface(s) from its peak to valley (in a direction orthogonal to the planar surface) can be less than about 0.05 mm, 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, or 0.09 mm. In some embodiments, the flatness of the surface(s) of the substrates from its peak to valley can be less than 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, or 0.09 mm. Each of the microfluidic pathway may fluidically connect a corresponding pathway inlet 3321 and a corresponding pathway outlet 3322 in the microfluidic chip 3300.

[0257] At least one of the microfluidic pathway 3320 may include an elongated portion 3323 between the corresponding pathway inlet 3321 and outlet 3322. At least part of the elongated portion may be straight. The elongate portion extends within the x-y plane, and may include a region that can bulge out from the top surface, the bottom surface, or both. In some embodiments, the bulging out portion may extends out from the x-y plane at least in a direction that is orthogonal to the x-y plane of the microfluidic chip.

[0258] At least one of the microfluidic pathway 3320 may include a curved portion 3324 between the corresponding pathway inlet 3321 and outlet 3322. Some part of the elongated portion 3323 and / or the curved portion 3324 may extend higher than the height of the microfluidic chip 3300 in the direction orthogonal to the x-y plane. In other words, some part of the elongated portion 3323 and / or the curved portion 3324 may bulge out from the top, bottom surface 3311, 3312, or both.

[0259] In some embodiments, the curved portion 3324 is curved within the x-y plane of the microfluidic chip, e.g., as shown in FIG. 31B. In some embodiments, the curved portion may extend out from the x-y plane at least in a direction that is orthogonal to the x-y plane ofthe microfluidic chip. For example, the curved portion may be of a 3D shape like a half or 3 / 4 donut, e.g., in FIG. 31B.

[0260] In some embodiments, at least one pathway lacks any curved portion between its corresponding inlet and outlet and within the x-y plane. In some embodiments, at least one pathway comprises a substantially circular shape between the corresponding inlet and outlet.

[0261] In some embodiments, some part of the elongated portion 3323, the circular portion, and / or the curved portion 3324 may be made of flexible or collapsible materials so that under pressure or force, the flexible material can collapse or otherwise deform to transfer such pressure or force to the fluid(s) within the microfluidic pathways, thereby moving the fluid(s) in one or both directions in the pathways. At least a part of the microfluidic chip may be made of various materials that are flexible or collapsible under pressure. As a non-limiting example, the surface of the chip that is configured to contact the push pin which exert pressure on the chip may be made of flexible plastic and the other surface may be made of non-flexible material such as glass.

[0262] The fluid dispensing device 3000 may comprise one or more actuators 3500 that actuate the movable pin, the plunger, or the syringe for pumping the fluidic reagents in one or both directions. In some embodiments, the reagents are pushed from the one or more compartments 3210 to the one or more dispensing tips 3400 through the one or more microfluidic pathways. In some embodiments, the reagents are pushed from the one or more dispensing tips 3400 to the one or more compartments 3210 through the one or more microfluidic pathways.

[0263] In some embodiments, the fluid dispensing device 3000 further comprises one or more movable pins 3600 that are configured to move and apply pressure or force on various portion(s) of the corresponding pathway 3320 (e.g., the curved portion or bulge portion) to move reagent therein in a predetermined direction. In some embodiments, the movable pin 3600 may be of various 3D size and geometrical shapes. For example, it may be of a disk shape in FIG. 30A.

[0264] The movable pin may comprise various movement to actuate the reagents, such as rotation, translation, or both. The movable pin may rotate about a rotational axis that is orthogonal to the x-y plane of the microfluidic chip to move fluid in the microfluidic pathway in one or both directions. Alternatively, a movable pin herein may translate toward the microfluidic chip to move fluid in the microfluidic pathway(s) e.g., in the curved portion, in one or both direction. In some embodiments, a movable pin herein may, without rotation, translate relative to the microfluidic chip to move fluid in the microfluidic pathway(s) e.g., inthe curved portion, in one or both direction. A movable pin may also, alone or in combination with other motions, translate in a direction that is parallel to the x-y plane of the microfluidic chip to move fluid in the curved portion in one or both direction.

[0265] In some embodiments, the movable pin(s) 3600 is removably coupled to the one or more actuators. In some embodiments, the movable pins may function to seal the fluid(s) in the microfluidic chip when it is physically coupled to the microfluidic chip. In some embodiments, the movable pin(s) is coupled to the microfluidic chip but separable from the actuator(s) to seal the corresponding fluid reagent in the one or more microfluidic pathways within the microfluidic chip to prevent leakage of the dispensing module. As such, no leakage occurs even if the dispensing module is physically separated from the actuators, as shown in FIG. 32B. In embodiments when the movable pins needs to be actuated and move relative to the microfluidic chip, it can be removably coupled to the one or more actuators. When the dispensing module is uncoupled from the sequencing system or the flow cell device and disposed, the movable pin may be disposed together the dispensing module is disposed.

[0266] In some embodiments, the movable pin(s) 3600 is fixedly coupled to the one or more actuator 3500. FIG. 32B shows an exemplary embodiment where the movable pin(s) stays integrated (e.g., physically coupled) with the microfluidic chip, and other elements of the dispensing module 3200. In this particular embodiment, the movable pin is not disposed with the dispensing module but stays coupled to the actuator(s).

[0267] In some embodiments, the one or more actuators may comprise a first number of actuators, and the first number is equal to or smaller than a second number of microfluidic pathways in the corresponding microfluidic chip. As a non-limiting example, the one or more actuators 3500 comprise only one actuator, as shown in FIG 31C. Alternatively, the one or more actuators 3500 may comprise 2, 3,4, or more actuators (e.g., as shown in FIG. 31A). The one or more actuators 3500 may be movable relative to the one or more microfluidic pathways to actuate corresponding movable pin(s), syringe, plunger, etc.. For example, with a single actuator, it may move relative to the microfluidic chip to actuate different movable pins 3600 of different pathways. Alternatively, the one or more actuators 3500 comprise exactly the same number of actuators as the number of movable pins, so that each actuator can be fixedly positioned relative to the corresponding moveable pin, e.g., immediately above it or beneath it to actuate the corresponding pin.

[0268] The one or more actuators 3500 may be fixedly coupled to a next generation sequencing (NGS) system, e.g., the sequencing system 100 herein. In some embodiments, the one or more actuators may be removably coupled to the sequencing system.

[0269] In some embodiments, the one or more actuators 3500 are configured to move to a corresponding spatial position the movable pin(s) to push a corresponding fluidic reagent from the cartridge 3200 to the corresponding dispensing tip 3400 via a corresponding microfluidic pathway 3320 on the microfluidic chip 3300.

[0270] The microfluidic chip 3300, the one or more actuators 3500, and the one or more movable pins 3600, or their combinations may be comprised in a pump.

[0271] The fluid dispensing device may include one or more dispensing tips 3400. The dispensing tips 3400 can be positioned below the reagent cartridge 3200, the microfluidic chip 3300, or both.

[0272] In some embodiments, the pump may include one or more syringe pumps. The syringe pumps are configured to direct reagent(s) from the one or more compartments to the one or more pathways.

[0273] In some embodiments, the pump may comprise one or more plunger and barrel pairs. FIGS. 33A-33B show an exemplary embodiment with plunger and barrel pairs 3340. Each plunger and barrel pair may be in fluidic communication with a corresponding compartment 3210 and a corresponding pathway 3320. The plunger may be movable relative to the corresponding barrel to move a corresponding fluidic reagent therewithin in a predetermined direction. The barrel(s) may be immobilized relative the reagent cartridge 3200, the microfluidic chip 3300, or both. The plunger(s) may be actuated by the one or more actuators 3500. The plunger may move in two opposite directions to move the reagent(s) accordingly in two opposite directions.

[0274] FIGS. 34A-34C show an exemplary embodiment with a single plunger and barrel pair 3340. The plunger and barrel pair may be in fluidic communication with a corresponding compartment 3210 (not shown, and / or can be positioned above or underneath the microfluidic chip 3300) and a corresponding pathway 3320. The plunger may be movable relative to the corresponding barrel to move a corresponding fluidic reagent therewithin. The plunger may move in two opposite directions to move the reagent(s) accordingly in two opposite directions. The barrel(s) may be immobilized relative the reagent cartridge 3200, the microfluidic chip 3300, or both. The plunger(s) may be actuated by the one or more actuators 3500. In some embodiments, one or more fluidic reservoirs are in communication with a single barrel and plunger pair. In some embodiments, individual reservoirs 3360 are fluidically connected to the barrel and plunger pair via a same connector 3361. In some embodiments, one or more fluidic reservoirs 3360 are each in communication with a corresponding barrel and plunger pair.

[0275] In some embodiments, the connector 3361 can be of various designs or structures. Non-limiting examples of the connector include an O-ring connector, a luer lock connector, and a luer slip connector. The connector structure can be molded directly or otherwise permanently fasten on the chip to provide a fluidly sealed connection of the barrel and plunger pair to the microfluidic chip 3300.

[0276] In some embodiments, the microfluidic chip may comprise one or more fluidic reservoirs or wells 3360 within the chip 3300. The one or more fluidic reservoirs can be of various sizes or volumes to fit within the microfluidic chip. In other words, the length, width, and height of the reservoirs is no greater than the length, width, and height of the microfluidic chip. By way of example, the fluidic reservoirs are of identical sizes with a same cross section within the x-y plane in FIG. 34A. In some embodiments, different reservoirs may be of different sizes or shapes. The one or more fluidic reservoirs or wells may be fluidly sealed except for the inlet and outlet that are in fluidic communication to one or more microfluidic pathways. The one or more fluidic reservoirs may be in fluidic communication with the one or more microfluidic pathways. The microfluidic chip 3300 may comprise a top film, a bottom film, or both bonded to a substrate positioned underneath the top film, above the bottom film, and / or between the top and bottom film, thereby forming the microfluidic pathways and / or the one or more reservoirs within the microfluidic chip. In some embodiments, the top film, bottom film, and / or the substrate in between the films may include polypropylene, cyclic olefin copolymer, cyclic olefin polymers, and / or various other materials.

[0277] For example, each reservoir can be positioned in a corresponding microfluidic pathway between the barrel and plunger pair 3340 and the dispensing tip 3400. More specifically, as shown in FIGS. 34A, each reservoir is positioned in a microfluidic pathway between the barrel and plunger pair 3340 and the three-way valve 3350. In this particular embodiment, the one or more movable pins comprises the plunger in the barrel and plunger pair.

[0278] In some embodiments, the microfluidic chip 3300 may include one or more valves 3350 in the one or more microfluidic pathways. The one or more valves can be 3-way valves that can switch between a first position and a second position. The valve(s) can be controlled by an electrical current or voltage, a mechanical motor, a pneumatic pressure, a magnetic force, or different actuating sources. Non-limiting examples of the valves include: a solenoid valve, a pneumatic valve, a rotary valve, or a membrane valve. Details of an exemplary membrane valve have been disclosed in U.S. Patent No. 10,830,362, is the contents of whichare incorporated herein by reference in their entirety. In some embodiments, the one or more valves may be replaced with other structures that can functionally enable: (1) fluidic communications from the reservoir via the inlets 3321 to the compartment(s) but not to the dispensing tip(s); and (2) fluidic communications from the reservoir not to the compartment(s) but to the dispensing tip(s) via the outlets 3322. The one or more valves may each be in fluidic communication with a corresponding one of the one or more dispensing tips, one or more compartments, and one or more fluidic reservoirs via the one or more microfluidic pathways. As shown in FIG. 34A, there are three different microfluidic pathways from the individual valve to the reservoir and the barrel and plunger pair, to the corresponding compartment of the cartridge, and to the dispensing tip. The valve can switch between the first and second position to connect the reservoir either with the cartridge or the dispensing tip. The one or more compartments and the one or more fluidic reservoirs may be in fluidic communication via the one or more microfluidic pathways when the corresponding valves are in a first position. The one or more dispensing tips 3400 and the one or more fluidic reservoirs may be in fluidic communication via the one or more microfluidic pathways when the corresponding valves are in a second position.

[0279] The fluid dispensing device 3000 may include one or more dispensing tips 3400 to openly dispense one or more reagents to a flow cell device 300, 400, 500, 700 (e.g., in FIG. 30A). The one or more dispensing tips 3400 are movable relative to an inlet of the flow cell device 300, 400, 500. For example, a first dispensing tip and a second dispensing tip may be moved above two different inlet leading to different microfluidic channels of the same flow cell device to simultaneously deliver two identical or different reagents. As another example, a first dispensing tip and move to a first inlet to deliver a washing buffer to a first channel of the flow cell device and then move to a second inlet to deliver the same washing buffer to a second channel of the same flow cell device.

[0280] In some embodiments, a various number of dispensing tips can be included, e.g., 1-100, 1-50, 1-40, 1-30, etc., or any range therebetween. In some embodiments, the number of dispensing tips can be identical to the number of compartments in the reagent cartridge. In some embodiments, the spatial arrangement of the dispensing tips can vary depending on different sequencing applications. For example, the first dispensing module may include 2, 3, or more rows of dispensing tips, while the second dispensing module may include a single row of dispensing tips spaced evenly from each other. The dispending tips may be customized in various sizes and shapes, for example, a cone shape as shown in FIG. 30A.

[0281] Although in embodiments shown in figures herein include the cartridge and it compartments and the motor on top of the microfluidic chip while the dispensing tips are underneath the microfluidic chip, in other embodiments, the cartridge and its compartment and / or the motor may be positioned in different locations relative to the chip, e.g., underneath the microfluidic chip or on a side of the microfluidic chip. Similarly, the relative position of the barrel and plunger pair(s), the moveable pin, and the dispensing tips to the microfluidic chip may be but is not limited to the positions disclosed herein relative to the figures.

[0282] In some embodiments, the flow cell system may include a flow cell device comprising: a support comprising one or more substrates; one or more channels defined by the one or more substrates, wherein the one or more channels are configured to allow fluids and a gas gap between the fluids to flow therethrough; an inlet in the one or more substrates, the inlet in fluidic connection with the one or more channels; and an outlet in the one or more substrates, wherein the one or more channels run from the inlet to the outlet. The flow cell system may further comprise a fluid dispensing device comprising a dispensing module comprising: a reagent cartridge with one or more compartments, wherein the one or more compartments are configured for holding fluidic reagents therein; a microfluidic chip in fluidic communication with each of the one or more compartments, wherein the microfluidic chip comprises one or more microfluidic pathways therewithin; and one or more dispensing tips; and one or more actuators that actuate a movable pin to push the fluidic reagents from the one or more compartments to the one or more dispensing tips through the one or more microfluidic pathways. Fluid Dispensing device with Transportation Valves

[0283] In some embodiments, the fluid dispensing device 3000 comprises a dispensing module 3100 comprising: a reagent cartridge 3200 with one or more compartments 3210, wherein the one or more compartments 3210 are configured for holding fluidic reagents therein; a microfluidic chip 3300 in fluidic communication with the one or more compartments 3210, the microfluidic chip 3300 having one or more fluidic pathways 3320, 3320-1, 3320-2, 3320-3 therewithin; a transportation valve 3700 having at least one open position and at least one closed position rotationally separated from the at least one open position, and wherein the transportation valve 3700 is positioned between the reagent cartridge 3200 and the microfluidic chip 3300; an actuator configured to actuate the transportation valve 3700 between the at least one open position and the at least one closed position; and one or more dispensing tips 3400, wherein the transportation valve 3700, in theat least one closed position, is configured to seal each of the one or more compartment 3210 and the microfluidic chip 3300.

[0284] In some embodiments, the fluid dispensing device 3000, comprises: a dispensing module 3100 comprising: a reagent cartridge 3200 with one or more compartments 3210, wherein the one or more compartments 3210 are configured for holding fluidic reagents therein; a microfluidic chip 3300 in fluidic communication with the one or more compartments 3210, the microfluidic chip 3300 having one or more fluidic pathways 3320 therewithin; a transportation valve 3700 having at least two open positions and at least one closed position that is rotationally separated from the at least two open positions; an actuator configured to actuate the transportation valve 3700 between the at least two open positions and the at least one closed position; and one or more dispensing tips 3400, wherein the transportation valve 3700, in the at least one closed position, is configured to seal each of the one or more compartment 3210 and the microfluidic chip 3300.

[0285] FIGS. 35A-35C, 36A-36B, and 37A-37D show an exemplary embodiment of the fluid dispensing device 3000 comprising the transportation valve 3700. FIGS. 38A-38B, 39A-39C, and 40 show another exemplary embodiment of the fluid dispensing device 3000 with the transportation valve 3700.

[0286] The transportation valve 3700 may be configured to seal the reagent(s) in the reagent cartridge 3200, and / or seal any fluidic that may be contained within the microfluidic chip 3300. The fluidic dispensing module 3100 can be pre-assembled as a disposable and integrated device with reagent(s) contained therein. Without the transportation valve 3700, the reagent(s) may leak from the one or more compartment(s) even if the in-chip valve(s) are closed, for example, during transportation of the dispensing module 3100, thereby causing undesired damage and / or contamination to the fluid dispensing device 3000. The transportation valve 3700 can be advantageously included in the fluid dispensing device 3000 to efficiently seal the one or more compartment(s) and prevent contamination or damage to the device. Further, transportation valve 3700 may also facilitate sealing of fluidic, if any, that are supposed to be contained within the microfluidic chip 3300.

[0287] In some embodiments, the transportation valve 3700 comprises a valve substrate 3710. The valve substrate 3710 may be a disk substrate or a ring substrate as shown in FIG. 35C and FIG. 38B. The valve substrate 3710 may comprise a plurality of open ports 3720. The plurality of open ports 3720 may be distributed on the valve substrate in various patterns. For example, the plurality of open ports 3720 may be distributed radially along a same circumference in the valve substrate, e.g., FIG. 35C. As another example, the plurality ofopen ports 3720 may be distributed radially along different circumferences in the valve substrate, e.g., FIG. 38B. In some embodiments, the open ports 3720 are distributed along a same radius as in FIG. 38B or along different radius as in FIG. 35C. The angular separation between two radius where two open ports are positioned may be customized. In some embodiments, the angular separation or angle between two adjacent open ports 3720 may be based on the size, shape, orientation, and other possible aspects of the cone or more compartments of the reagent cartridge 3200. In some embodiments, the angular separation or angle between two adjacent open ports, e.g., angle a in FIG. 35C, can be in the range from 10 degrees to 160 degrees. In some embodiments, the angular separation or angle between two adjacent open ports may not be the same as the angular separation or angle between another two adjacent open ports, e.g., as shown in FIG. 35C.

[0288] In some embodiments, a total number of open ports matches the total number of compartments of the reagent cartridge, and each open port corresponds only to a corresponding compartment for allowing fluidic communication from the corresponding compartment to the open port and then to the microfluidic chip thereby avoid cross contamination between reagents. The total number of open ports can be in the range from 1 to 20 or more. In some embodiments, two or compartments may share the same open port. For example, the compartment for holding washing solution may share a single open port with one or more other compartment containing reagents since the cross-contamination level may be controlled to satisfy a predetermined contamination threshold.

[0289] In some embodiments, the transportation valve 3700 comprises an over-mold or a gasket 3730 around each of the plurality of open ports 3720. The over-mold or gasket 3730 may be around each of the plurality of open ports 3720 on a first side facing the reagent cartridge 3200 and a second side facing the microfluidic chip 3300, e.g., a top side and a bottom side of the open ports 3720. In some embodiments, each over-mold or gasket, e.g., a first side thereof, is configured to compress the corresponding outlet of the reagent compartment 3210 thereby sealing the reagent within the compartments. In some embodiments, each over-mold or gasket, e.g., a second side thereof, is configured to block the corresponding pathway in the microfluidic chip thereby sealing any fluidic within the pathway of the microfluidic chip. The compression may be provided using various methods. For example, as shown in FIG. 35B, the microfluidic chip and the transportation valve can be mounted to the bottom of the reagent cartridge using mounting hardware like screws. The size and thickness of the over-mod or gasket can be customized based on the size and / orshape of the compartments and the microfluidic chip. In some embodiments, the over-mold or gasket extends at least 5 to 45 degrees along a circumference, e.g., angle b in FIG. 35C.

[0290] In some embodiments, the transportation valve 3700 comprises an actuation arm 3740 configured to be actuated by an actuator thereby causing rotation of the transportation valve 3700. The transportation valve 3700 may be configured to rotate about an axis that is orthogonal to the valve substrate 3710. In some embodiments, instead of the actuation arm, the transportation valve 3700 may be actuated in various other ways to rotate. In some embodiments, the transportation valve is configured to rotate 3 to 45 degrees from the at least one open position to the at least one closed position in a first direction, e.g., clockwise. The transportation valve is configured to rotate 3 to 45 degrees from the at least one closed position to the at least one open position in a second direction opposite to the first direction, e.g., counter-clockwise. FIG. 36A shows the at least one closed position of the transportation valve. The at least one closed position may correspond to a first compartment of the reagent cartridge. In some embodiments, one or more compartments are simultaneously sealed when the transportation valve 3730 is in the at least one closed position. In some embodiments, the at least one closed position comprises a single closed position so that all the compartments are sealed in the single closed position. For example, when the dispensing module is in transportation, the transportation valve is in the at least one closed position, and all the compartments are sealed to prevent reagent leakage during transportation.

[0291] The transportation valve 3700 may comprise at least one open position. The at least one open position may include one or more open positions. FIG. 36B shows a first open position in which the corresponding compartment is open, and reagent can flow from the corresponding compartment through the open port into the microfluidic chip. In other words, at least one compartment is open when the transportation valve is in the first open position. In some embodiments, more than one compartment, e.g., all can be simultaneously open when the transportation valve is in the first open position, different reagent flows from different compartments are not blocked by the transportation valve, but may still be blocked by in-chip valves 3351, 3352 of the microfluidic chip 3300.

[0292] In some embodiments, the first compartment is open when the transportation valve is in the first open position while the other compartments are closed, and a second compartment is open when the transportation valve is in the second open position while the other compartments are closed. In some embodiments, the first compartment is open when the transportation valve is in the first open position while the other compartments are open or closed.

[0293] In some embodiments, the transportation valve 3700 is mounted to the bottom of the reagent cartridge 3200. The microfluidic chip 3300 may be mounted to the bottom of the transportation valve.

[0294] In some embodiments, the microfluidic chip 3300 comprises a plurality of pairs of dispensing ports 3322. Each dispensing port 3322 may be in fluidic communication with a corresponding dispending tip 3400. A distance between each pair of the dispensing ports is determined based on the distance between two open landing areas of a flow cell device so that the pair of dispensing ports may allow simultaneously dispensing to the different lanes of the flow cell device, e.g., FIG. 37A. In some embodiments, each pair of dispensing ports corresponds to only a corresponding in-chip well and a corresponding compartment. In some embodiments, the number of dispensing ports in fluidic communication with a corresponding in-chip well and reagent compartment can increase when the flow cell device comprises more than 2 lanes.

[0295] FIGS. 37A-37B show an exemplary embodiment of the microfluidic chip 3300 in relation to the transportation valve 3700. The microfluidic chip 3300 may comprise a first in- chip well or reservoir 3360 corresponding to a first pair of the dispensing ports 3322. The first in-chip well 3360 may be in fluidic communication with an off-chip pump 3340 via a first fluidic pathway 3320_1. The off-chip pump may pump air into the first in-chip well or pull air out from the first in-chip well to facilitate dispensing or aspiration of reagents. The off-chip pump may be in fluidic communication with the first in-chip well 3600 via a first fluidic pathway 3320_1. The off-chip pump may pump in or aspirate washing solution.

[0296] The first in-chip well 3360 may be in fluidic communication with the reagent cartridge 3200 via a first in-chip valve 3351 and a second fluidic pathway 3320_2. The first in-chip well 3360 may be in fluidic communication with a first pair of the dispensing ports 3322 via a second in-chip valve 3352. The second in-chip valve 3352 may be in fluidic communication with the first pair of the dispensing ports 3322 via a third fluidic pathway 3320_3. In some embodiments, the first in-chip well 3360 may be in fluidic communication only with the first pair of the dispensing ports 3322 but no other dispensing ports to avoid cross contamination of reagents.

[0297] In some embodiments, the first fluidic pathway, the second fluidic pathway, the third fluidic pathway (3320-1, 3320-2, 3320-3), the first in-chip well 3360, the first in-chip valve 3351, the second in-chip valve 3352, the first pair of dispensing ports 3322, or a combination thereof correspond to only the first compartment 3210 of the reagent cartridge 3200, but not other compartments to avoid cross contamination of reagents.

[0298] In some embodiments, the first or second in-chip valve 3351, 3352 may have various shapes. Each of the first or second in-chip valve may comprise at least an open position and a closed position. The first or second in-chip valve may be configured to shift between the open position and the close position when a pressure exerted on the first or second in-chip valve is altered.

[0299] For example, the first or second in-chip valve may be a three-way valve as shown in FIGS 34A-34C, so that it may shift to connect two of the three different pathways to allow aspiration or dispensing. As another example, each in-chip valve may comprise a dimple dome between two non-connected in-chip fluidic pathways, e.g., FIG. 37B. When the dimple dome is depressed, the flow is blocked, when the dimple dome is released, the flow between two non-connected in-chip fluidic pathways is connected.

[0300] In some embodiments, when the transport valve 3700 is in the at least one open position, the first in-chip valve 3351 is open, and the second in-chip valve 3352 is closed, and the dispensing module 3100 is configured to aspirate reagent from the first compartment 3210 to the in-chip well 3360. When the transport valve 3700 is in the at least one open position, the first in-chip valve 3351 is closed, and the second in-chip valve 3352 is open, and the dispensing module is configured to dispense reagent from the in-chip well 3360 to only the first pair of the plurality of pairs of dispending ports 3322.

[0301] In some embodiments, each of the plurality of open ports 3720 comprises an elongated shape extending along a circumference of the valve substrate 3710 as shown in FIG. 38B. In some embodiments, the transportation valve 3700 may further comprise a central open port 3721 having the elongated shape extending along a radius of the valve substrate, e.g., in FIG. 38B. The central open port 3721 may be in fluidic communication with the off-chip pump 3340 external to the transportation valve 3700.

[0302] In some embodiments, the microfluidic chip 3300 is positioned between the transportation valve 3700 and the reagent cartridge 3200, e.g., FIG. 39A-39C. The microfluidic chip 3300 may comprise one or more in-chip wells 3360. Each well may be configured to contain a different reagent to avoid cross contamination. The total number of wells may be based on the total number of reagents required in a sequencing application.

[0303] Each in-chip well may comprise a first well opening 3365 and a second well opening 3366. The microfluidic chip 3700 may comprise a first through-hole or outlet 3322 in fluidic communication with the first pair of dispensing tips 3400, optionally via a third fluidic pathway 3320_3. In some embodiments, the microfluidic chip comprises the firstthrough-hole that is in fluidic communication with only the first pair of dispensing tips but no other dispensing tips to avoid cross contamination of reagents.

[0304] In some embodiments, the fluidic pathways in the microfluidic chip, 3320, 3320- 1, 3320_2, 3320_3 may include a lumen between a top and a bottom surface of the chip, e.g., the cross-sectional view at line AA’ in FIG. 38A.

[0305] The microfluidic chip 3700 may comprise a second through-hole 3321 or inlet in fluidic communication with a first compartment 3210 of the reagent cartridge, optionally via a second fluidic pathway 3320_2. In some embodiments, the microfluidic chip comprises the second through-hole or inlet that is in fluidic communication with only the first compartment of the reagent cartridge to avoid cross contamination of reagents, optionally via a second fluidic pathway 3320_3.

[0306] The transportation valve 3700 may be in a first open position of the at least two open positions when a first open port 3720 connects the first through-hole 3322 and a first well opening 3365 of a first in-chip well 3360, e.g., in FIG. 39C. The transportation valve may be in the first open position of the at least two open positions to allow reagent dispensing from the first in-chip well 3600 to the first pair of dispensing tips 3400. The transportation valve 3700 may be in the first open position when the first open port 3720 is positioned above the first through-hole 3322 and the first well opening 3365, e.g., in FIG. 39C.

[0307] The transportation valve 3700 may be in a second open position of the at least two open positions when a first open port 3720 connects the second through-hole 3321 and a first well opening 3365 of a first in-chip well 3360. The transportation valve 3700 may be in the second open position of the at least two open positions to allow reagent aspiration from the first compartment 3210 of the reagent cartridge 3200 to the first in-chip well 3360. The transportation valve is in the second open position when the first open port 3720 is positioned above the second through-hole 3321 and the first well opening 3365, e.g., in FIG. 39B.

[0308] In some embodiments, when the transportation valve 3700 is in the first or second open position, the central open port 3721 connects a second well opening 3366 to the pump 3340 via a first fluidic pathway 3320_1. Correspondingly, the pump may be pulled to facilitate reagent aspiration from the compartment to the in-chip well. The pump may be pushed to facilitate reagent dispensing from the in-chip well to the dispensing tip(s).

[0309] In some embodiments, the microfluidic chip 3700 comprises a chip substrate 3710. The chip substrate comprises a circular shape as shown in FIG. 39A with more than one circumferences shown as dotted lines. In some embodiments, the first and secondthrough-holes 3322, 3321 correspond to the first in-chip well 3600, but not other in-chip wells to avoid cross contamination of reagents.

[0310] In some embodiments, the transportation valve 3700 further comprises third and fourth through-holes corresponding to a second in-chip well. The first and second through- holes correspond only to a first open port, and the third and fourth through-holes correspond only to a second open port that is different from the first open port to avoid cross contamination.

[0311] In some embodiments, the first and second through-holes are positioned along a circumference of the chip substrate 3710. The third and fourth through-holes may be positioned along the same circumference or a second circumference of the chip substrate. The first and third through-holes may be 5 to 85 degrees apart by rotation about an axis orthogonal to the valve substrate, e.g., angle c in FIG. 38A. The first and second through- holes may be 1 to 45 degrees apart by rotation about an axis orthogonal to the valve substrate

[0312] In some embodiments, the at least two open positions correspond to the first open port 3720 that corresponds to a first compartment 3210. When the transportation valve is in the least two open positions, only the first in-chip well 3600 is in fluidic communication with the first pair of dispensing tips or the first compartment, the other in-chip well(s) is sealed from the reagent cartridge and the plurality of dispensing tips.

[0313] In other embodiments, when the transportation valve is in the least two open positions, the first in-chip well is in fluidic communication with the first pair of dispensing tips or the first compartment, and the second in-chip well may also be in fluidic communication with the reagent cartridge or the plurality of dispensing tips, so that reagent aspiration from two different compartments may occur simultaneously and / or reagent dispensing from two different in-chip wells to different dispensing tips may occur simultaneously. Such simultaneous dispending may be used for simultaneous aspiration of different reagent to save fluidic operation time during a sequencing run. Further, such simultaneous dispending may be used for simultaneous dispensing to multiple flow cells, for example flow cells that are arranged along different radius and separated by a rotational angle from each other to further reduce fluidic operation time during a sequencing run and enable improved sequencing system throughput.

[0314] The transportation valve 3700 may be in a closed position when the first open port 3720 is positioned not directly above the first through-hole 3322, the second through-hole 3321, and the first well opening 3365, e.g., as shown in FIG. 39A. The transportation valve 3700 may be in the closed position to seal all the compartments and prevent reagent leakage.Methods of Using the Flow Cell Devices

[0315] Disclosed herein are methods of using the flow cell devices 200, 300, 400, 500, 700 for performing, or facilitating, or combinations thereof, sequencing analysis using the sequencing system 110. Disclosed herein are also methods of manufacturing the flow cell devices 200, 300, 400, 500, 700 that can be used to perform, or to facilitate, or combinations thereof, sequencing analysis. The methods herein can include some or all of the operations disclosed herein. The operations may be performed in, but is not limited to, the order that is described herein.

[0316] The operations herein may be performed manually. The operations may be automatically performed by a robotic arm or the like (not shown). The robotic arm can be controlled by a computer system, e.g., 126 in FIG. 1, to automatically perform some or all of the operations disclosed herein. Alternatively, the computer system 126, dedicated processors, 118, the FPGA(s) 120, or their combinations, may be programmed to control the robotic arm. The computer system of the robotic arm can have installed on it software, firmware, hardware, or their combinations that in operation cause the computer system to perform the operations or actions disclosed herein.

[0317] The methods can be performed by one or more processors in the computer system, e.g., 126, disclosed herein. In some embodiments, the processor can include one or more of: a processing unit, an integrated circuit, or their combinations. For example, the processing unit can include a central processing unit (CPU), or a graphic processing unit (GPU), or combinations thereof. The integrated circuit can include a chip such as a field-programmable gate array (FPGA). In some embodiments, the processor can include the computing system.

[0318] In some embodiments, some or all operations in the methods can be performed by the FPGA(s) 120. In embodiments when some operations are performed by FPGA(s), the data after an operation performed by the FPGA(s) can be communicated by the FPGA(s) to the CPU(s) so that the CPU(s) can perform subsequent operation(s) in method using such data. Similarly, data can also be communicated from the CPU(s) to the FPGA(s) for processing by the FPGA(s). In some embodiments, all the operations in the method can be performed by CPU(s). Alternatively, the operations performed by CPU(s) can be performed by other processors such as the dedicated processors, or GPU(s). In some embodiments, all the operations in method can be performed by FPGA(s).

[0319] The methods of manufacturing the flow cell devices disclosed herein can comprise an operation of obtaining the one or more substrates. The operation of obtaining theone or more substrates can comprises obtaining the one or more substrates separately so that the one or more substrates are not yet physically coupled to each other.

[0320] The methods disclosed herein can comprise an operation of generating one or more channels in the one or more substrates. In some embodiments, the channels are generated as holes completely in the middle substrates. In some embodiments, generating a channel comprises generating a groove in the top or bottom substrates and generating a hole in the middle substrate, and the channel can be formed by stacking the groove and the hole together. In some embodiments, generating a channel comprises generating a groove in each of the two adjacent substrates and combining the groves together to form the channel, via etching or any other mechanisms. The present disclosure does not limit the mechanisms by which the hole, groove, or cavity, can be formed in the substrates. The hole, groove, or cavity can form a lumen that allows fluids and a gas gap between the fluids to flow therethrough, when the substrates are fixedly coupled together, e.g., bonded.

[0321] The methods disclosed herein can comprise an operation of forming an inlet. The operation of forming an inlet can comprise forming a hole or a void in at least one of the one or more substrates and forming an open landing area. The hole or void can be at or near one end of the substrates, or the channels, or combinations thereof. For example, forming the inlet can comprise forming a cylinder hole in the top substrate and forming an open landing area in the middle substrate that matches the location of the cylinder hole, e.g., at the same location, so that when the two substrates are stacked together, the hole is directly above the landing area or at least partly above the landing area.

[0322] The methods disclosed herein can comprise an operation of forming an outlet. The operation of forming an outlet can comprise forming a hole or a void in at least one of the one or more substrates. For example, forming the outlet can comprise forming a cylinder hole in the bottom substrate at or near the opposite end of the substrates, or channels, or combinations thereof, from the inlet.

[0323] In some embodiments, the inlet and outlet are in fluidic connection with the one or more channels.

[0324] The methods disclosed herein can comprise an operation of fixedly coupling the substrates together. The coupling operation can be achieved via chemical, mechanical, or laser bonding, but is not limited to such bonding techniques.

[0325] The methods disclosed herein can comprise coating at least a portion of a surface of the one or more channels with a first coating, as disclosed herein. The surface can beinterior surface defining the lumen(s) of the one or more channels. For example, the surface can include a top or bottom interior surface.

[0326] The methods disclosed herein can comprise coating at least a portion of a surface of the one or more channels with an additional coating to the first coating, e.g., a third coating of fluorescent beads.

[0327] The methods disclosed herein can comprise an operation of covering at least a portion of the open landing area with a second coating as disclosed herein. The second coating can be different from or identical to the first coating in the channels. In some embodiments, the process of applying the second coating can be different from or identical to applying the first coating in the channels. In some embodiments, at least some actions in the entire process of applying the second coating can be different from or identical to applying the first coating in the channels.

[0328] In some embodiments, coating the open landing area comprises impregnating lubricants in one or more porous surfaces. In some embodiments, coating the open landing area comprises acid-catalyzed graft polycondensation of one or more saline monomers.

[0329] In some embodiments, the methods of manufacturing the flow cell devices further comprises an operation of forming a cleaning outlet in the one or more substrates. The operation of forming the cleaning outlet can comprise forming the cleaning outlet in fluidic connection with the inlet and positioning the cleaning outlet so that it is closer to the inlet than to the outlet. The operation of forming the cleaning outlet can further comprise forming the cleaning outlet in a predetermined size and shape. For example, the size and shape of the cleaning outlet can be approximately the same as the outlet. The operation of forming the cleaning outlet can further comprise forming the cleaning outlet in the bottom substrate, the top substrate, the middle substrate, or their combinations. As an example, the cleaning outlet can be a side port formed by a half groove in the middle substrate and the bottom substrate.

[0330] The methods of manufacturing the flow cell devices can comprise coating a surface of the flow cell device (e.g., an interior surface) with one or more one or more hydrophilic polymer coating layers as described herein. The one or more hydrophilic polymer coating layers can comprise a plurality of oligonucleotide molecules, such as the surface capture primers described herein, attached to at least one hydrophilic polymer coating layer.

[0331] The methods of using the flow cells disclosed herein can comprise an operation of dispensing a first reagent openly to an open landing area of an inlet of the flow cell device. The dispensing operation can be performed manually or automatically by a robotic arm. The dispensing of the first reagent can be from a dispensing tip of a dispenser of the fluidiccontrol device disclosed herein. In some embodiments, a dispensing tip is used for dispensing the first reagent but not any other reagents to avoid unintended mixing of reagents in the dispensing tip.

[0332] The methods can further comprise an operation of moving the dispensing tip to a specific location before dispensing. The specific location can be above the hole of the inlet of the flow cell. The specific location can be that the tip is at least part inside the hole of the inlet. In some embodiments, at least part of the tip is in contact with the wall of the hole or the open landing area at the bottom of the hole. In some embodiments, the dispensing tip may comprise a shock absorbing portion that contacts the open landing area without exerting damaging force to the open landing area or the substrate(s). The dispending operation can last for a predetermined period of time to ensure a predetermined amount of first reagent is dispensed into the outlet. The predetermined dispensing time can be on the scale of sub seconds to less than a minute.

[0333] The methods can further comprise an operation of retrieving the dispensing tip from the specific dispensing location.

[0334] The methods can further comprise an operation of flowing at least part of the first reagent from the open landing area to one or more channels of the flow cell device. This operation of flowing the reagents can be driven passively without actively adding any mechanical force on the reagents. Alternatively, the operation can be facilitated by adding a mechanical force to transfer the reagent from one end of the channels in direct contact with the open landing pad to the opposite end of the channels that is in contact with the outlet. For example, the force can be applied by a pump or a vacuum at the outlet. The sequencing reactions can occur when the first reagent flows through the channels.

[0335] The methods can further comprise an operation of cleaning residuals of the first reagent from the one or more channels by driving an air gap before dispensing any second reagent to the flow cell device. The air gap can be driven by a mechanical force applied by a pump or a vacuum at the outlet. The air gap may also help clean some of the residuals on the landing pad. The mechanical force can be adjusted so that the air gap can occupy about 30% to about 80% volume of each channel in a predetermined time window. Channels with a larger lumen may need a larger air gap for similar cleaning effect as compared to channels with smaller lumens.

[0336] The methods can further comprise an operation of washing the channels before dispensing any second reagents to achieve a cleaning effect.

[0337] The methods can further comprise an operation of dispensing a second reagent openly to the open landing area via a different dispensing tip from that of the first reagent when the second reagent is different from the first reagent.

[0338] Before dispensing the second reagent, the methods can further comprise an operation of confirming that the channels have been cleaned and a predetermined cleaning threshold has been met. For example, the predetermined cleaning threshold can be a contamination level that is required for the second reagent that is going to be administered.

[0339] The methods can further comprise an operation of facilitating cleaning of residuals of the first reagent off the open landing area by using a coating on at least part of the open landing area. The residuals of the first reagent on the open landing pad may also contaminate the second reagent to be administered subsequent to the first reagent. Cleaning of such residuals can also help reduce contamination level of the second reagent and thus improve accuracy and reliability of the sequencing reactions based on the second reagent. The coating, e.g., liquid repelling or slippery, on the open landing pad can passively facilitate transfer of the first reagent to the channels and reduce residuals on the landing pad.

[0340] The methods can further comprise an operation of cleaning residuals of the first reagent from at least part of the open landing area by driving the residuals through the cleaning outlet. An active mechanical force can be applied via the cleaning outlet, e.g., by a pump or a vacuum, to suck the residuals from the open landing pad to the cleaning outlet. The active mechanical force can be combined with a passive second coating on the landing pad to facilitate cleaning of the open landing pad before administration of the second reagent.

[0341] FIGS. 24A-24E and FIGS. 25A-25E show embodiments of the flow cell devices disclosed herein. Structural elements of the flow cell devices disclosed herein can have varying sizes. Such structural elements can include, but are not limited to, the inlet, the open landing area, the outlet, the tapered transition portion from the cleaning outlet to the open landing area or the inlet, the tapered transition portion from the inlet to the corresponding channel, and the tapered transition portion from the channel to the corresponding outlet. FIGS. 26A-26C shows embodiments of flow cell devices in which the sizes of the open landing area, the tapered transition portion from the cleaning outlet to the inlet or the open landing area, the tapered transition portion from the inlet or the opening landing area to the channel, or their combinations, are altered from the flow cell devices in FIGS. 24A-24E and FIGS. 25A-25E.

[0342] FIGS. 27A-27G show embodiments of the flow cell device disclosed herein. In these particular embodiments, as shown in FIG. 27A, the total thickness of the flow cell device is 2.07 mm. The top and the bottom substrates have thicknesses of 1 mm.

[0343] Various embodiments of the methods may be implemented, for example, using one or more computer systems, such as computer system 800 shown in FIG. 8. One or more computer systems 800 may be used, for example, to implement any of the embodiments discussed herein, as well as combinations and sub-combinations thereof. The flatness of surface A in FIG.27A from its peak to valley, e.g., the difference between the highest and lowest points on the surface, is less than 0.02 mm. The flatness of surface B in FIG. 27B from its peak to valley, e.g., the difference between the highest and lowest points on the surface, is less than 0.02 mm. FIG. 27B shows that each channel edge to the edge of the flow cell device along x axis can be about 2.24 mm. The channels can have a width of 8.5 mm. The gap between the two channels along the x axis can be 3.5 mm. Each lane starts at 11 mm away from the closest edge of the flow cell device along the y axis. FIG. 27C shows that the cleaning outlet is 6.5 mm away from the edge of the flow cell device along the x axis, and the two cleaning outlets are 12 mm apart from each other along x axis. The cleaning outlets are 3 mm away from the closest edge of the flow cell device along the y axis. The diameter of the cleaning outlet is 0.81 mm. Alignment element “1” in FIG. 27C is configured to align the flow cell devices to the moving stage that can hold the flow cell device and move it relative to the optical system, which is positioned 11.25 mm from one edge of the flow cell device and 13.75 mm from the other edge of the flow cell device along the x axis, and positioned between the opening landing areas. As shown in FIG. 27C, the outlets are of the same dimension as the cleaning outlets. The total width of the flow cell device is 25 mm. The total length of the flow cell device is 75 mm. The cleaning outlets are 3 mm away from one edge of the flow cell device along the y axis. The outlets are 3 mm away from the opposite edge of the flow cell device along the y axis. FIG. 27G shows the middle substrate of 0.07 mm. FIGS. 27E and 27F shows that the open landing area has a circular shape with a diameter of 8.52 mm. The tapered transition from the outlet to the body of the channel includes a curved portion that is a portion of a circular shape with a radius of 0.5 mm, and the angle defined between the tapered transition portion is 50.2 degrees.

[0344] In some embodiments, the curved portion of the tapered transition portion, as shown in FIG. 27E can be a portion of a circular shape with a radius in the range of about 0.2 mm to about 1.5 mm. In some embodiments, the curved portion can be a portion of a circular shape with a radius in the range of about 0.3 mm to about 0.9 mm. In some embodiments, thecurved portion can be a portion of a circular shape with a radius in the range of about 0.4 mm to about 0.7 mm.

[0345] In some embodiments, the curved portion can be a portion of a circular shape with a radius in the range of 0.2 mm to 1.5 mm. In some embodiments, the curved portion can be a portion of a circular shape with a radius in the range of 0.3 mm to 0.9 mm. In some embodiments, the curved portion can be a portion of a circular shape with a radius in the range of 0.4 mm to 0.7 mm.

[0346] Disclosed herein are methods of using a fluid dispensing device 3000 for dispensing fluidic reagents to various nucleic acid molecules on a support, e.g., the flow cell devices. The methods of using a fluid dispensing device herein may advantageously enable or facilitate sequencing analysis using the sequencing system 110. Methods of using a fluid dispensing device 3000 for dispensing fluidic reagents can be advantageously more convenient, easy to use, and efficient than existing methods with comparable or even reduced contamination. The methods herein can include some or all of the operations disclosed herein. The operations may be performed in, but is not limited to, the order that is described herein.

[0347] The operations herein may be performed manually. The operations may be automatically performed by a robotic arm or the like (not shown). The robotic arm can be controlled by a computer system, e.g., 126 in FIG. 1, to automatically perform some or all of the operations disclosed herein. Alternatively, the computer system 126, dedicated processors, 118, the FPGA(s) 120, or their combinations, may be programmed to control the robotic arm. The computer system of the robotic arm can have installed on it software, firmware, hardware, or their combinations that in operation cause the computer system to perform the operations or actions disclosed herein.

[0348] The methods can be performed by one or more processors in the computer system, e.g., 126, disclosed herein. In some embodiments, the processor can include one or more of: a processing unit, an integrated circuit, or their combinations. For example, the processing unit can include a central processing unit (CPU), or a graphic processing unit (GPU), or combinations thereof. The integrated circuit can include a chip such as a field-programmable gate array (FPGA). In some embodiments, the processor can include the computing system.

[0349] In some embodiments, some or all operations in the methods can be performed by the FPGA(s). In embodiments when some operations are performed by FPGA(s), the data after an operation performed by the FPGA(s) can be communicated by the FPGA(s) to the CPU(s) so that the CPU(s) can perform subsequent operation(s) in method using such data.Similarly, data can also be communicated from the CPU(s) to the FPGA(s) for processing by the FPGA(s). In some embodiments, all the operations in method 500 can be performed by CPU(s). Alternatively, the operations performed by CPU(s) can be performed by other processors such as the dedicated processors, or GPU(s). In some embodiments, all the operations in method can be performed by FPGA(s).

[0350] In some embodiments, the methods of using the fluid dispensing device 3000 may comprise: providing a dispensing module comprising: a reagent cartridge with one or more compartments, wherein the one or more compartments are configured for holding fluidic reagents therein; a microfluidic chip in fluidic connection with each of the one or more compartments, wherein the microfluidic chip comprises one or more microfluidic pathways therewithin; and one or more dispensing tips; and providing one or more actuators that actuate a movable pin to push the fluidic reagents from the one or more compartments to the one or more dispensing tips through the one or more microfluidic pathways.

[0351] In some embodiments, the methods of using the fluid dispensing device 3000 may comprise an operation of fluidically connecting the one or more compartments of the reagent cartridge with the microfluidic chip. The fluidic connection can be permanently sealed to prevent liquid leakage from the connection, and the fluidic connection can be permanent, e.g., by welding, by molding as a single piece, etc., so that the user cannot removably separate the cartridge and the microfluidic chip and reconnect them. Instead, the user may dispose of them together when needed. The one or more compartments may be configured for holding fluidic reagents therein. The microfluidic chip may include one or more microfluidic pathways therewithin. In some embodiments, the operation of fluidically connecting the one or more compartments of the reagent cartridge with the microfluidic chip comprises fluidically connecting each outlet of the compartment(s) to a corresponding inlet of a corresponding microfluidic pathway of the microfluidic chip. Each different reagent may have its own compartment and microfluidic pathway. Two or more compartments and microfluidic pathways may contain identical liquid reagent therewithin.

[0352] In some embodiments, the methods of using the fluid dispensing device 3000 may comprise an operation of fluidically connecting one or more dispensing tips with the one or more microfluidic pathways of the microfluidic chip. In some embodiments, the operation of fluidically connecting the one or more dispensing tips with the one or more microfluidic pathways of the microfluidic chip comprises fluidically connecting each outlet of the microfluidic pathway to a corresponding dispensing tip.

[0353] In some embodiments, the methods of using the fluid dispensing device 3000 may comprise an operation of removably coupling the dispensing module to the following: the one or more actuators; the flow cell device, the sequencing system, or their combinations. The dispensing module may comprise: the reagent cartridge with the one or more compartments; the microfluidic chip in fluidic connection with the one or more compartments; and the one or more dispensing tips. Such operation of removably coupling the dispensing module to the device(s) disclosed herein can be after removably uncouple an old dispensing module for disposal.

[0354] In some embodiments, the methods of using the fluid dispensing device 3000 may comprise an operation of applying, by the one or more actuators, a first force or pressure on the curved portion of a first microfluidic pathway for a first predetermined time to actuate a first reagent from a first compartment of the reagent cartridge to a first dispensing tip via a first microfluidic pathway. Such operation of applying the first force or pressure can be during a sequencing run in a predetermined flow cycle. Such operation of applying the first force or pressure can be during a sequencing run in a predetermined flow cycle. Such operation of applying the first force or pressure can be repeated for a number of times at a predetermined rate, e.g., washing the microfluidic channel in each flow cycle. Such operation of apply the first force or pressure can be for a single occurrence, e.g., for dispensing specific library molecules to the flow cell device. The one or more actuators may be controlled automatically by instructions executable on the computer system disclosed herein.

[0355] In some embodiments, the methods of using the fluid dispensing device 3000 may comprise an operation of receiving the first reagent from the first dispensing tip openly at an inlet of the flow cell device. The first reagent then may travel to and contact the nucleotide acid molecules immobilized on surface(s) of a microfluidic channel of the flow device.

[0356] In some embodiments, the methods of using the fluid dispensing device 3000 may comprise an operation of applying a second force or pressure on the curved portion of second first microfluidic pathway for a second predetermined time to actuate a second reagent from a second compartment to a second dispensing tip via a second microfluidic pathway.

[0357] In some embodiments, the methods of using the fluid dispensing device 3000 may comprise an operation of receiving the second reagent from the second dispensing tip openly to the inlet of the flow cell device.

[0358] In embodiments where only a smaller number of actuators are included in comparison to the number of microfluidic pathways (e.g., a single actuator), the methods of using the fluid dispensing device 3000 may comprise moving the actuator(s) relative to theflow cell device or the microfluidic pathways in order to exert force or pressure on a specific microfluidic pathway. For example, the single actuator may move from right underneath the first microfluidic pathway to be underneath the fourth microfluidic pathway in order to push fluid(s) therewithin.

[0359] In some embodiments, the methods of using the fluid dispensing device 3000 may comprise an operation of switching one or more valves into a first position. When the one or more valves are in the first position, the method 3000 may further comprise applying a first force or pressure for a first period of time on the plunger to move fluids in one or more compartment(s) through the one or more valves into the reservoir. In some embodiments, the first force or pressure and / or the first period of time can be predetermined so that the fluid does not contaminate any common line that is shared. In some embodiments, the microfluidic chip may include one or more sensors. The fluid position during pulling and / or pushing may be detected by a sensor. The sensor may provide a feedback of the fluid position to the actuator that actuates the plunger so that the actuator may stop or continue to actuate the plunger based on the feedback. In some embodiments, the contamination in one or more of the microfluidic paths is controlled to be below a predetermined threshold hold level. In some embodiments, the methods of using the fluid dispensing device 3000 does not require any washing of the common line and / or the individual microfluidic pathways leading to the dispensing tips. As shown in FIG. 34B, the common line 3345 is between the connector 3361 and the barrel 3340. In this particular embodiment, the first force or pressure pulls the plunger away from the connector 3361.

[0360] In some embodiments, the methods of using the fluid dispensing device 3000 may comprise an operation of switching one or more valves into a second position. When the one or more valves are in the second position, the method 3000 may further comprise applying a second force or pressure for a second period of time on the plunger to move fluids in the one or more valves to the one or more dispensing tips 3400. In some embodiments, the second force or pressure and / or the second period of time can be predetermined so that the fluid does not contaminate any common line that is shared and possible air bubbles does not reach the dispensing tips. As shown in FIG. 34C, the common line is between the connector 3361 and the barrel 3340. In this particular embodiment, the second force or pressure pushes the plunger toward the connector 3361. The second force or pressure may or may not empty the corresponding reservoir.

[0361] In some embodiments, the methods of using the fluid dispensing device 3000 may comprise removing at least part of the first reagent or second reagent from the flow celldevice. In some embodiments, such operation of removing the reagent(s) may be performed by some or all elements of the fluid operation device disclosed herein. In some embodiments, the removed first or second reagent may be recycled back to the reagent cartridge, in particular, the corresponding compartment of the reagent. Such removal and recycling operations may be repeated for various times, e.g., for each flow cycle in the sequencing run.

[0362] In some embodiments, the methods of using the fluid dispensing device 3000 may comprise an operation of providing a first plurality of nucleic acid template molecules immobilized on the flow cell device, wherein each nucleic acid template molecule comprise: a first insert sequence and a first sample index sequence, wherein the first sample index sequence comprises a first universal sample index sequence identifying a sample source of the insert sequence.

[0363] In some embodiments, the methods of using the fluid dispensing device 3000 may further comprises an operation of conducting, by the sequencing system, one or more cycles of sequencing reactions of the first insert sequence before conducting one or more cycles of the sequencing reactions of the first sample index sequence to generate flow cell images comprising the first flow cell image and the second flow cell image in the first flow cycle. In some embodiments, the methods of using the fluid dispensing device 3000 may comprise an operation of conducting, by the sequencing system, one or more cycles of sequencing reactions of the first sample index sequence before conducting one or more cycles of the sequencing reactions of the first insert sequence to generate flow cell images.

[0364] In some embodiments, the operation of conducting the one or more cycles of sequencing reactions of the first insert sequence or the first sample index sequence comprises an operation of dispensing, by the fluid dispensing system, a first reagent comprising a first plurality of soluble sequencing primers that hybridize to the first plurality of nucleic acid template molecules to one or more inlets of a flow cell device. The operation of conducting the one or more cycles of sequencing reactions of the first insert sequence or the first sample index sequence may further comprise an operation of allowing the first reagent to travel to and contact the plurality of nucleic acid template molecules at the flow cell device.

[0365] After the operation of conducting the one or more cycles of sequencing reactions, the methods of using the fluid dispensing device 3000 may further comprise an operation of reversibly removing the dispensing module without removing the one or more actuators relative to the sequencing system. Subsequently, the methods of using the fluid dispensing device 3000 may further comprise an operation of removably coupling a second dispensing module to the one or more actuators and to the sequencing system.

[0366] In some embodiments, the operation of conducting the one or more cycles of sequencing reactions of the first insert sequence or the first sample index sequence may comprise an operation of dispensing, by at least a first dispensing tip of the fluid dispensing device, a first plurality of sequencing primers, a first plurality of polymerases and a first mixture of different types of labeled nucleotides or multivalent molecules to one or more inlets of the flow cell device. In some embodiments, the first plurality of sequencing primers, the first plurality of polymerases and the first mixture of different types of labeled nucleotides or multivalent molecules may be a mixture that can be dispensed from only a single dispensing tip. In some embodiments, the first plurality of sequencing primers, the first plurality of polymerases and the first mixture of different types of labeled nucleotides or multivalent molecules may be dispensed sequentially, in various orders, by different dispensing tips. Subsequently, the operation of conducting the one or more cycles of sequencing reactions may comprise: allowing first plurality of sequencing primers, the first plurality of polymerases and the first mixture of different types of labeled nucleotides or multivalent molecules to travel from the one or more inlet to the surface(s) and contact the nucleotide acid template modules for sequencing reactions. In some embodiments, the operation of conducting the one or more cycles of the sequencing reactions of the first insert sequence of the first sample index sequence may comprise dispensing by at least a first dispensing tip of the fluid dispensing device, one or more buffers (e.g. a buffer that allows for dissociation of the first sequencing polymerases), one or more reagents to remove a chain terminating moiety, and / or a plurality of polymerases and a plurality of nucleotides under conditions suitable to incorporate the nucleotides into 3’ ends of the first plurality of sequencing primers.

[0367] In some embodiments, the operation of conducting the one or more cycles of sequencing reactions of the first insert sequence or the first sample index sequence may comprise an operation of dispensing, by at least a second dispensing tip, a second plurality of sequencing primers, a second plurality of polymerases and a second mixture of different types of labeled nucleotides or multivalent molecules). In some embodiments, the second plurality of sequencing primers, the second plurality of polymerases and the second mixture of different types of labeled nucleotides or multivalent molecules may be a mixture that can be dispensed from only a single dispensing tip. In some embodiments, the second plurality of sequencing primers, the second plurality of polymerases and the second mixture of different types of labeled nucleotides or multivalent molecules may be dispensed sequentially, in various orders, by different dispensing tips. In some embodiments, the operation ofconducting the one or more cycles of the sequencing reactions of the first insert sequence of the first sample index sequence may comprise dispensing by at least a second dispensing tip of the fluid dispensing device, one or more buffers (e.g. a buffer that allows for dissociation of the second sequencing polymerases), one or more reagents to remove a chain terminating moiety, and / or a plurality of polymerases and a plurality of nucleotides under conditions suitable to incorporate the nucleotides into 3’ ends of the second plurality of sequencing primers.

[0368] The first and second plurality of sequencing primers can be different or identical. The first and second plurality of polymerases can be different or identical. The first and second mixture of different types of labeled nucleotides or multivalent molecules can be different or identical.

[0369] In some embodiments, the operation of conducting the one or more cycles of sequencing reactions of the first insert sequence or the first sample index sequence may comprise an operation of performing one or more operations of the two-stage methods for sequencing as disclosed herein. In some embodiments, the operation of conducting the one or more cycles of sequencing reactions of the first insert sequence or the first sample index sequence may comprise an operation of performing one or more operations of the sequencing-by-binding methods as disclosed herein. In some embodiments, the reagent(s) can be dispensed by one or more dispensing tips using the fluid dispensing device herein. In some embodiments, the reagent(s) can be removed using the fluid dispensing device herein.

[0370] In some embodiments, the methods of using the fluid dispensing device 3000 may further comprise one or more operation associated with the transportation valve and its corresponding microfluidic chip.

[0371] In some embodiments, the methods of using the fluid dispensing device 3000 may further comprise an operation of switching the transportation valve into a first open position from a closed position. Such operation may comprise: rotating, by one or more actuators, the transportation valve about an axis orthogonal to the valve substrate for a predetermined rotational angle in a second direction, e.g., counter-clockwise or clockwise. The predetermined rotational angle may be in a range from 3 degrees to 45 degrees. Such operation may open the transportation valve and its seal on one or more compartments of a reagent cartridge. As a result, such an operation may allow a first reagent from the first compartment through the transportation valve and flow to the microfluidic chip, e.g., into a first in-chip well via a fluidic pathway for a predetermined duration, e.g., FIG. 36B.

[0372] In some embodiments, the methods of using the fluid dispensing device 3000 may further comprise an operation of switching a first in-chip valve 3351 into an open position. The first in-chip valve corresponds to a first compartment of the reagent cartridge. Such operation may occur before or after opening the transportation valve. The second in-chip well may be closed while switching on the first in-chip valve.

[0373] In some embodiments, the methods of using the fluid dispensing device 3000 may further comprise an operation of switching the transportation valve into a second open position by rotating, by one or more actuators, the transportation valve about an axis orthogonal to the valve substrate for a predetermined rotational angle in the second direction or the first direction. The predetermined rotational angle may be in a range from 3 degrees to 45 degrees. Such operation may remove the seal of a second compartment thereby allowing a second reagent from the second compartment to flow to the microfluidic chip, e.g., into a second in-chip well via a fluidic pathway for a predetermined duration.

[0374] In some embodiments, the methods of using the fluid dispensing device 3000 may further comprise an operation of switching the transportation valve into the at least open position from a closed position. Such operation may comprise: rotating, by one or more actuators, the transportation valve about an axis orthogonal to the valve substrate for a predetermined rotational angle in a second direction, e.g., counter-clockwise or clockwise. The predetermined rotational angle may be in a range from 3 degrees to 45 degrees. Such operation may open the transportation valve and its seal on all compartments of a reagent cartridge. As a result, such an operation may allow reagents to flow through the transportation valve and to the microfluidic chip, e.g., into corresponding in-chip wells via corresponding fluidic pathways for a predetermined duration, e.g., FIG. 36B. In some embodiments, the corresponding in-chip valves connecting the corresponding compartments to their in-chip wells are open to enable the flow to the in-chip wells.

[0375] In some embodiments, the methods of using the fluid dispensing device 3000 may further comprise an operation of switching transportation valve into a closed position, by rotating, by the one or more actuators, the transportation valve for the predetermined rotational angle in a first direction opposite the second direction, to seal the first compartment and / or the second compartment from the microfluidic chip.

[0376] In some embodiments, the methods of using the fluid dispensing device 3000 may further comprise an operation of switching a second in-chip valve into an open position to allow the first reagent to flow from the first in-chip well to a first pair of dispensing tips. The first in-chip well may be closed while switching on the second in-chip valve.

[0377] In some embodiments, the methods of using the fluid dispensing device 3000 may further comprise an operation of switching a third in-chip valve 3351 into an open position. The third in-chip valve corresponds to a second compartment of the reagent cartridge. The fourth in-chip well may be closed while switching on the third in-chip valve.

[0378] In some embodiments, the methods of using the fluid dispensing device 3000 may further comprise an operation of switching a fourth in-chip valve into an open position to allow the second reagent to flow from the second in-chip well to a second pair of dispensing tips. The third in-chip well may be closed while switching on the fourth in-chip valve.

[0379] In some embodiments, the methods of using the fluid dispensing device 3000 may further comprise an operation of switching the transportation valve into a second open position from a closed position by rotating, by one or more actuators, the transportation valve for a first predetermined rotational angle in a first direction, to allow a first open port to connect a first compartment to a first in-chip well via a fluidic pathway, e.g., from FIG. 39A to FIG. 39B.

[0380] In some embodiments, the fluid dispensing device 3000 may be in a closed position during shipping, and it may be switched to the second open position for aspirating reagents from a first compartment to a first in-chip well. In some embodiments, such operation simultaneously connects the central open port to the first in-chip well. In some embodiments, the methods of using the fluid dispensing device 3000 may further comprise an operation of operating the pump 3340, e.g., pulling the plunger, to facilitate fluid aspiration into the first in-chip well. In some embodiments, the first predetermined rotational angle is in a range from 5 degrees to 60 degrees. In some embodiments, the first predetermined rotational angle is in a range from 15 degrees to 45 degrees. In some embodiments, the transportation valve may stay in the first open position for a predetermined time, e.g., 0.1 second, 0.5 seconds or more to allow a predetermined amount of the first reagent to travel to the first in-chip well.

[0381] In some embodiments, the methods of using the fluid dispensing device 3000 may further comprise an operation of switching the transportation valve into a first open position by rotating, by the one or more actuators, the transportation valve for a second predetermined rotational angle in a second direction opposite the first direction, to allow the first open port to connect the first in-chip well to the first pair of dispensing tips thereby enabling reagent dispensing from the first in-chip well to the first pair of dispensing tips, e.g., from FIG. 39B to FIG. 39C. In some embodiments, such operation simultaneously connects the central open port to the first in-chip well. In some embodiments, the methods of using the fluid dispensingdevice 3000 may further comprise an operation of operating the pump 3340, e.g., pushing the plunger, to facilitate reagent dispensing. In some embodiments, the second predetermined rotational angle may be in a range from 5 degrees to 60 degrees. In some embodiments, the second predetermined rotational angle may be in a range from 15 degrees to 45 degrees. In some embodiments, the transportation valve may stay in the first open position for a predetermined time, e.g., 0.1 second, 0.5 seconds or more to allow a predetermined amount of the first reagent to be dispensed.

[0382] Subsequent to the dispensing of the first reagent, in some embodiments, the methods of using the fluid dispensing device 3000 may further comprise an operation of switching the transportation valve into a fourth open position by rotating, by one or more actuators, the transportation valve for a third predetermined rotational angle in the first direction or the second direction, to allow a second open port to connect a second compartment to a second in-chip well via a fluidic pathway. In some embodiments, the third predetermined rotational angle is in a range from 5 degrees to 90 degrees. In some embodiments, the transportation valve may stay in the second open position for a predetermined time, e.g., 0.1 second, 0.5 seconds or more to allow a predetermined amount of the second reagent to travel to the second in-chip well.

[0383] In some embodiments, the methods of using the fluid dispensing device 3000 may further comprise an operation of switching the transportation valve into a third open position by rotating, by the one or more actuators, the transportation valve for a fourth predetermined rotational angle in a first or second direction, to allow the second open port to connect the second in-chip well to the second pair of dispensing tips thereby enabling reagent dispensing from the second in-chip well to the second pair of dispensing tips. In some embodiments, the fourth predetermined rotational angle may be in a range from 5 degrees to 80 degrees. In some embodiments, the transportation valve may stay in the second open position for a predetermined time, e.g., 0.1 second, 0.5 seconds or more to allow a predetermined amount of the second reagent to be dispensed. Sample Preparation Systems and Methods

[0384] In some embodiments, the flow cell devices and samples immobilized thereon can be prepared using a sample preparation system disclosed herein. The sample(s) immobilized on the flow cell device can be on multiple surfaces of the flow cell device, e.g., multiple interior surfaces forming the microfluidic channels. The sample(s) can be in 2D or 3D.

[0385] In some embodiments, disclosed herein are sample preparation systems configured to allow a user to prepare samples on flow cell substrate(s) with customized distribution of the sample(s) with various densities of the analytes in sample(s). The sample preparation systems may comprise: a plate with one or more wells extending from a top surface to a bottom surface thereof; a flow cell substrate configured to hold the sample(s) immobilized on a top surface or a bottom surface of the flow cell substrate; a seal that seals a gap between the plate and the flow cell substrate; and a cover that protects the flow cell substrate and the sample(s) from external contamination. The flow cell substrate(s) may be used to form a flow cell device that can be used in a sequencing system. The sample preparation systems herein may advantageously allow preparation of 2D (e.g., nucleic acid libraries immobilized on a surface of the flow cell) or 3D samples (e.g., cells, tissues or other in situ samples) with protection from contamination and leakage as well as facilitating sample incubation, imaging, or other sample analysis to determine whether the sample(s) has met predetermined sample quality or not before sequencing starts. The sample preparation system may advantageously allow preparation of different 3D samples and 2D samples, e.g., on a single flow cell substrate. The sample preparation system may advantageously allow preparation of samples on multiple flow cell substrate(s) sequentially or in parallel. The prepared flow cell substrate(s) with samples immobilized thereon may be used to form a flow cell device disclosed herein that is compatible with open fluidic dispensing, closed fluidic communication, or both. The prepared flow cell substrate(s) with samples immobilized thereon may be used to form a flow cell device that may be used for DNA sequencing of the samples using the sequencing system herein.

[0386] The sample preparation system herein may advantageously allow a user to customize the flow cell substrate, and the customized flow cell substrate may be used to form a flow cell device that is customized and can be used for sequencing the prepared samples using a next generation sequencing (NGS) system.

[0387] In some embodiments, the user may customize the size, shape, number, and / or distribution of wells of the sample preparation system for containing samples therewithin. In some embodiments, the user may then use the sample preparation system to customize the size, shape, number, and / or distribution of the samples on the flow cell substrate to be sequenced. As a result, the sample distribution on the flow cell substrate prepared using the sample preparation system may correspond to the well distribution of the plate of the sample preparation system. In other words, the sample may only be distributed in areas that are directly underneath the bottom opening of the wells of the sample preparation system.

[0388] As a non-limiting example, the wells may be of an identical shape, size and may be distributed on 1, 2, 3, 4, or more columns / rows in the plate of the sample preparation system, and the wells may be distributed with an identical spacing from the adjacent wells. The wells that are at the edge may or may not have an identical spacing to the nearest edge of the flow cell substrate as the spacing between adjacent wells. As another non-limiting example, the users may use the sample preparation system to customize the number of wells. For example, the number of wells may be 6, 12 wells (e.g., FIG. 42A) 24, or 48 wells. The wells may be distributed on a 1 by 6, 2 by 6 matrix, a 4 by 6 matrix, or a 6 by 8 matrix. In some other embodiments, the wells may be distributed to be on a 3 by 4 matrix or a 2 by 12 matrix. The spacing between 2 adjacent wells in the same column may be identical. The spacing between two adjacent columns may or may not be identical to the spacing between 2 adjacent wells in the same column. In some embodiments, the number of cells in the prepared sample per well, e.g., of the 12-well plate or 24-well plate and the corresponding flow cell substrate, may be at least 2 x104, 3 x104, 4 x104, 5 x104, 6 x104, 8 x104, 1 x105, 2 x105, 3 x105, 4 x105, 5 x105, 6 x105, 7 x105, 8 x105, 9 x105, or 1 x106cells. In some embodiments, the number of cells in the prepared sample per well, e.g., of the 12-well plate or 24-well plate and the corresponding flow cell substrate, may be between about 2 x104and about 1 x107cells, between about 3 x104and about 1 x106cells, between about 4 x104and about 9 x105cells, between about 8 x104and about 5 x105cells, or any range therebetween. In some embodiments, the maximal number of cells per well may have to be limited to avoid over- crowded sample that may reduce the quality of the sequencing analysis. However, the sample preparation systems herein may allow a greater number of cells (than what traditional patterned flow cell with comparable sample area) to be prepared and still satisfy a predetermined quality of sequencing analysis using the sequencing system disclosed herein. For example, the predetermined quality of sequencing may be signal to noise ratio (SNR), contrast to noise (CNR), base calling quality such as quality score, purity, or flow cell occupancy level. As a non-limiting example, the flow cell occupancy level may be determined as a percentage calculated by the pixels occupied by sample(s) divided by the total number of pixels within the wells.

[0389] The predetermined quality of sequencing analysis may be determined using various quality metrics including but not limited to the quality score. In some embodiments, the maximal number of cells per well may be at least 1.5x, 2x, 4x, 6x, 8x, 10x, 15x, 20x, 25x, 30x, 40x, or 50x greater than the maximal number of cell per well (e.g., of a same well area or volume) of traditional flow cell devices when the quality score of base calling of thesample is no less than that using traditional flow cell device and sequencing systems. In some embodiments, the maximal number of cells per well may be at least 1.5x, 2x, 4x, 6x, 8x, 10x, 15x, 20x, 25x, 30x, 40x, or 50x greater than the maximal number of cell per well of traditional flow cell devices when the quality score of base calling of such samples is at least Q30, Q35, Q38, Q40, Q45, Q50, Q55, Q60, or Q65.

[0390] In some embodiments, the sample preparation system herein may allow a polony density (or equivalently, a cluster density) of greater than 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 70,000, 80,000, 90,000, 100,000,150, 000, or 200,000 polonies per mm2to be prepared within a well-determined area (e.g.,area underneath the bottom surface of the corresponding well) of the flow cell substrate. In some embodiments, the sample preparation system herein may allow a polony density (or equivalently, a cluster density) of between about 10,000 and about 200,000, between about 20,000 and about 150,000, between about 50,000 and about 100,000 polonies per mm2or any range therebetween to be prepared within a well-determined area of the flow cell substrate.

[0391] In some embodiments, the sample preparation system herein may allow a polony density (or equivalently, a cluster density) of greater than 100, 200, 500, 800, 1000, 2000, 5000, 8000, 10,000, 100,000, or 1000,000 cells per mm2to be prepared within a well- determined area (e.g., area underneath the bottom surface of the corresponding well) of the flow cell substrate.

[0392] In some embodiments, the plate of the sample preparation system may include a single well (e.g., a mega well). In some embodiments, the single well may include customized sizes. In some embodiments, the single well may have a length along y axis to be in a range from 30 mm to 150 mm. In some embodiments, the single well may have a width along x axis to be in a range from 10 mm to 100 mm. In some embodiments, the single well may have a height along z axis to be in a range from 5 mm to 50 mm. As a nonlimiting example, the single well may have a size of 50 mm by 19 mm by 9 mm. As another nonlimiting example, the single well may have a size of 42 mm by 16 mm by 10 mm.

[0393] In some embodiments, the plate of the sample preparation system may include 2, 4, 6, 8, 10, or 12 wells. The wells can be ranged in one or more columns or rows. For example, a 6-well plate may have a distribution of the wells in 1 by 6 or 2 by 3. In some embodiments, plate of the sample preparation system may include various numbers of wells ranging from 2 to 1024 wells. In some embodiments, plate of the sample preparation system may include various numbers of wells ranging from 1 to 96 wells. In some embodiments, each well of the 12-well plate may include customized sizes. In some embodiments, eachwell of the 12-well plate may have a length along y axis to be in a range from 3 mm to 20 mm. In some embodiments, each well of the 12-well plate may have a width along x axis to be in a range from 3 mm to 20 mm. In some embodiments, each well of the 12-well plate may have a height along z axis to be in a range from 5 mm to 50 mm. As a nonlimiting example, each well of the 12-well plate may have a size of 8 mm by 8 mm by 9 mm. As another nonlimiting example, each well of the 12-well plate may have a size of 7 mm by 6 mm by 10 mm.

[0394] In some embodiments, each well of the 48-well plate may include customized sizes. In some embodiments, each well of the 48-well plate may have a length along y axis to be in a range from 2 mm to 15 mm. In some embodiments, each well of the 48-well plate may have a width along x axis to be in a range from 2 mm to 15 mm. In some embodiments, each well of the 48-well plate may have a height along z axis to be in a range from 5 mm to 50 mm. As a nonlimiting example, each well of the 48-well plate may have a size of 3 mm by 4 mm by 9 mm. As another nonlimiting example, each well may have a size of 2 mm by 3 mm by 8 mm.

[0395] In some embodiments, each well of the 96-well plate may include customized sizes. In some embodiments, each well of the 96-well plate may have a length along y axis to be in a range from 2 mm to 12 mm. In some embodiments, each well may have a width along x axis to be in a range from 2 mm to 12 mm. In some embodiments, each well may have a height along z axis to be in a range from 5 mm to 50 mm. As a nonlimiting example, each well of the 96-well plate may have a size of 2 mm by 3 mm by 9 mm. As another nonlimiting example, each well may have a size of 2 mm by 2 mm by 8 mm.

[0396] In some embodiments, sequencing of samples prepared with the sample preparation system disclosed herein, e.g., with 1, 2, 4, or more wells, advantageously enables greater sequencing throughput than sequencing using traditional sample preparation systems and traditional flow cell devices. The throughput enabled by the sample preparation system and flow cell devices herein may be 2x, 4x, 8x, 10x, 12x, 16x, 20x, 30x, 40x, 50x, 60x, 80x, or 100x greater than using traditional flow cell devices. In some embodiments, sequencing of samples using the sample prepared with the sample preparation system disclosed herein, e.g., with 1, 2, 4, or more wells, advantageously allow faster sequencing analysis of the same volume of sample(s) or same number of cells than sequencing using traditional flow cell devices. The total sequencing time enabled by the sample preparation system and flow cell devices herein may be 2x, 4x, 8x, 10x, 12x, 16x, 20x, 30x, 40x, or 50x, less than the sequencing using traditional flow cell devices.

[0397] In some embodiments, the samples prepared with the sample preparation system disclosed herein and sequenced with NGS systems with ultrawide field of views (FOVs) advantageously enable greater sequencing throughput than sequencing samples prepared with traditional sample preparation systems and sequenced using traditional NGS systems. The details of the NGS systems that allows imaging of ultrawide FOVs (e.g., greater than 5, 10, or 20 mm2) are disclosed in WO2024158927 which is incorporated by reference in its entirety herein. The throughput enabled by sequencing samples on flow cell devices prepared using the sample preparation systems herein using the NGS systems disclosed herein may be 2x, 4x, 8x, 10x, 12x, 16x, 20x, 30x, 40x, 50x, 60x, 80x, or 100x greater than using traditional flow cell devices and NGS systems. In some embodiments, sequencing of samples prepared with the sample preparation system disclosed herein may advantageously allow faster sequencing analysis of the same volume of sample(s) or same number of cells than using traditional flow cell devices. The total sequencing time enabled by sequencing samples on flow cell devices prepared by the sample preparation system herein may be 2x, 4x, 8x, 10x, 12x, 16x, 20x, 30x, 40x, or 50x, less than same volume of samples or same number of cells using traditional flow cell devices.

[0398] Disclose herein are sample preparation systems for preparing one or more samples on a flow cell device. The sample preparation systems may comprise: a plate with one or more wells extending from a top surface to a bottom surface thereof; a flow cell substrate configured to hold the one or more samples immobilized thereon; a seal that seals a gap between the plate and the flow cell substrate; and a cover that protects the flow cell substrate from contamination.

[0399] As shown in FIG. 41 and FIG. 42A, the sample preparation system 4100 comprises a plate 4110 with a plate body 4116 and one or more wells 4111 extending from a top surface 4112 to a bottom surface 4113 of the plate body 4116.

[0400] The plate can have a thickness extending from the top surface to the bottom surface. The top surface and bottom surface are orthogonal to the z (axial) direction as shown in FIGS. 42B-42D. Each well may extend from the top surface to the bottom surface with either uniform or different cross-sectional area at different z-levels. In some embodiments, each of the one or more wells have an identical cross-sectional area along an axial direction. In some embodiments, at least one of the one or more wells have different cross-sectional areas at two different axial levels. For example, the well cross section at the top surface may or may not be identical to that at the bottom surface.

[0401] In some embodiments, to accommodate the size or volume of a sample, the sample preparation system may allow a user to customize the number, size, and / or shape of the one or more wells. For example, for a large volume of samples to be immobilized on the flow cell substrate, the user may select the plate that may have a single well that encompasses the area enclosed by the walls extending from the top surface 4112 to the bottom surface 4113 of the plate 4110. FIG. 42A (bottom right) shows non-limiting exemplary embodiments of the single well plate. The single well can have a top opening at its top surface and a bottom opening at its bottom surface, and the top opening and bottom opening may have various shape and / or size at its top and / or bottom surfaces. In one or more embodiments, each well has a uniform cross-sectional area along the axial direction (e.g., the z-axis). For example, the plate 4110 in FIG. 41 has a uniform cross-sectional area extending from the top surface of the plate 4110 to the bottom surface of the plate 4110. In one or more embodiments, each well has a non-uniform cross-sectional area along the axial direction (e.g., the z-axis). For example, a well can have a tapered cross-sectional area. As shown in FIG. 42A, with the plates having 12 wells, each well has a tapered cross-sectional area from the top surface to the bottom surface. The cross-sectional area of the well at the top surface is larger than the cross-sectional area of the well at the bottom surface, providing for a funnel to direct sample to the flow cell substrate.

[0402] In some embodiments, the user may customize the number, size, and / or shape of the one or more wells to accommodate different sample characteristics, including but not limited to the sample volume. For example, a user may want to sequence a larger volume of samples or a large number of cells (e.g., 1 million cells) that may not fit into a traditional flow cell device with multiple separated wells. Using the customized well(s) herein, the sample prepared may comprise a customized distribution on the flow cell substrate. After the flow cell substrate is included in a flow cell device, it may result in a flow cell device with customized sample distribution therewithin (e.g., with a larger distribution area than what traditional flow cell device may allow).

[0403] In some embodiments, each well of the one or more wells may have walls extending from the top surface to the bottom surface of the plate. Each well may have rounded corners to facilitate sample distribution and avoid possible residuals accumulation or other possible interferences to sample distribution on the flow cell substrate. Correspondingly, the walls may have rounded connections that when projected along the z axis (i.e., the axial direction), the rounded connections aligns with the rounded corners on the flow cell substrate.

[0404] In some embodiments, sample preparation may occur on a first surface of the flow cell substrate (e.g., top surface of the flow cell substrate) facing the plate. In some embodiments, sample preparation may also occur on a second surface of the flow cell substrate (e.g., bottom surface of the flow cell substrate). Such sample preparation may occur after turning the second surface to face the plate above the flow cell substrate, optionally after sample preparation has been completed on the first surface. In some embodiments, the flow cell substrate with sample(s) prepared only on the first surface can be considered a single surface flow cell substrate. In some embodiments, the flow cell substrate with sample(s) prepared on the first and second surface can be considered a dual surface flow cell substrate. Both the single and dual surface flow cell substrate may be coupled to another substrate to form a flow cell device.

[0405] In some embodiments, a well 4111 comprises a corresponding cross-sectional area that matches the cross-sectional area of the corresponding microfluidic channel of the flow cell device. For example, the top left panel in FIG. 42A has two wells that each match the corresponding cross-sectional areas of the microfluidic channel of the flow cell in FIG. 43A. The microfluidic channel in this particular embodiment includes the tapered regions.

[0406] In some embodiments, a well comprises a cross-sectional area that is less than ±5%, ±10%, ±15%, or ±20% different from a corresponding cross-sectional area of a microfluidic channel of the flow cell device. In some embodiments, each of the one or more wells comprises a cross-sectional area that is less than ±5%, ±10%, ±15%, or ±20% different from a corresponding cross-sectional area of a microfluidic channel of the flow cell device at the bottom surface of the plate. For example, the wells in FIG. 42 may comprise a cross- sectional areas that is smaller than that of the microfluidic channel(s) of the flow cell device shown in FIG. 43A.

[0407] In some embodiments, the one or more wells comprises a single well, e.g., in FIG. 42A and 42C. The single well may be used on the flow cell substrate that may then have 1, 2, 3, or even more microfluidic channels. In some embodiments, the one or more wells comprises a first number of wells that matches a second number of microfluidic channels of the flow cell device, e.g., as shown in FIGS. 42A-42B. In some embodiments, each single well comprises a bottom area that is identical to a surface area of a corresponding microfluidic channel of the flow cell device, and the flow cell device may have 1 or multiple microfluidic channels.

[0408] In some embodiments, the sample preparation system with a single-well plate may advantageously allow preparation of tissue samples that may be distributed across an arealarger than the bottom area of one or more wells in a multiple-well plate. If prepared using multiple well plate, such larger sample(s) may need to be separated into smaller pieces to fit in the bottom area of individual wells thereby causing damages spatial context and integrity of the sample and in accuracy in subsequent sequencing analysis. The single-well plate may allow preparation of such tissue sample(s) without disruption or damage to the sample(s).

[0409] In some embodiments, the sample preparation system may comprise a single well plate (top right of FIG. 42A) or a single well per channel plate (top left of FIG. 42A) with the difference that samples may be distributed to a larger area when prepared with the single well plate but not with the single well per channel plate.

[0410] In some embodiments, the sample preparation system with a single well per channel plate may advantageously allow preparation of samples without changes fluidic dynamics in each channel during sequencing from a 2-channel flow cell device. Such fluidic dynamics may include using a corresponding inlet for introducing fluidics to the channel, and using a corresponding outlet for allowing the fluidics to exit the channel during sequencing. The flow direction can be bidirectional between the inlet and corresponding outlet. Such fluidic dynamics may also include a total volume of reagent(s), reagent flow rate, reagent flow speed, washing or rinsing protocol, or the like during sequence applications.

[0411] In some embodiments, the sample preparation system with a single well plate may allow sample distribution expanding to two microfluidic channels and the area 4117 between the two channels that should be underneath the plate body when the same samples are prepared using a single well per channel plate (top left vs top right in FIG. 42A). In some embodiments, two different inlet and outlet pairs may be used during sequencing for introducing fluids and allow them to exit the flow cell device. In some embodiments, sample under area 4117 may not be as efficiently exposed to reagents or otherwise fluids as other areas due its relative location with respects to the inlets and outlets. In some embodiments, the reagent volume and / or other flow parameters (flow rate) may be adjusted to allow efficient flow of reagents into such areas, e.g., underneath 4117.

[0412] In some embodiments, the sample preparation system with a single well plate may allow more samples, e.g., more cells with a same cell density, to be prepared on the flow cell substrate for sequencing analysis than the sample preparation system with a single well per channel plate and the sample preparation system with multiple wells, e.g., 12 wells or 48 wells.

[0413] In some embodiments, the sample preparation system herein allows sample preparation in some but not all the areas underneath the wells. For example, for samplepreparation using a 48-well plate, areas of the flow cell substrate underneath 10, 20, 30 or more wells may be free from the samples after the preparation and only areas underneath the rest of the wells may have samples prepared thereon. Such areas free of samples may be used for other operations, e.g., later sample preparation operations of a different cell type or of different cell culturing protocol, or for other uses. As another example, for sample preparation using single well per channel plate, a first area underneath a first single well corresponding to a first microfluidic channel of the flow cell device may have a first cell type cultured thereon using a first culturing protocol, and a second area underneath a second single well corresponding to a second microfluidic channel may have a different cell type suspended thereon using a different protocol. The cell culturing and suspension protocols may be carried out by the user simultaneously or sequentially. After the sample preparation operations, the different cell types can be sequenced simultaneously while positioned on a same flow cell device, thereby minimizing variations during sequencing and sequencing analysis across the two different cell types and enabling comparison of sequencing data of the different cell types.

[0414] In some embodiments, the plate of the sample preparation system with the single well (top right of FIG. 42A) or a single well per microfluidic channel (top left of FIG. 42A) advantageously allow a user to distribute the samples at a larger surface area, e.g., without at least party of the plate body 4116 in between adjacent wells, comparing with a plate of the same dimensions but with multiple wells. In some embodiments, the sample preparation system with the single well or a single well per microfluidic channel allow more efficient imaging of the sample and improved utilization of the field of view of the image sensor during sequencing e.g., when the optical system may allow a wide field of view (e.g., greater than 3mm2, 5mm2or more). In other words, with multiple wells, sample are distributed in areas underneath the multiple wells and are separated by area(s) underneath the plate body. It is difficult not to include such areas underneath the plate body 4116 within the field of view, .e.g., with a wide field of view, but such areas contains no sample(s), therefore wasting effective field of view and causes delay in the sequencing run.

[0415] In some embodiments, the sample preparation system with a single well plate (top right of FIG. 42A) or a single well per microfluidic channel plate (top left of FIG. 42A) advantageously allow a user to prepare more samples per flow cell area and / or save reagent consumption comparing with preparing samples using sample reparation with multiple wells. In some embodiments, a first total reagent volume required for sequencing a first amount of samples prepared using a sample preparation system with a single well may be less than asecond total reagent volume required for the same first amount of samples prepared using a sample preparation system with multiple wells, e.g., 12, 24, or 48 wells. In some embodiments, the first and second total reagent volumes are used with a same sequencing system in a sequence run with identical sequencing chemistry and identical number of sequencing cycles to achieve a same contamination level of reagents during the sequencing run. In some embodiments, the first total reagent volume may be at least 5%, 8%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, or 50% less than the second total reagent volume. In some embodiments, the first total reagent volume may be at least 50%, 60%, 70%, 80%, or 90% less than the second total reagent volume.

[0416] For example, a first flow cell substrate is prepared using a sample preparation system with a single well plate and 200,000 cells are immobilized thereon. A second flow cell substrate is prepared using a sample preparation system with a 48-well plate of the same dimensions as the single well plate (e.g., same width, length, and height but with different number of wells) and 100,000 cells are immobilized thereon. Cells are distributed less than or equal to a threshold density on the flow cell substrate to ensure sequencing accuracy and reliability. A corresponding flow cell device is formed with the first or the second flow cell substrate and positioned into a same sequencing system for an identical sequencing run with 30 sequencing cycles. The identical sequencing run may use identical sequencing chemistry on the samples of the first and second flow cell device, simultaneously, or sequentially. A first total volume of reagents may be used to sequence each 100,000 cells of the first flow cell device during the sequence run. A second total volume of same reagents may be used to sequence 100,000 cells, and the first amount of reagents may be at least 10%, 15%, 20%, 30%, or 40% less than the second amount of reagents to achieve the same contamination level of reagents or sequencing quality during the sequencing run.

[0417] In some embodiments, a first amount of reagent is used in sequencing a first number of cells, e.g., in situ cells, prepared on a first flow cell substrate using the sample preparation system with a single well plate, a second number of cells, prepared on a second flow cell substrate using the sample preparation system with a single well per channel plate, and a third number of cells, prepared on a third flow cell substrate using the sample preparation system with multiple wells, e.g., 12 wells, 24 wells, or 48 wells. The first, second, and third flow cell substrates may form corresponding flow cell devices and be positioned on a same sequencing system for sequencing of the cells. The first, second, and third number of cells may be sequenced using a same sequencing protocol with a same reagent consumption and sequencing chemistry. The first, second, and third number of cells are of the same celltype. The cell density of the first, second, and / or third number of cells distributed on the flow cell substrate may be identical. The first number of cell may be at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more than the second and / or third number of cells. The second number of cell may be at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more than the third number of cells. In some embodiments, the first, second, or third number of cells is in a range from 10 to 109cells. In some embodiments, the first, second, or third number of cells is in a range from 100 to 107cells. In some embodiments, the first, second, or third number of cells is in a range from 200 to 106cells.

[0418] In some embodiments, the one or more wells comprises multiple wells in a predetermined spatial pattern, e.g., as in FIG. 42A. The predetermined spatial pattern may be repeated along x, along y, or along any other axis in the x-y plane. The mid panels in FIG. 42A shows wells that have multiple repetitions of the same shape and / or size along x and along y directions.

[0419] In some embodiments, the one or more wells facilitate delivery of sample(s) randomly so that the surface(s) of the microfluidic channel has randomly distributed samples, e.g., top and bottom panels in FIG. 42A. In some embodiments, the one or more wells facilitate delivery of sample(s) in a patterned manner, e.g., mid panels in FIG. 42A.

[0420] Each individual well can be of various sizes that are approximately identical or smaller than one or multiple channels along a same direction. For example, the well can be as wide as two channels along x. The well can be as long as a single channel along y or much smaller than a single channel along y. It is worth noting that each well may be customized in its shape and / or size to allow efficient delivery of samples to the flow cell substrate and should not be limited to the disclosure herein.

[0421] In some embodiments, two of the multiple wells comprises a different cross- sectional area or cross-sectional shape at the bottom surface of the plate. In some embodiments, the multiple wells comprises an identical cross-sectional area or cross- sectional shape at the bottom surface of the plate.

[0422] In some embodiments, the cross-sectional area of the wells (e.g., opening of the wells at the bottom surface) may determine where the sample(s) are distributed on the flow cell substrate. In some embodiments, a seal between the plate and the flow cell substrate facilitates leakage prevention so that minimum or no sample(s) is distributed in any areas of the flow cell substrate that are not directly underneath the cross-sectional area of the well at the bottom surface. In some embodiments, the seal between the plate and the flow cell substrate prevents samples distributed underneath the cross-sectional area of the well at thebottom surface from exiting the area of the corresponding well and / or entering the area(s) underneath adjacent well(s).

[0423] The seal is positioned between the plate and the flow cell substrate, extending around the one or more fluidic channels to form a closed perimeter. The seal can be composed of one or more materials that are impermeable to liquid. In one or more embodiments, the seal is composed of an elastomer (e.g., polydimethylsiloxane (PDMS)) for elasticity in forming a leakproof barrier around the perimeter of the fluidic channels. In some embodiments, the seal is composed of one or more materials that are chemically resistant, able to withstand degradation by reagents used during nucleic acid sequencing. The seal prevents leakage and cross-contamination by maintaining a fluid-tight barrier that confines samples and sequencing buffers within the flow cell’s channels.

[0424] It is worth noting that each well may be customized in its shape and / or size to allow efficient delivery of samples in different applications. The size and shape may be based on the size, shape, and / or number of microfluidic channels of the corresponding flow cell device, but different well size and / or shape may be used for delivery of samples onto the flow cell device with same channels. For example, top panels of the plate 4111 may be used to randomly distribute the samples to the flow cell device, while the mid panels of plates may be used to distribute different samples to different locations of the flow cell device, e.g., cells with different densities.

[0425] In some embodiments, the microfluidic channel may include a channel cavity or indentation that may be formed when the flow cell substrate is coupled, e.g., adhered, bonded, etc., with another substrate to form the corresponding flow cell device. In some embodiments, the flow cell device herein may comprise at least two substrates, e.g., a top substrate and a bottom substrate. The flow cell substrate may be the top substrate or bottom substrate depending on its relative position to another substrate of the flow cell device. In some embodiments, the channel cavity or indentation may be comprised in only one substrate. For example, the channel cavity or indentation may be comprised in only the top substrate of the flow cell device when the flow cell substrate with sample is the bottom substrate, and the sample(s) is prepared on the top surface of the flow cell substrate facing the channel cavity. As another example, the channel cavity or indentation may be comprised in only the bottom substrate of the flow cell device when the flow cell substrate with sample is the top substrate and the samples are prepared on the bottom surface of the flow cell substrate facing the bottom substrate of the flow cell device. In some embodiments, the channel cavity or indentation may be partially comprised in at least two substrates so that when the twosubstrates are coupled, e.g., adhered, bonded, etc., to form the corresponding flow cell device, the microfluidic channel(s) is formed.

[0426] FIGS. 46A-46B show exemplary embodiments of the flow cell substrate 4130 and the corresponding flow cell device 112 that may be used for sequencing the samples prepared on the flow cell substrate. Prepared samples are immobilized on the flow cell substrate within a sample distribution region 4131. The size and shape of the sample distribution region may be identical to the bottom opening area of the one or more wells 4111 of the plate 4110. As shown in FIG. 46A, the flow cell substrate 4130 is the top substrate of the corresponding flow cell device, and the sample distribution region 4131 faces downward toward the bottom substrate of the flow cell device. The bottom substrate, in this case, includes at least partly the indentation or cavity to form the microfluidic channels of the flow cell device. The bottom substrate may also include inlet(s) and / or outlet(s) that allow fluidic communication to the microfluidic channels, e.g., via a fluidic pathway connecting, from reagent or buffer reservoir(s). The inlet(s) and / or outlet(s) may include a through hole of a customized size in the bottom substrate.

[0427] As shown in FIG. 46B, the flow cell substrate 4130 is the bottom substrate of the corresponding flow cell device, and the sample distribution region 4131 faces upward toward the bottom substrate of the flow cell device. The top substrate, in this case, includes at least partly the indentation or cavity to form the microfluidic channels of the flow cell device. The top or bottom substrate may also include inlet(s) and / or outlet(s) that allow fluidic communication to the microfluidic channels, e.g., via a fluidic pathway connecting, from reagent or buffer reservoirs. The inlet(s) and / or outlet(s) may include a through hole of a customized size in the top or bottom substrate.

[0428] FIGS. 46C- 46D show an exemplary embodiment of the flow cell substrate 4130 and the corresponding flow cell device 112 that is formed by at least the flow cell substrate that may be used for sequencing the samples prepared on the flow cell substrate. Prepared samples may be immobilized on the flow cell substrate 4130 within one or more sample distribution regions 4131. The size, shape, and / or location of the sample distribution region on the flow cell substrate may be customized by a user to advantageously allow flexibility in sample distribution on identical flow cell devices in various sequencing applications. For example, the user may customize wells with the bottom opening as a square. The four corners of the square may be rounded to allow more efficient sample administration to the flow cell substrate and less residual accumulation in the wells.

[0429] In some embodiments, e.g., FIGS.46C-46D, the flow cell device may include an open landing area (e.g., 341, 441, 541) in one of its substrate, e.g., the bottom substrate. The open landing area may correspond to a cavity, of various geometric shapes in another substrate of the flow cell device when coupled to form the flow cell device. The open landing area and the corresponding cavity may allow open dispensing of reagents or other solutions via the cavity to the open landing area, and then into the microfluidic channel(s). As shown in FIGS. 46C-46D, the cavity is circular, and the open landing area is on the bottom substrate of the flow cell device. The sample distribution regions 4131 are also on the bottom substrate and may not overlap with the open landing area when the flow cell substrate is coupled to another substrate with the open landing area to form a flow cell device. As shown in FIG. 46C, the flow cell device may have each sample distribution area corresponding to a microfluidic channel, and may have a total number of 6 sample distribution areas. As shown in FIG. 46E, the flow cell device may have each sample distribution area corresponding to a microfluidic channel, and may have a single sample distribution area on the flow cell substrate. It is worth noting that the number of sample distribution regions does not have to be identical to the number of microfluidic channels, and can vary in range from 1 to 500.

[0430] Although not shown, the open landing area may be on the bottom surface of the top substrate of the flow cell, and the sample distribution region(s) may be on the same top substrate. In such embodiments, the cavity corresponding to the open landing area may be at the bottom substrate facilitating administration of fluids via the cavity to the open landing area.

[0431] In some embodiments, the sample distribution region(s) may be on the same substrate with the open landing area. In some embodiments, the sample distribution regions may be on a same substrate with the cavity. In some embodiments, the sample distribution region(s) may be on the top and bottom substrates of the flow cell device. In some embodiments, there may be an additional mid substrate or an adhesive layer between the top and bottom substrate, e.g., FIG 46D, the sample distribution area may be on the top substrate, the bottom substrate or both.

[0432] In some embodiments, the flow cell substrate or another substrate of the flow cell device may contain adhesive materials to provide adhesion and prevent fluidic leak from between the substrates after forming the flow cell device. FIG. 46A shows exemplary embodiments in which the bottom substrate may comprise the adhesive material surrounding the microfluidic channel. In some embodiments, the adhesive material, alone or in combination, may be surrounding the sample distribution region 4131. FIG. 46B shows anexemplary embodiment in which the top substrate may comprise the adhesive material surrounding the microfluidic channel. In some embodiments, the adhesive material, alone or in combination with the adhesive material on a different substrate, may be surrounding the sample distribution region 4131.

[0433] In some embodiments, there may be a sample distribution region in each individual substrate of the flow cell device. For example, as shown in FIG. 43A, the sample distribution region may be at the bottom surface of the top substrate of the flow cell device within the microfluidic channel(s), and the sample distribution region may also be at the top surface of the bottom substrate of the flow cell device. In particular embodiments, the sample distribution region may be the top and bottom surfaces of the microfluidic channel, and each substrate with the sample distribution region may be prepared using the sample preparation systems and methods herein.

[0434] Traditional flow cell devices may have adjacent sample distribution regions separated by a gap without any samples, e.g., patterned sample distribution based on the well distribution. The sample preparation system and methods herein advantageously allow user to customize the size, shape, area, and pattern of sample distribution areas on the flow cell substrate. In some embodiments, the sample preparation system and methods herein allow the flow cell substrate to be prepared using a plate with only one or two wells, e.g., as shown in bottom row in FIG. 42A.

[0435] In some embodiments, the prepared sample may be in situ samples of cells and / or tissue that can have a volume so that flow cell images at different z-levels may be needed in order to cover the entire volume of the prepared sample(s). In some embodiments, the prepared sample may be traditional 2D samples that can have a thickness along the z-axis, so that a volume of the prepared sample(s) may be determined based on the area of the prepared sample(s) and thickness of the prepared sample. The flow cell substrate may have a total area of samples that is at least 1.5x, 2x, 4x, 6x, 8x, 10x, 15x, 20x, 25x, 30x, 40x, or 50x greater than the total sample area of traditional flow cell devices. The flow cell substrate may have a total volume of samples that is at least 1.5x, 2x, 4x, 6x, 8x, 10x, 15x, 20x, 25x, 30x, 40x, or 50x greater than the total sample volume of traditional flow cell devices.

[0436] In some embodiments, the prepared sample may be samples including cells and / or tissue. The sample prepared using the sample preparation system with only one or two wells may have a greater number of cells compared with that of traditional flow cell devices. In some embodiments, the sample prepared using the sample preparation system with only one or two wells may have a cell number per well that is at least 1.5x, 2x, 4x, 6x, 8x, 10x, 15x,20x, 25x, 30x, 40x, or 50x greater than the cell number per well of the traditional flow cell devices. In some embodiments, In some embodiments, the sample prepared using the sample preparation system with only one or two wells may have a cell number per well that is at least 0.1 million, 0.5 million, 1 million, 1.5 million, 2 million, 4 million, 5 million, 8 million, 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 50 million, 80 million, or 100 million. In some embodiments, the sample prepared using the sample preparation system with only one or two wells may have a cell number per well that is between about 0.1 million and about 100 million, between about 1 million and about 50 million, or between about 10 million and about 30 million, or any range therebetween.

[0437] In some embodiments, the flow cell substrate prepared using a plate within only one or two wells may advantageously allow more data to be prepared under a same sample preparation condition and to be sequenced under similar sequencing conditions if not identical sequencing conditions. In some embodiments, the flow cell substrate prepared using a plate within only one or two wells may advantageously allow more sample(s) to be sequenced in a same flow cell device such that the sequencing may provide more spatial context or information to the biological analytes within the sample(s). In some embodiments, the biological analytes that may be located at or near the edge of a circular sample distribution region in a patterned flow cell with limited connection to other biological analytes in the same circular region or other circles spaced apart, but with the flow cell device prepared with only a single well or larger wells, the same biological analytes may be located with possibly more biological analytes in its adjacency, such spatial context of being adjacent to other biological analytes may provide additional information, e.g., morphological information, that can be used in sequencing applications.

[0438] In some embodiments, the microfluidic channels are configured to allow fluidic communication between an open landing area and the samples immobilized on the flow cell device, e.g., as shown in FIGS. 3-5.

[0439] In some embodiments, the microfluidic channels are configured to allow fluidic communication between a closed fluidic pathway in fluidic connection with the inlet(s) of the microfluidic channels and the samples immobilized on the flow cell device, e.g., as used in existing NGS systems.

[0440] The bottom surface of the plate may be flat or planar so that it facilitate coupling of the plate to the flow cell substrate 4130. In some embodiments, the flatness of the bottom surface of the plate can be measured as the height from its peak to valley in an direction orthogonal to the surface. The height can be less than about 0.01 mm, 0.02 mm, 0.03 mm,0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, or 0.09 mm, e.g., along a direction orthogonal to the surface. In other words, the flat surface(s) of the substrates may fit between two parallel planar 2D planes that is less than 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, or 0.09 mm apart from each other. In some embodiments, the flatness of the surface(s) of the substrates can include a height from its peak to valley that is less than 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, or 0.09 mm. However, the bottom surface do not have to be planar in certain embodiments. Alternatively, a part or the entirety of bottom surface can be curved or ramped.

[0441] In some embodiments, the plate is positioned above the flow cell substrate. In some embodiments, the plate 4110 is positioned directly above the flow cell substrate and abutting the top surface of the flow cell substrate.

[0442] The sample preparation system 4100 may further comprise the seal 4120 that can be positioned in between the plate and the flow cell substrate 4130. In some embodiments, the plate is positioned above the flow cell substrate, and a seal is positioned in between and contacting both the plate and the flow cell substrate. In some embodiments, the seal comprises a gasket. In some embodiments, the seal comprises a shape that surrounds at least part of each of the one or more microfluidic channels, e.g., as shown in FIG.43B. In some embodiments, the seal is preassembled to a bottom surface of the plate. In some embodiments, the seal comprises a layout (or shape) in the x-y plane identical to the layout of the bottom surface of the plate layout, e.g., with cutouts where the wells are positioned on the plate. Some exemplary layouts of the one or more wells in the x-y plane are shown in FIGS. 42B-42C. In some embodiments, the seal protects a top surface 4231 of the flow cell substrate from mechanical impact from the plate 4110. A layout of a layer may be the planar shape of the layer, which may include an outline and one or more inner cutouts aligned to the one or more wells. In some embodiments, the sample preparation system 4100 may include a plurality of seals, e.g., wherein each seal is positioned to form a leakproof barrier around one or more of the channels in the flow cell substrate. In some embodiments, the seal can be machine cut from a sheet of material. In some embodiments, the seal can be three- dimensionally printed, e.g., via an additive manufacturing process. In some embodiments, the seal can be formed via a mold, e.g., injection molding.

[0443] In some embodiments, the sample preparation system comprises a flow cell substrate 4130. The flow cell substrate can be a top substrate or a bottom substrate disclosed herein in relation to the flow cell devices herein, e.g., FIGS. 2-6 and FIGS. 24-26.

[0444] In some embodiments, the flow cell substrate is a bottom substrate of the flow cell device. In some embodiments, the flow cell substrate 4130 is a top substrate of the flow cell device. In other words, the sample preparation system can be used to prepare samples on multiple surfaces of the flow cell device. For example, for flow cell devices with 4 surfaces having samples immobilized thereon, and the surface are axially displaced from each other, the sample preparation system may prepare all 4 surfaces of the flow cell devices in series using the same sample preparation module.

[0445] In some embodiments, the sample preparation system advantageously allow preparation of samples on multiple substrates in parallel when it includes multiple modules of the system. A single module of the sample preparation system is shown in FIG. 41. Multiple modules can be arranged in an array of various number of rows or columns depending on the sample preparation need. For example, as shown in FIG. 45, 4 modules can be included in a single sample preparation system. Each module may or may not use identical plates. For example, same sample(s) may be delivered simultaneously using different dispensing tips to different substrates using the system in FIG.45. As another example, different samples may be delivered using different dispensing tips using plates of different wells to enable various distribution of samples on different flow cell substrate. The sample preparation system therefore can advantageously increase the efficiency and reduce time consumption in substrate preparation, and improve flexibility in preparing different samples with different distribution patterns on the flow cell substrates.

[0446] In some embodiments, the sample preparation system further comprises a removable clamp 4140 configured to hold the plate 4110, the seal 4120, and the flow cell substrate 4130 in a stack along z direction (i.e., axial direction) during sample preparation. In some embodiments, the plate or the cover comprises a groove or tongue 4114 configured to reversibly couple to the removable clamp so that the plate, the seal, and the flow cell substrate can be fixedly or removably stacked together. The groove provides a contour in the plate for a corresponding hook of the removable clamp to grip the plate. In some embodiments, the groove is linear and extends along an edge of the plate. The removable clamp may include the corresponding hook that can slide along the groove from either end of the groove. Once the removable clamp is slid on, in some embodiments, the seal comprises an adhesive layer configured to attach to the plate, the flow cell substrate, or both. In some embodiments, the adhesive layer 4120 allows reversible attachment of the plate and seal to the flow cell substrate which can be removed after sample preparation. Other features may also be used to reversibly couple the plate, the seal, and the flow substrate. Reversiblecoupling refers to the ability of two components to be couplable together and decouplable apart.

[0447] In some embodiments, the sample preparation includes all the procedures or operations before the flow cell substrate is removed from the sample preparation system for coupling into a flow cell device. In some embodiments, sample preparation includes: delivery of samples onto the flow cell substrate, sample incubation, sample imaging (e.g., under a microscope), sample analysis, or other various processes. In some embodiments, the sample preparation includes examining quality of the sample(s) to determine whether the sample(s) has met a predetermined criterion or not. The predetermined criterion can be customized based on characteristics of the sample(s), and / or various imaging and sequencing applications. For example, cell density, morphology, polony density, or other various factors can be included in the predetermined criterion, should not be limited by the disclosure herein.

[0448] In some embodiments, the sample preparation system comprises a cover 4150. The cover may be positioned above the plate 4110, i.e., coupled to a top surface of the plate 4110. The cover 4150 may also be fixed coupled to the plate 4110, the seal 4120, the flow cell substrate or some combination thereof. The cover 4150 may be transparent to enable sample imaging, e.g., using an objective lens from above the sample preparation module. The cover 4150 may allow protection of the sample during the sample preparation process from contamination.

[0449] In some embodiments, sample preparation using the sample preparation system herein may include one or more sample preparation operations disclosed herein. In some embodiments, the one or more sample preparation operations are performed on the samples located on the flow cell substrate(s). In some embodiments, the one or more sample preparation operations includes: applying a surface coating to the flow cell substrate(s); delivering the sample(s) to the flow cell substrate; culturing the sample(s) on the flow cell substrate; growing the sample(s) on the flow cell substrate; treating some or all of the sample(s) by one or more biological or chemical compounds (e.g., treating some of the samples with a drug while leaving some of the samples as controls); and fixing the cells. In some embodiments, the one or more sample preparation operations includes cell capturing. In some embodiments, the sample(s) include cells and / or tissue. In some embodiments, the one or more sample preparation operations include applying one or more coating(s) (e.g., the first coating disclosed herein) on the flow cell substrate(s). In some embodiments, the one or more sample preparation operations includes: applying a cell culture coating on the flow cell substrate; applying a cell capture coating on the flow cell substrate; cell capturing by cellsuspension; and cell fixing. In some embodiments, the sample(s) may include adhering cells and / or non-adhering cells. Exemplary embodiments of coatings on the flow cell substrate(s) for preparing different types of sample(s) are disclosed in WO2025147667, and is incorporated herein by reference in its entirety.

[0450] After sample preparation on the flow cell substrate, the flow cell substrate is configured to be fixedly coupled to a top substrate, a middle substrate or both to form a flow cell device with one or more microfluidic channels. In some embodiments, the flow cell device comprises an adhesive layer 4160 configured to fixedly attach the top substrate or the bottom substrate to the flow cell substrate.

[0451] In some embodiments, the adhesive layer comprises only areas that does not overlap with the area of the one or more microfluidic channels on a x-y plane. In other words, the adhesive layer does not block any cross-sectional area of the microfluidic channels. FIG.43B shows a top view of an exemplary embodiment of the flow cell device, which includes a seal 4120 around each microfluidic channel, and an adhesive layer 4160 that directly adjoins and surrounds the gasket to seal the substrates fixedly together. There is no or minimum overlap of the adhesive layer and the seal in this particular embodiment. In some embodiments, the seal 4120 and adhesive layer 4160 can be at the same z-level, and the top view is shown in FIG. 43B. In some embodiments, the seal 4120 and adhesive layer 4160 can each be a layer that stacks together along the z axis. In some embodiments, the seal 4120 and adhesive layer can have at least some overlap in the x-y plane but at different z-levels.

[0452] After sample preparation and coupling of the flow cell substrate into the flow cell device, the flow cell device may be configured to be inserted into a flow cell cartridge or a manifold that is configured to hold the flow cell device and enable fluidic communication between the flow cell device and a fluidic dispensing device, a fluidic reservoir, a waste container for collected used reagents or other solutions, or a combination thereof. The cartridge or manifold may also provide unique identification of the flow cell device and sample(s) contained therewithin. The cartridge or manifold may also contain an alignment feature configured to position the flow cell device relative to a reference, e.g., relative to an objective lens. Exemplary alignment feature can include a grove, a post, a slot, etc., An exemplary cartridge is shown in FIG. 44.

[0453] In some embodiments, the flow cell device, the manifold, or both enables open fluidic communication from a fluidic reservoir to the one or more microfluidic channels. In some embodiments, the flow cell device, the manifold, or both enables open dispensing of a reagent to the open landing area of the flow cell device. In some embodiments, the flow celldevice, the manifold, or both enables closed fluidic communication of a reagent between the fluidic reservoir and the one or more microfluidic channels. In some embodiments, the closed fluidic communication is through one or more fluidic pathways connecting the manifold or flow cell cartridge to the fluidic reservoir.

[0454] In some embodiments, the open landing area in flow cell devices herein are at least open when the dispensing tip is not dispensing fluids to the open landing area.

[0455] In some embodiments, disclosed herein are methods for preparing one or more samples on a flow cell device using the sample preparation system 4100. In some embodiments, the one or more samples are immobilized on a single flow cell substrate or separately on different substrates. In some embodiments, the samples are 3D samples, e.g., cells, tissue, etc. In some embodiments, the method for preparing the flow cell device with sample comprises: \coupling a plate with one or more wells on top of a flow cell substrate, wherein the flow cell substrate is configured to hold the one or more samples immobilized thereon, and wherein the one or more wells extending from a top surface to a bottom surface thereof. The coupling operation can be by using a reversible clamp, an adhesive layer between the plate and the flow cell substrate or other possible variations. In some embodiments, the method for preparing the flow cell device with sample further comprises: openly delivering one or more samples through the one or more wells to the flow cell substrate, wherein the one or more samples comprises cells or tissues. In some embodiments, the one or more wells facilitate the delivery by limiting the delivery by its cross-sectional area at the bottom surface of the plate. In some embodiments, the cell samples are only distributed to some or all of the areas on the substrate that are exposed to the dispensing tip(s) via the cross-sectional areas of the well at the bottom surface of the plate. In some embodiments, the method for preparing the flow cell device with sample further comprises: positioning a cover that protects the flow cell substrate from contamination on top of the plate; incubating or imaging the one or more samples. In some embodiments, during incubating or imaging operation, the cover rand the plate remains coupled to the flow cell substrate. In some embodiments, the method for preparing the flow cell device with sample further comprises: in response to determining that a predetermined criterion of the sample(s) has been met, removing the flow cell substrate from the plate and the cover; and forming a flow cell device comprising one or more microfluidic channels by coupling the flow cell substrate to a top substrate or a bottom substrate, wherein the one or more fluidic channels enables fluidic communication from a dispensing device to the one or more samples. The predetermined criterion can be customized based on characteristics of the sample(s), and / orvarious imaging and sequencing applications. For example, cell density, morphology, polony density, or other various factors can be included in the predetermined criterion, should not be limited by the disclosure herein.

[0456] In some embodiments, the method for preparing the flow cell device with sample comprises comprising providing a sample preparation system disclosed herein. In some embodiments, the method for preparing the flow cell device with sample comprises: comprising providing a plate with one or more wells extending from a top surface to a bottom surface thereof; a flow cell substrate configured to hold the one or more samples immobilized thereon; a seal that seals a gap between the plate and the flow cell substrate; and a cover that protects the flow cell substrate from contamination.

[0457] In some embodiments, the method for preparing the flow cell device with sample further comprises allowing a user to couple the plate on top of the flow cell substrate; allowing the user to openly deliver one or more samples through the one or more wells to the flow cell substrate, wherein the one or more samples are three-dimensional (3D); allowing the user to remove the flow cell substrate from the plate in response to determining that a predetermined criterion has been met; allowing the user to form a flow cell device comprising one or more microfluidic channels by coupling the flow cell substrate to a top substrate or a bottom substrate, wherein the one or more fluidic channels enables fluidic communication from a dispensing device to the one or more samples. In some embodi...

Claims

CLAIMS What is claimed is:

1. A system for preparing one or more samples on a flow cell device, comprising: a plate with one or more wells extending from a top surface of the plate to a bottom surface of the plate; a flow cell substrate configured to receive the one or more samples through the one or more wells and to immobilize the one or more samples on the flow cell substrate; a seal that seals a gap between the plate and the flow cell substrate; and a cover that protects the flow cell substrate from contamination.

2. The system of claim 1, wherein the bottom surface of the plate is coupled to the seal, with the plate being positioned above the flow cell substrate.

3. The system of claim 1 or 2, wherein the seal is preassembled to the bottom surface of the plate.

4. The system of any one of the preceding claims, wherein the seal comprises a layout identical to a layout of the bottom surface of the plate.

5. The system of any one of the preceding claims, wherein the seal protects a top surface of the flow cell substrate from mechanical impact from the plate.

6. The system of any one of the preceding claims, wherein the seal comprises one or more adhesive layers for attaching to the plate, the flow cell substrate, or both.

7. The system of any one of the preceding claims, wherein the flow cell substrate is a bottom substrate of the flow cell device.

8. The system of any one of the preceding claims, wherein the flow cell substrate is a top substrate of the flow cell device.

9. The system of any one of the preceding claims, wherein the flow cell substrate is configured to be fixedly coupled to a top substrate, a middle substrate, or both to form one or more microfluidic channels.

10. The system of any one of the preceding claims, wherein the flow cell substrate is configured to be fixedly coupled to a top substrate or a bottom substrate to form the one or more microfluidic channels.

11. The system of any one of the preceding claims, wherein the one or more samples are received and immobilized on a top surface of the flow cell substrate, a bottom surface of the flow cell substrate, or both.

12. The system of any one of the preceding claims, wherein the flow cell substrate is configured to be fixedly coupled to another flow cell substrate to form the flow cell device.

13. The system of any one of the preceding claims, wherein the flow cell substrate comprises glass or plastic.

14. The system of any one of the preceding claims, wherein the flow cell device comprises an adhesive layer configured to fixedly attach the top substrate or the bottom substrate to the flow cell substrate.

15. The system of any one of the preceding claims, wherein the adhesive layer comprises an area that does not overlap with the area of the one or more microfluidic channels along a x-y plane that is orthogonal to a z axis along which the plate, the seal, and the flow cell substrate are coupled.

16. The system of any one of the preceding claims, wherein the seal comprises a gasket.

17. The system of any one of the preceding claims, wherein the seal comprises a shape that surrounds at least part of each of the one or more microfluidic channels.

18. The system of any one of the preceding claims, wherein the adhesive layer comprises a shape that surrounds at least part of each of the one or more microfluidic channels.

19. The system of any one of the preceding claims, wherein the adhesive layer comprises a shape that surrounds the gasket.

20. The system of any one of the preceding claims, wherein the one or more microfluidic channels provide fluidic communication between a landing area and the samples immobilized on the flow cell device.

21. The system of any one of the preceding claims, wherein the one or more microfluidic channels provide fluidic communication between a landing area and the samples immobilized on a surface of the one or more microfluidic channels.

22. The system of any one of the preceding claims, wherein the cover is coupled to a top surface of the plate.

23. The system of any one of the preceding claims, wherein the system further comprises a removable clamp configured to hold the plate, the seal, and the flow cell substrate in a stack for sample preparation, sample examination, or both.

24. The system of any one of the preceding claims, wherein the plate comprises a groove or a tongue configured to reversibly couple the plate to the removable clamp.

25. The system of any one of the preceding claims, wherein the plate comprises a height along a z axis in a range from 0.5 mm to 5 cm, from 0.5 mm to 3 cm, or from 1 mm to 3 cm.

26. The system of any one of the preceding claims, wherein each of the one or more wells comprises a corresponding cross-sectional area that matches a size and a shape of the cross- sectional area of the corresponding microfluidic channel of the flow cell device.

27. The system of any one of the preceding claims, wherein each of the one or more wells comprises a cross-sectional area that is less than ±5%, ±10%, ±15%, or ±20% different from a corresponding cross-sectional area of a microfluidic channel of the flow cell device.

28. The system of any one of the preceding claims, wherein each of the one or more wells has a uniform cross-sectional area along the z axis.

29. The system of any one of the preceding claims, wherein at least one of the one or more wells has a non-uniform cross-sectional area along the z axis.

30. The system of any one of the preceding claims, wherein the one or more wells comprise a single well.

31. The system of any one of the preceding claims, wherein the one or more samples comprise at least 1 x 106, 2 x 106, 3 x 106, 4 x 106, 5 x 106, 6 x 106, 8 x 106, or 1 x 107cells received through the single well and immobilized on the flow cell substrate.

32. The system of any one of the preceding claims, wherein the one or more wells comprise two, three, or four wells.

33. The system of any one of the preceding claims, wherein the one or more wells comprise 12 wells.

34. The system of any one of the preceding claims, wherein the one or more samples comprise cells, tissue, or organoids.

35. The system of any one of the preceding claims, wherein the one or more samples comprise at least 5 x 103, 1 x 104, 5 x 104, or 1 x 105cells received through each of the one or more wells and immobilized on the flow cell substrate.

36. The system of any one of the preceding claims, wherein the one or more wells comprise a first number of wells that matches a second number of microfluidic channels of the flow cell device.

37. The system of any one of the preceding claims, wherein the one or more wells comprise multiple wells arranged in a predetermined spatial pattern.

38. The system of any one of the preceding claims, wherein the one or more wells comprise at least two wells comprising a different cross-sectional area or cross-sectional shape at the bottom surface of the plate.

39. The system of any one of the preceding claims, wherein the one or more wells comprise at least two wells comprising an identical cross-sectional area or cross-sectional shape at the bottom surface of the plate.

40. The system of any one of the preceding claims, wherein the flow cell device is configured to be inserted into a flow cell cartridge or a manifold that is configured to hold the flow cell device and enable fluidic communication between the flow cell device and a fluidic dispensing device, a fluidic reservoir, a waste container, or a combination thereof.

41. The system of any one of the preceding claims, wherein the flow cell device, the manifold, or both enables open fluidic communication to the one or more microfluidic channels.

42. The system of any one of the preceding claims, wherein the flow cell device, the manifold, or both enables open dispensing of a reagent to the landing area of the flow cell device.

43. The system of any one of the preceding claims, wherein the flow cell device, the manifold, or both enables closed fluidic communication of a reagent between the fluidic reservoir and the one or more microfluidic channels.

44. The system of any one of the preceding claims, wherein the flow cell device, the manifold, the flow cell cartridge, or a combination thereof enables closed fluidic communication of a reagent between the fluidic reservoir and the one or more microfluidic channels.

45. The system of any one of the preceding claims, wherein the closed fluidic communication is through one or more fluidic pathways connecting the manifold or flow cell cartridge to the fluidic reservoir.

46. The system of any one of the preceding claims, wherein each of the one or more wells comprises a cross-sectional area that is less than ±5%, ±10%, ±15%, or ±20% different from a corresponding cross-sectional area of a microfluidic channel of the flow cell device at the bottom surface of the plate.

47. The system of any one of the preceding claims, wherein the adhesive layer comprises one or more selected from the group comprising: a pressure sensitive adhesive, a double- sided tape, a photo-crosslinkable adhesive, thermo-crosslinkable adhesive, a chemically- cured adhesive, an adhesive made from a monomer, and an adhesive gel.

48. A method for preparing one or more samples on a flow cell device, comprising: coupling a plate with one or more wells to a flow cell substrate, wherein the flow cell substrate is configured to hold the one or more samples immobilized on theflow cell substrate, and wherein the one or more wells extend from a top surface of the plate to a bottom surface of the plate; delivering one or more samples through the one or more wells to the flow cell substrate, wherein the one or more samples are three-dimensional (3D); coupling a cover to the top surface of the plate to protect the flow cell substrate from contamination from the top surface of the plate; incubating or imaging the one or more samples immobilized on the flow cell substrate; analyzing image data of the one or more samples to determine characteristics describing the one or more samples; determining whether a predetermined criterion has been met by comparing the characteristics against the predetermined criterion; in response to determining that the predetermined criterion has been met, removing the flow cell substrate from the plate and the cover; and forming a flow cell device comprising one or more microfluidic channels by coupling the flow cell substrate to a top substrate or a bottom substrate, wherein the one or more microfluidic channels enables fluidic communication from a dispensing device to the one or more samples.

49. A method for preparing one or more samples on a flow cell device, comprising: providing: a plate with one or more wells extending from a top surface of the plate to a bottom surface of the plate; a flow cell substrate configured to immobilize the one or more samples; a seal that seals a gap between the plate and the flow cell substrate; and a cover that protects the flow cell substrate from contamination; coupling the plate to the flow cell substrate with the seal disposed between the plate and the flow cell substrate; openly delivering the one or more samples through the one or more wells to the flow cell substrate, wherein the one or more samples are three-dimensional (3D); removing the flow cell substrate from the plate, in response to determining that a predetermined criterion has been met; forming a flow cell device comprising one or more microfluidic channels by coupling the flow cell substrate to a top substrate or a bottom substrate, wherein the oneor more fluidic channels provides fluidic communication from a dispensing device to the one or more samples.

50. The method of claim 48 or 49, wherein coupling the flow cell substrate to the top substrate or the bottom substrate comprises providing a pressure, a vacuum, a temperature change, or a photonic energy to fixedly couple the flow cell substrate to the top substrate or the bottom substrate.

51. A method for detecting leakage of a flow cell device, comprising: positioning a flow cell device on a sequencing system for a sequencing run, comprising: connecting an outlet of the flow cell device to a pump in a first sealed connection; connecting an inlet of the flow cell device to a valve in a second sealed connection, wherein the valve is in a closed position; pumping, by the pump, gas via the outlet to the flow device for a first predetermined period of time at a predetermined pressure level; sensing, by a pressure sensor, a gas pressure in a microfluidic channel of the flow cell device during the predetermined period of time; in response to detecting that the gas pressure is over a predetermined threshold pressure over a threshold duration, switching the valve to an open position; introducing liquid via the valve or the inlet to the flow cell device for a second predetermined period of time; acquiring, by an image sensor of the sequencing system, a first image of the flow cell device at a first z-level and at a first time point and a second image of the flow cell device at the first z-level and at a second time point while introducing gas via the valve or the outlet to the flow cell device; in response to determining that the first image and the second image are different, determining that the microfluidic channel has leakage; and in response to determining that the first image and the second image are not different, determining that the microfluidic channel lacks leakage.

52. A method for detecting leakage of a flow cell device, comprising: positioning a flow cell device on a sequencing system for a sequencing run, comprising:connecting an outlet of the flow cell device to a pump in a first sealed connection; connecting an inlet of the flow cell device to a valve in a second sealed connection, wherein the valve is in a closed position; pumping, by the pump, gas via the outlet to the flow cell device for a first predetermined period of time at a predetermined pressure level; sensing, by a pressure sensor, gas pressure in a microfluidic channel of the flow cell device during the predetermined period of time; and in response to detecting that the gas pressure is below a predetermined threshold pressure over a threshold duration, determining, by a processor of the sequencing system, that the microfluidic channel has leakage.

53. A method for detecting leakage of a flow cell device, comprising: positioning a flow cell device on a sequencing system for a sequencing run, comprising: connecting an outlet of the flow cell device to a pump in a first sealed connection; connecting an inlet of the flow cell device to a valve in a second sealed connection, wherein the valve is in an open position; acquiring, by an image sensor of the sequencing system, a first image of the flow cell device at a first z-level and at a first time point and a second image of the flow cell device at the first z-level and at a second time point while introducing liquid via the valve or the outlet to the flow cell device for a predetermined period of time; in response to determining that the first image and the second image are different, determining that the microfluidic channel has leakage; and in response to determining that the first image and the second image are not different, determining, by a processor of the sequencing system, that the microfluidic channel lacks leakage.

54. The method of any one of claims 48-53, wherein the microfluidic channel lacks any fluid therewithin.

55. The method of any one of claims 48-54, wherein the microfluidic channel comprises a surface on which the one or more samples are immobilized.

56. The method of any one of claims 48-55, wherein the flow cell device comprises a flow cell substrate on which the one or more samples are immobilized.

57. The method of any one of claims 48-56, wherein each of the first and the second sealed connection is liquid-sealed.

58. The method of any one of claims 48-57, wherein at least one of the first and the second sealed connection is not gas-sealed.

59. The method of any one of claims 48-58, wherein connecting the outlet of the flow cell device to the pump in the first sealed connection comprises: sealing a landing area of the flow cell device with a sealing element external to the sequencing system.

60. The method of any one of claims 48-59, wherein connecting the outlet of the flow cell device to the pump in the first sealed connection comprises: connecting the outlet of the flow cell device to the pump via one or more first closed fluidic pathways.

61. The method of any one of claims 48-60, wherein the one or more first closed fluidic pathways comprise tubing and a common line.

62. The method of any one of claims 48-61, wherein connecting the inlet of the flow cell device to the valve in the second sealed connection comprises: connecting the inlet to the valve via one or more second closed fluidic pathways.

63. The method of any one of claims 48-62, wherein the pressure sensor is located in the one or more first closed fluidic pathways.

64. The method of any one of claims 48-63, wherein the pressure sensor is located in the one or more second closed fluidic pathways.

65. The method of any one of claims 48-64, wherein the gas comprises air.

66. The method of any one of claims 48-65, wherein the first image of the flow cell device is at the first time point when the microfluidic channel lacks any gas bubbles at the first z-level and wherein the second image of the flow cell device at the second time point when the microfluidic channel has gas bubbles at the first z-level.

67. The method of any one of claims 48-66, wherein the first image or the second image comprises light intensities of a light source reflected by the flow cell device.

68. The method of any one of claims 48-67, wherein the valve is in fluidic connection with a buffer reservoir.

69. The method of any one of claims 48-68, wherein the pump is a syringe pump in fluidic connection with a reagent reservoir or a buffer reservoir.

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