EP4695419A1

EP4695419A1 - Methods for sensor device sample incubation loading

Info

Publication number: EP4695419A1
Application number: EP24725279.4A
Authority: EP
Inventors: Coleen NEMES; Scott Parker
Original assignee: Life Technologies Corp
Current assignee: Life Technologies Corp
Priority date: 2023-04-14
Filing date: 2024-04-08
Publication date: 2026-02-18
Also published as: CN121241152A; KR20260006587A; WO2024215601A1

Abstract

For various sequencing systems, the effective loading of template beads into a sensor device is an essential step in a number of essential steps in providing high quality sequencing data. The loading of various types of template beads into a microwell array of a sensor device can be effectively accomplished via incubation loading. The time required for incubation loading of a sensor device can be well within a desired device loading workflow for sequencing of between about ten minutes to about an hour.

Description

METHODS FOR SENSOR DEVICE SAMPLE INCUBATION LOADING

BACKGROUND

[0001] For various sequencing systems, the effective loading of template beads into a sensor device is an essential step in a number of essential steps in providing high quality sequencing data. A loading solution can contain hundreds of millions of hydrophilic polymer beads to be loaded into a reaction chamber of a sensor device. Given the size of various hydrophilic polymer beads in relationship to the cross-sectional area of the opening of a reaction chamber, instrument-mediated methods of loading a sensor device with template beads have been utilized to ensure optimal loading is performed in a target time frame.

[0002] According to the present disclosure, optimal loading in a target time frame can be achieved under selected conditions of incubation of a sensor device with a sample of beads. The time required for incubation loading of a sensor device can be well within a desired device loading workflow for sequencing of between about ten minutes to about an hour.

BRIEF DESCRIPTION OF THE DRAWINGS

[0003] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of what is disclosed herein will be obtained by reference to the following detailed description that sets forth illustrative examples, in which the principles of the present disclosure are utilized, and the accompanying drawings of which:

[0004] FIG. 1 and FIG. 2 are a perspective views illustrating generally an exemplary sensor device of the present disclosure.

[0005] FIG. 3 is a schematic depiction illustrating generally a physicomechanical approach to loading of beads into microwells of a sensor device.

[0006] FIG. 4 is a schematic depiction illustrating generally using centrifugation for loading of beads into microwells of a sensor device.

[0007] FIG. 5 is a schematic depiction illustrating conceptually a first step of incubation loading of sample of beads in a flow cell of a sensor device such as shown in FIG. 1 and FIG. 2.

[0008] FIG. 6 is a schematic depiction illustrating generally a sensor device loaded with a sample of beads. [0009] FIG. 7 is a series of micrographs that illustrate generally a sensor device loading study showing bead loading of a sensor device as a function of bead input and temperature.

[0010] FIG. 8 is a graph and table of data from the loading study of FIG. 7.

[0011] FIG. 9 is a graph and table illustrating generally a sensor device loading study showing bead loading of a sensor device as a function of time and temperature.

[0012] FIG. 10 is a graph illustrating generally a relationship between bead diameter per cross-sectional diameter of a microwell opening for various sensor devices of the present disclosure.

[0013] FIG. 11 is a schematic illustrating generally variables that impact loading more than one bead per well.

[0014] FIG. 12 is a schematic representation that illustrates generally a sequencing system using a sensor device of the present disclosure.

[0015] FIG. 13A is a schematic representation that illustrates generally another sequencing system using a sensor device of the present disclosure.

[0016] FIG. 13B is a perspective view that illustrates generally a sequencing system, such as a sequencing system of FIG. 13A

[0017] FIG. 13C is a perspective view that illustrates a container cabinet of the sequencing system of FIG. 13B.

[0018] FIG. 14 is a schematic section view that illustrates generally a flow cell chamber of various sensor devices of the present disclosure.

[0019] FIG. 15 is a schematic depiction illustrating conceptually a section of a sensor device, including a microwell array over a top portion of a sensor device.

[0020] FIG. 16 is a schematic depiction generally illustrating a method of preparation of amplified beads.

[0021] FIG. 17 is a schematic depiction generally illustrating a method of preparation of single-template beads.

[0022] FIG. 18 is a schematic depiction generally illustrating a method of on-device preparation of template beads from single-template beads. DETAILED DESCRIPTION

[0023] For various sequencing systems, the effective loading of template beads into a sensor device is an essential step in a number of essential steps in providing high quality sequencing data. A loading solution can contain hundreds of millions of beads to be loaded into microwells of a sensor device. Various sensor devices of the present disclosure can have microwells with a cross-sectional diameter of the microwell opening of between about 0.65 pm to about 1.30 pm, where a bead can have a diameter of between 0.8 pm for a single-copy template bead to 1.2 pm for an amplified or template bead. As will be described in more detail herein, various apparatuses have been utilized for the loading of a sensor device with templated beads that enhance the loading process, for example, to ensure optimal loading is performed in a target time frame. In contrast, according to the present disclosure, optimal loading in a target time frame can be achieved under selected conditions of incubation of a sensor device with a sample of beads.

[0024] FIG. 1 illustrates generally an exemplary sensor device of the present disclosure. As depicted in FIG. 1, sensor device 100 includes substrate 110, upon which sensor die 120 is mounted; substrate 110 providing a walled structure seating over substrate 110, as well as providing electrical contacts between sensor device sensor die 120 and substrate 110. As will be described in more detail herein, substrate 110 with sensor die 120 mounted therein provide a bottom and walls for a flow cell. Sensor die 120 can be fabricated within a semiconductor substrate. Sensor die 120 can include a microwell array formed over a sensor array. In FIG. 1, flow cell 160 includes flow cell cover 130, which is sealably attached over sensor die 120. For example, flow cell cover 130 can be adhered to sensor die 120 using an adhesive. Flow cell cover 130 includes fluid ports 133 and 135, which are in fluid communication with flow cell chamber 140. Flow cell chamber 140 is defined between flow cell cover 130 and the sensor die 120. Either fluid port may be used as an inlet port, while the other as an outlet port, as they are functionally interchangeable. As such, fluid applied to either port can flow through flow cell chamber 140 and out the opposite port.

[0025] FIG. 2 illustrates generally an exemplary multilane sensor device of the present disclosure. As depicted in FIG. 2, multilane sensor device 200 includes substrate 210, upon which sensor die 220 is mounted. Sensor die 220 can include a microwell array formed over a sensor array. In a similar fashion as described for sensor device 100 of FIG. 1, flow cell cover 200, the flow cell volume sensor die 220 is divided in multiple discrete flow cells using, for example, a compressible gasket that is formed between sensor die 220 and flow cell cover 230. By way of a non-limiting example, multilane sensor device 200 can be divided into flow cells 260A-260D. Flow cells 260A-260D, also referred to as flow cell lanes 260A-260, where each flow cell lane is individually in fluid communication with a set of fluid ports. As depicted in FIG. 2 for multilane sensor device 200, flow cell cover 230 includes 4 sets of first fluid ports 233A-233D and 4 sets of second fluid ports 235A-235D. Either set of fluid ports of 233A-D and 235A-D may be used as inlet ports, while the other set as an outlet ports, as they are functionally interchangeable. As such, fluid applied to either set of ports can flow through each respective flow cell chamber 240A-240B and out the opposite set of ports. Finally, alignment pins 260A and 260B are used for mounting multilane sensor device 200 into an analysis system.

[0026] As illustrated in FIG. 2, multilane sensor device 200 includes four flow cell lanes, however, a multilane sensor device can include less than four flow cell lanes or more than four flow cell lanes. For example, multilane sensor device 200 can include between 2 and 10 flow cell lanes, such as between 2 and 8 flow cell lanes, or 4 to 6 flow cell lanes. Finally, as will be described in more detail herein, given that each lane of multilane sensor device is effectively a sensor device, similar to sensor device 100 of FIG. 1, the flow cell lanes, such as lanes 260A-260C of FIG.2, can be used at separate times or concurrently, depending on a user- defined sequencing run plan.

[0027] Table 1 and Table 2 summarize attributes of various exemplary sensor devices of the present disclosure, such as sensor device 100 of FIG. 1 and sensor device 200 of FIG. 2:

Table 1: Attributes of some exemplary FIG. 1 sensor devices

Table 2: Attributes of some exemplary FIG. 2sensor devices

[0028] As can be seen through inspection of Table 1 and Table 2, there are many similarities between device types presented in the tables. For example, a 1-1 type device and a type 2-1 have many of the same attributes, but are differentiated by the nature of the multilane structure of a FIG. 2 device. Additionally, there are some differences between the top cross-sectional diameter (TXSD) of the opening of microwells for type 1 versus type 2 devices.

[0029] Currently, apparatuses and related methods for loading a sample of beads into a microwell array are predicated on minimizing loading time through instrument-mediated facilitation of loading beads into microwell array. For example, FIG. 3 conceptually depicts a physicomechanical approach to bead loading using magnetic loading in sensor device 150, which can be either sensor device sensor device 100 of FIG. 1 or sensor device 200 of FIG. 2. As depicted, sensor device 150 includes flow cell cover 130 and microwell array 300, which bound flow cell chamber 140. In FIG. 3, the magnet creates magnetic loading bead pile 60_P from a combination of beads and magnetic loading beads 60, which have a substantially larger radius than each bead 50. As depicted, magnetic loading bead pile 60p pushes the beads toward microwell array 300, enhancing the probability that a bead will load into a microwell. Though shown in a horizontal disposition, in keeping with the modeling, magnetic loading as depicted in FIG. 3 can also be performed in a vertical orientation.

[0030] By way of an additional example, FIG. 4 conceptually depicts loading beads into sensor device 150 using centrifugation. Sensor device 150 of FIG. 4 is as described for FIG. 3, and includes flow cell cover 130 and microwell array 300, which bound flow cell chamber 140. As depicted in FIG. 4, bead sample 55 that includes a population of a hydrophilic polymer bead 50 is loaded into flow cell chamber 140. Sensor device 150 can be inserted into a centrifuge, so that bead sample is subjected to defined centripetal force for a defined time, driving the movement of beads to the surface of microwell array 300, thereby facilitating loading of beads into the microwells. [0031] An ordinary definition of incubation is to maintain a chemical or biochemical system under specific conditions favorable for development or reaction. As such, the particular development of the present disclosure is the interaction of hydrogel beads with a microwell array surface of a sensor device leading to loading of microwell sites. The present inventors performed initial experiments in which loading of hydrogel beads into a microwell array of a sensor device was done without using any apparatus-mediated method, but moreover surprisingly by incubation of a sensor device with a sample of beads. After the initial experiments, the present inventors have identified conditions in which the time required for incubation loading of a sensor device can be well within a desired device loading workflow for sequencing of between about ten minutes to about an hour.

[0032] Incubation loading as depicted conceptually in FIG. 5, in a first step, can include the introduction of a bead sample 55 into flow cell chamber 140 of sensor device 150. The sensor device can be oriented horizontally or vertically. Each bead 50 is bounded in a first orientation between flow cell cover 130 and microwell array 300, and bounded in a second orientation by the flow cell walls (not shown). A bead proximal to microwell array 300 can encounter an interstitial surface of the microarray, such as each interstitial surface 321 depicted in FIG. 5, or can encounter well opening 312, and load into a well. As will be described subsequently herein, hydrogel beads with a diameter of between about 60% to about 120% of the cross-sectional diameter of the opening of a microwell can be loaded via incubation loading into a microwell array of various sensor devices of the present disclosure without loading two beads into the same well. As depicted in FIG. 5, when aligned with opening 312 of microwell 310, instead, for example, interstitial surface 321, a hydrogel bead can load into microwell 310. Without being bound by theory, the modeling of spatial movement of beads in a flow chamber performed by the present inventors suggests that once a bead encounters a surface reversibly, it has 98% probability of finding the surface again within 5 minutes under typical experimental conditions as described herein. As such, even if a bead 50 first encounters an interstitial space, as it is proximal to the microwell array, there is a high probability that it will interact within a defined period of time to load within a microwell.

[0033] FIG. 6 is a schematic depiction illustrating generally, in which bead sample 55 including a population of hydrophilic polymer beads, such as bead 50, is loaded into microwell array 300 of sensor device 150. Though all methods of loading described begin with introducing a bead sample into the flow cell as depicted in FIG. 5 and end with a loaded sensor device as depicted in FIG. 6, incubation loading is the only method described herein with no additional handling in between introduction of beads into the flow cell and a loaded sensor device. For incubation loading of beads with reduced radius, a solution of appropriate change in ionic strength or dielectric constant can be introduced into flow cell chamber 140, providing for beads of an expanded radius to be seated in a respective microwell.

[0034] FIG. 7 is a compilation of micrographs that illustrate generally an investigative sensor device loading study based on initial observations, in which bead loading was investigated as a function of bead input and temperature at a constant time interval. The study was conducted using untemplated hydrogel beads. In the study, a sample of beads was sequentially loaded into each of three multilane devices, such as described for FIG. 2 and device 2-1 of Table 2. Each of four preparations of a loading solution varied the amount of hydrogel beads from a loading solution having 100 million beads (100M) per loading solution to a loading solution having 400 million beads (400M) beads per loading solution. Each loading solution was prepared by mixing 20 pL of a solution containing the target number of beads with 6 pL of a solution of a 0.2M tris buffer, pH 8.0, also including 1.0M potassium chloride, 0.23M magnesium chloride and 0.1% Triton®X-100. Under these conditions, the ratio of the bead diameter relative to the cross-sectional diameter of the opening of the well was 0.8 pm. Accordingly, each lane of the three multilane devices was filled using 26 pL of each preparation of the loading solutions. Given that the each lane in device 2-1 of Table 2 has a volume of 12 pL, the excess volume of the prepared solution was used to flush the flow cell to ensure all bubbles were removed. Each devices was then incubated for 10 minutes at the designated temperature, after which the residual un-loaded beads are rinsed away by injecting 100 pL of a phosphate buffered saline (PBS) with 0.2% Tween® into each lane. Though not required for effective loading, the beads were seating deeper into the wells by pulling vacuum on the lane for 10 seconds before and after a 50 pL injection of mixture of 60% by volume of PBS with 0.2% Tween® and 40% by volume isopropyl alcohol.

[0035] As can be seen from the visual inspection of FIG. 7, bead concentration and temperature have a clear impact on the degree of loading at a set time. FIG. 8 provides a graph and table of data from the loading study of FIG. 7. By inspection of the graph and table of FIG. 8, bead concentration and temperatures in the 10 minute interval ranged from 14% loading (4°C/100M beads) to 78% loading (50°C/400M beads). This early study demonstrated the feasibility of achieving a target loading for sequencing of 65% to 95% within a desired workflow for sequencing.

[0036] FIG. 9 is a graph and table illustrating generally a sensor device loading study showing bead loading of a sensor device as a function of time and temperature at a selected concentration of beads. The study was conducted using single-template hydrogel beads and sequentially loading each of two multiplane devices, such as described for FIG. 2 and device 2-1 of Table 2. For this study, a loading solution having 300 million beads (300M) per loading solution was used. For each lane that was loaded in the two devices, a solution was prepared by mixing 20 pL of a solution containing 300M beads with 6 pL of a solution of a 0.2M tris buffer, pH 8.0, also including 1.0M potassium chloride, 0.23M magnesium chloride and 0.1% Triton®X-100. Under these conditions, the ratio of the bead diameter relative to the cross- sectional diameter of the opening of the well was 0.8 pm. For each time point, one lane of each device was filled with the loading buffer and then incubated at the designated time at the designated temperature.

[0037] By inspection of the graph and table of FIG. 9, sensor loading as a function of time and temperatures using the 300M bead loading solution ranged from 71% loading (35°C/20 minutes) to 99.5% loading (50°C/20 minutes). In general, in order to achieve a quality sequencing run, loading of between about 65% to about 95% is desirable. As provided in the results presented in FIG. 9, those results were readily achieved using incubation loading. The lower boundary of desired loading is predicated on achieving a desirable number of reads, while on the higher boundary of desired loading to predicated on avoiding excessive loading. Excessive loading is defined by loading high enough to negatively impact sequencing performance. For example, excessive loading can result in elimination of desired number of empty wells that can be used as reference wells used in signal processing. The threshold for excessive loading depends on a variety of factors and is typically 97% or greater. Excessive loading is a concern with magnetic loading and loading using centrifugation, and can be readily avoided using incubation loading by using shorter incubation times and/or lower incubation temperature. [0038] FIG. 10 is a graph illustrating generally a relationship between the ratio of bead diameter to the cross-sectional diameter of a microwell opening for various sensor devices of the present disclosure. Bead diameters are determined by spinning a dilute concentration of beads down onto a glass coverslip in a solution of PBS. The solution surrounding the beads can then be exchanged with the solvent of interest. Bead diameters can then be measured utilizing light microscopy by comparing their apparent diameter to a calibration curve generated using hard-sphere polystyrene particles of known diameters. Given the variation in the diameter of a microwell openings and therefore top cross-sectional diameter of microwells for various devices, beads can be produced in accordance with the size of the microwell opening and microwell dimensions. Factors that can impact a selection of bead diameter for use with any given device as shown in Table 1 and Table 2 include bead diameter as a result of method of templating, changes in bead diameter as a result of various buffers, diluents and the like that are used during loading and sequencing, and a consideration of bead having a diameter large enough to load one bead per microwell.

[0039] For the study used to generate the graph of FIG. 10, devices in Table 2 were used and loaded with single template beads. Additionally, devices of the 1-3 type were used and loaded with fully-templated beads. The 2-1 type devices used in the study have a top cross- sectional diameter of 1.35pm and were loaded with single-template beads in a first buffer, Bl, with composition of 1.6x PBS plus 0.32% Tween®20 or in a second buffer, B2, with composition of 0.036 mM Tris-HCI, pH 8.0, 180 mM KCI, 41.4 mM MgCI₂ and 0.02% Triton®X- 100. The average diameter of the beads was 1.06pm in the Bl buffer and 1.04 in the B2 buffer, yielding ratios of 0.78 and 0.77, respectively. The 2-2 type devices used in the study have a top cross-sectional diameter of 1.08pm and were loaded with single-template beads in the Bl buffer or in the B2 buffer. The average diameter of the beads was 0.86pm in the Bl buffer and 0.85 in the B2 buffer, yielding ratios of 0.80 and 0.79, respectively. The 3-2 type devices used in the study have a top cross-sectional diameter of 0.65pm and were loaded with singletemplate beads in the Bl buffer or in the B2 buffer. The average diameter of the beads was 0.63pm in the Bl buffer and 0.62 in the B2 buffer, yielding ratios of 0.97 and 0.95, respectively.

[0040] In comparison to a 660 megapixel 2-3 type device loaded with single-template beads, a 1-3 type sensor as described in Table 1 and FIG. 1, was loaded with fully-amplified beads. As will be disclosed subsequently herein, such beads can be prepared using emulsion PCR and result in highly negatively charged beads. For the study used to generate the data that is presented in FIG. 10, fully-templated beads were used having a diameter of 0.80 pm in a buffer, B3, with composition 0.33x PBS with 0.07% Tween®20, also including 32 mM Tris HCI, pH 8.0, and additionally 160 mM KCI, 37 mM MgCb with 0.02% Triton®X-100. Given that the top cross-sectional diameter of a 3-1 type chip is 0.65pm, this yields a ratio of bead diameter/MW opening diameter of 1.2. As such, an upper limit of 120% for a ratio of bead diameter to the cross-sectional diameter of a microwell opening is clearly indicated by the data for the 1-3 type chip presented in FIG. 10.

[0041] The consideration for the lower limit for bead diameter to the cross-sectional diameter of a microwell opening is predicated on analysis of a bead diameter size limit that would exclude the loading of more than one bead per well. FIG. 11 is a schematic illustrating generally variables that impact loading more than one bead per well. An estimate of a lower limit can be derived from the following equation in reference to the schematic of FIG. 11:

[0042] Using Eq. 1, a minimum bead diameter can be estimated, for example for bead 50i of FIG. 11, that would exclude a second bead, such as bead 502 of FIG. 11 from loading into a microwell, such as microwell 10 of FIG. 10. Table 3 summaries the results of the analysis:

Table 3: Ratio of bead diameter to top cross-sectional area of a microwell

[0043] Based on the estimates provided by Eq. l for a minimum bead diameterthat would exclude a second bead from loading is provided in Table 3. In consideration of the tolerances of the measurements, a lower limit of 60% for a ratio of a minimum bead diameter to the cross-sectional diameter of a microwell opening is indicated by the analysis performed as summarized in Table 3. [0044] Additionally, a lower limit based on the estimates provided by Eq. 1, can be provided a ratio of minimum bead diameter that would exclude a second bead from loading to the height of a microwell. In consideration of the tolerances of the measurements, a lower limit of 65% for a ratio of a minimum bead diameter to the height of a microwell is indicated by the analysis performed as summarized in Table 4.

Table 4: Ratio of bead diameter to microwell height

[0045] With respect to the preparation of a loading solution, taking into account the nature of the bead starting material, and in light of the target % loading of microwells of between 65% to 95% within specified loading conditions of time and temperature, a target ratio of 2-4 beads per microwell was determined. Table 5 below displays the ratio of the number of beads per microwell calculated for a target number of beads (millions) in a loading solution for some exemplary devices of Table 1 and Table 2. As can be seen by inspection of Table 5, in order to provide the lower limit of 2 beads per microwell, the preparation of the loading solution must be adjusted accordingly. For example, for loading a single lane of a 2-1 type device, the flow cell volume for a lane is about 12 pL. A preparation of 150 million beads in a volume sufficient for handling, for example, 26 pL, can be prepared. Given the number of microwells per lane as provided in Table 2, such a loading solution would provide a ratio of 2 beads per microwell for loading the lane. In contrast, for a type 1-1 device, where the flow cell volume is about 45 pL, given the number of microwells as provided in Table 1, a loading solution prepared using 350 million beads in 50 pL would provide a ratio of 2 beads per microwell for loading the device.

Table 5: Ratio of the beads/microwell for various loading solutions SEQUENCING SYSTEMS, DEVICES AND REAGENTS

[0046] FIG. 12 is system used for performing sequencing using a sensor device of the present disclosure. IG. 12 is a block diagram that illustrates generally a chemFET-based analysis system of the present disclosure; depicting the integration of various elements of fluidic system 1020 with fluidic multiplexer devicel030 and sensor device 150, which could be a sensor device 100 of FIG. 1 or multilane sensor device 200 of FIG. 2. As will be described in more detail herein, sensor device 150 can include a microwell array cooperatively engaged over a sensor or pixel array or pixel array; each microwell is capacitively coupled to at least one pixel. Various devices can be fabricated so that each microwell is coupled to between one to four sensors or pixels. As recited herein, the terms "sensor" and "pixel," as well the terms "device" and "chip" and derivatives of these terms can be used interchangeably. Additionally, "sensor array" and "chemFET sensor array," and derivatives thereof can be used interchangeably.

[0047] As depicted in FIG. 12, reagent and solution containers 1040A-40E of fluidic system 1020, are in fluid communication with fluidic multiplexer device 1030. During use, sensor device 150 is sealably attached to fluidic multiplexer device 1030. Microfluidic multiplexer circuit 1032 of fluidic multiplexer device 1030 permits various user-selected reagents and solutions from reagents and solution containers 1040A-1040E to be controllably delivered to sensor device 150 via reagent values 1043, which are controlled through system controller 1010. A selected reagent or solution can be placed in fluid communication with each of an inlet port, such as fluidic multiplexer circuit inlet ports 1034 of FIG. 12, then directed to sensor device 150 via fluidic multiplexer device inlet channel 1036 of fluidic multiplexer device 1030. Effluent flowing from sensor device 150 is returned to fluidic multiplexer device 1030 via fluidic multiplexer device outlet channel 1038, which can be placed into fluid communication with any of fluidic manifold fluidic manifold waste lines 1041A-1041B to waste container 1044. Accordingly, reagents and solutions from the fluidic system 1020 can be selectively driven through fluidic multiplexer device 1030 by control of reagent valves 1043, which receive signals from system controller 1010, and then through sensor device 150 to waste container 1044.

[0048] System controller 1010 of FIG. 12 provides control for wash solution valves 1043A and 1043B, as well as control for wash solution valves 1045A and 1045B. Reference electrode 1046 is an important component for providing a stable reference voltage to the sensor device, as each sensor of a sensor device generates an output signal that depends on the value of a stable reference voltage. As depicted in FIG. 12, reference electrode 1046 is in fluid communication with wash solution container 1040F through wash solution fluid line 1047 and in contact with sensor device 150 via fluidic multiplexer device 1030. Wash solution container 1040F contains a wash solution of known electrolyte composition. As such, the constant electrolyte fluidic environment of reference electrode 1046 provides a constant and stable reference voltage to sensor device 150. As depicted in FIG. 12, a tee in the wash solution line provides a controllable flow path for wash solution to microfluidic multiplexer circuit 1032, as well as to fluidic multiplexer device inlet channel 1036. When a reagent or solution from reagents and solution containers 1040A-1040E is controllably selected, wash solution valve 1045B is closed with reference electrode 1046 in contact with the wash solution. If wash solution is the solution selected, the wash solution valve 1045B is opened. During the delivery of a reagent or solution through the system, the reference electrode 46 remains in constant contact with wash solution, and a stable reference potential is applied to sensor device 150. According to the present disclosure, reference electrode 1046 can be a hollow cylindrical structure, for example, of an inert metal, non-limiting examples of which include stainless steel, platinum or titanium. Such hollow cylindrical metal structures, can provide effective ohmic contact with a fluid in a flow stream. Alternatively, the reference electrode could be a wire or a flat plate.

[0049] FIG. 13A is a block diagram that illustrates generally a sequencing system of the present teachings, which can be a sequencing system incorporating a sample preparation platform. As depicted in FIG. 13A, sequencing system 2000 can include controller 2002 in communication with sample preparation deck 2004, loading station 2006, and sequencing station 2008. Sample preparation deck 2004 can include pipetting robot 2012, which can be a three-axis pipetting robot. Pipetting robot 2012 can access samples 2014, reagents and solutions 2016, thermocycler 2018 and other devices 2020, such as a magnetic separator or a centrifuge. Target sequences of a sample to be analyzed on sequencing system 2000 can be prepared at sample preparation deck 2004, and then can be provided to the loading station 2006. For example, sample preparation deck 2004 can provide library preparation of a sample to be analyzed, as well as preparation of target sequences from a library, which can be used to prepare a sample of particles or beads. Such a sample of particles or beads can then be provided to loading station 2006 to be loaded onto a sensor device, such as sensor device 200 of FIG. 2. Loading can be performed using magnetic loading, such as described for FIG. 3. Alternatively, loading can be performed using incubation loading as described herein for FIG. 5 through FIG. 11.

[0050] Once loaded, the sensor device can be transported to sequencing station 2008 using slide mechanism 2007, which can move a sensor device from a loading position to a sequencing position. Sequencing station 2008 can include both fluidic and electronic interfaces to automatically process a sample loaded on a sensor device during a sequencing run. Container cabinet 2010 can house containers holding various reagents and solutions used in a sequencing run, as well housing various waste containers. Data gathered from the sensor device can be provided to sequencing computer 2022, which can perform base calling, read alignment, and variant calling.

[0051] Controller 2002 can further communicate with a user interface, such as a monitor, keyboard, mouse, touchscreen, or any combination thereof, among other interfaces, such as user interface 2024 of FIG. 13A. Further, controller 2002 can communicate with a network interface that may access a local area network, wide area network, or global network. Network interface 2026 can be a wired interface or a wireless interface using various standard communication protocols. Sequencing system 2000 can be powered by power source 2028.

[0052] FIG. 13B is a perspective view that illustrates generally a sequencing system of the present teachings. Sequencing system 2500 of FIG. 13B can be a sequencing system with various components as described for sequencing system 2000 of FIG. 13A. Sequencing system 2500 can include upper portion 2502 and container cabinet 2510, such as container cabinet 2010 of FIG. 13A. Upper portion 2502 can include door 2506 to access sample preparation deck 2504 on which samples to be analyzed, reagent containers, and other consumables can be placed, for example, as described for FIG. 13A. In addition, various examples of a sequencing system, such as sequencing system 2500, can include a user interface, such as a touchscreen display 2508.

[0053] FIG. 13C illustrates generally container cabinet 2510, which can be a component of a sequencing system, such as sequencing system 2000 of FIG. 13A and sequencing system 2500 of FIG. 13B. Container cabinet 2510 can be useful in the management of fluidic processing for a sequencing system. For example, as depicted in FIG. 13C, container cabinet 2510 includes reagent cartridge loading interface 2512 for loading a reagent concentrate cartridge. Moreover, container cabinet 2510 can house various containers for holding reagents, and solutions. For example, a wash solution and a cleaning solution can be held in containers of first container assembly 2514 of FIG. 13A. Further, bulk nucleotide reagents, as well as a bulk calibration solution can be held in containers of second container assembly 2516 of FIG. 13C. Additionally, a container cabinet can house various sample preparation waste, sensor waste and main waste containers for collecting effluent. For example, first waste container 2518A can collect effluent generated during sample preparation, such as effluent generated from sample preparation deck 2004 of sequencing system 2000 of FIG. 13A and sample preparation deck 2504 of FIG. 13B. Additionally, second waste container 2518B of FIG. 13C can collect effluent generated from a system fluidic system, for example, during a sequencing run.

[0054] FIG. 14 illustrates generally a section view of a sensor device 150, which could be a section of sensor device 100 of FIG. 1 or any of sensors 1-1, 1-2 and 1-3 of Table 1. Sensor device 150 can include substrate 10, which in addition to providing a walled structure for mounting sensor die 20, can include wire bond elements including wire bond pad 26 and wire bond 22. Each wire is connected to sensor die 20, so that substrate 10 provides for electrical interconnection between a sensor device and one or more interface boards that are part of an analysis system, such as a sequencer. The wire bonds are protected by an encapsulant 24. Flow cell cover 30 is sealably attached over sensor device 150, forming flow cell chamber 40. The flow cell chamber height, HFC, can be adapted to provide uniform flow through the flow cell by having flow cell cover 30 formed with a concave face, so that the height of the flow cell chamber in the center, H FC-C, is less than the height of the flow cell chamber at the edges, HFC E- For example, in various sensor devices the height of the flow cell chamber in the center, HFC-C, can be about 60 pm, while the height of the flow cell chamber in the edges, HFC-E, is 100 pm. For sensor device 200 of FIG. 2 or any of the sensors 2-1, 2-2, and 2-3 of Table 2, the height of the flow cell chamber is uniform and can be about 125 pm for each lane.

[0055] FIG. 15 is a schematic depiction illustrating conceptually a section of sensor device loaded with beads 50, which could be a section of sensor device 100 of FIG. 1 or a section view of multilane sensor device 200 of FIG. 2. It should be noted that the sizes of features depicted in FIG. 15 are not drawn to scale, but moreover depicted in a fashion to dynamically visualize features of a sensor device having vastly different sizes. For example, each microwell 310 of microwell array 300 includes an opening, such as opening 312. Each opening has a top cross-sectional diameter of a microwell, such as diameter 312D of microwell 310 of FIG. 15. For sensor device 100 of FIG. 1, such as provided in Table 1, a top cross-sectional diameter of a microwell can range from about 0.65 pm to about 1.30 pm, while the height of the flow cell chamber in the center, HFC-C, can be about 60 pm, and the height of the flow cell chamber in the edges, HFC-E, can be about 100 pm. For sensor device 200 of FIG. 2, such as provided in Table 2, a top cross-sectional diameter of a microwell can range from about 0.65 pm to about 1.35 pm, while for each lane the flow cell height is uniform at about 125 pm.

[0056] With respect to the conceptual section of a sensor device of FIG. 15, for either sensor die 120 of sensor device 100 of FIG. 1 or sensor die 220 of sensor device 200 of FIG. 2, an array of microwells, such as microwell array 300 of FIG. 15, can be formed over an array sensors, such as sensor array 330 of FIG. 15. In FIG. 15, sensor array 330 is the top structure of an array of chemically-sensitive field effect transistor (chemFET) sensors, in which each microwell can be capacitively coupled to at least one sensor. Various devices can be fabricated so that each microwell is coupled to between one to four sensors. As such, microwell array 300 and sensor array 330 of FIG. 15 are the top portions of sensor die 120 of FIG. 1 or sensor die 220 of FIG. 2.

[0057] As depicted in FIG. 15, sensor array 330 includes the top metal layer 342 of floating gate 340. Top metal layer 342 is also a sensing plate. Floating gate 340 also includes metal via 341, which joins top metal layer 342 to metal layer 344. As will be described in more detail herein, a floating gate of a chemFET of the present disclosure can include four to five metal layers connected by metal vias, which overlay the channel region (not shown) of a chemFET . Floating gate 340 is formed in dielectric layer 332. Dielectric layers 320 and 322 formed over dielectric layer 332 of sensor array 330, and are dielectric layers in which a microwell array is formed, such as microwell array 300 of FIG. 15. Each microwell 310 of microwell array 300 includes an opening, such as opening 312, sidewalls, such as sidewalls 314, and a bottom or floor, such as bottom or floor 316. Microwell sidewalls 312 and microwell bottom 316 form a continuous surface over which a metal layer, such as metal layer 318 is formed. As such, microwell bottom is a conductive metal layer formed over sensor plate 342, while microwell sidewalls 314 are part of the continuous metal layer 318, effectively providing a sensing surface, such as sensing surface 315. Metal layer 318, can be selected from, for example, but not limited by, titanium, zirconium, ruthenium, vanadium and tantalum, a thin film of a metal oxide naturally forms under atmospheric conditions.

[0058] Reactions carried out in microwell 310 can be analytical reactions to identify or determine characteristics or properties of an analyte of interest. Such reactions can generate directly or indirectly byproducts that affect the amount of charge adjacent to sensor plate 342. If such byproducts are produced in small amounts or rapidly decay or react with other constituents, then multiple copies of the same analyte may be analyzed in microwell 310 at the same time in order to increase an output signal generated in response to the change in the amount of charge adjacent to sensor plate, such as sensor plate 342 of FIG. 15. According to the present disclosure,, multiple copies of an analyte may be attached to a bead 50, either before or after deposition into the microwell 301. As recited herein, the terms "bead", "solid phase support", or "particle" and derivatives thereof may be used interchangeably. Briefly, various beads of the present disclosure are hydrophilic polymer solid phase supports used for providing multiple copies of the same analyte for increasing the output signal. In particular, the solid phase support can include copies of polynucleotides. For example, such hydrophilic particles can immobilize a plurality of copies of a polynucleotide for sequencing using a sequencing system such as shown in FIG. 12.

[0059] In general, a sample of beads can be treated to include biomolecules, for example, such as nucleosides, nucleotides, nucleic acids (oligonucleotides and polynucleotides), polypeptides, saccharides, polysaccharides, lipids, or derivatives or analogs thereof. A terminal end or any internal portion of a biomolecule can bind or attach to a polymeric particle. A polymeric particle can bind or attach to a biomolecule using linking chemistries. A linking chemistry includes covalent or non-covalent bonds, including an ionic bond, hydrogen bond, affinity bond, dipole-dipole bond, van der Waals bond, and hydrophobic bond. A linking chemistry can include affinity between biorecognition complementary elements, for example between an avidin moiety and a biotin moiety; an antigenic epitope and an antibody or immunologically reactive fragment thereof or a hapten, a lectin and a polysaccharide and an enzyme and a substrate.

[0060] Regarding the preparation of amplified beads, as illustrated in method 400 of FIG. 16, a plurality of polymeric particles 404 can be placed in a solution along with a plurality of polynucleotides 402. The plurality of particles 404 can be activated or otherwise prepared to bind with the polynucleotides 402. For example, the particles 404 can include an oligonucleotide complementary to a portion of a polynucleotide of the plurality of polynucleotides 402. In another example, the polymeric particles 404 can be modified with target polynucleotides 404 using techniques such as biotin-streptavidin binding.

[0061] The hydrophilic particles and polynucleotides are subjected to polymerase chain reaction (PCR) amplification or recombinase polymerase amplification (RPA). For example, dispersed phase droplets 406 or 408 are formed as part of an emulsion and can include a hydrophilic particle or a polynucleotide. In the example of method 400 of FIG. 16, the polynucleotides 402 and the hydrophilic particles 404 are provided in low concentrations and ratios relative to each other such that a single polynucleotide 402 is likely to reside within the same dispersed phase droplets as a single hydrophilic particle 404. Other droplets, such as a droplet 408, can include a single hydrophilic particle and no polynucleotide. Each droplet 406 or 408 can include enzymes, nucleotides, salts or other components sufficient to facilitate duplication of the polynucleotide. An enzyme such as a polymerase is present, bound to, or is in close proximity to the hydrophilic particle or hydrogel particle of the dispersed phase droplet. In the example of FIG. 16, a polymerase is present in the dispersed phase droplet, such as droplet 408, to catalyze the duplication of the polynucleotide. The polymerase enzyme used can be a naturally-occurring polymerase, recombinant polymerase, mutant polymerase, variant polymerase, fusion or otherwise engineered polymerase, chemically modified polymerase, synthetic polymerase, or analog, derivative, fragment or subunit thereof.

[0062] Following PCR or RPA, amplified or template particles are formed, such as amplified or template bead 410 of FIG. 16, which can include hydrophilic particle 412 and a plurality of copies 414 of the polynucleotide. In particular, amplified bead 410 has a monoclonal population of target polynucleotides. While the polynucleotides 414 are illustrated as being on a surface of hydrophilic particle 412, the polynucleotides can extend within hydrophilic particle 412. Hydrogel and hydrophilic particles having a low concentration of polymer relative to water can include polynucleotide segments on the interior of and throughout amplified bead 410 or polynucleotides can reside in pores and other openings. In particular, amplified bead 410 can permit diffusion of enzymes, nucleotides, primers and reaction products used to monitor the reaction. A high number of polynucleotides per particle produces a better signal.

[0063] Various polymeric particles from an emulsion-breaking procedure can be collected and washed in preparation for sequencing. Collection can be conducted by contacting biotin moieties (e.g., linked to amplified polynucleotide templates which are attached to the polymeric particles) with avidin moieties, and separation away from polymeric particles lacking biotinylated templates. Collected polymeric particles that carry double-stranded template polynucleotides can be denatured to yield single-stranded template polynucleotides for sequencing. Denaturation steps can include treatment with base (e.g., NaOH), formamide, or pyrrolidone.

[0064] Amplified bead 410 of FIG. 16 can be loaded in sensor device 150, which can be sensor device 100 of FIG. 1 or sensor device 200 of FIG. 2, that can include microwell 310, as previously described herein. For example, amplified bead 410 can be loaded into sensor device 150 using incubation loading. A primer can be added to the wells of a microwell array, such as microwell array 300 of FIG. 15 or the amplified bead 410 can be pre-exposed to the primer prior to placement in the microwell 310. In particular, the amplified bead 410 can include bound primer. The primer and polynucleotide form a nucleic acid duplex including the polynucleotide (e.g., a template nucleic acid) hybridized to the primer. The nucleic acid duplex is an at least partially double-stranded polynucleotide. Enzymes and nucleotides can be provided to microwell 310 to facilitate detectible reactions, such as nucleotide incorporation.

[0065] Sequencing can be performed by detecting nucleotide addition. For example, in reference to sequencing system 1000 of FIG. 12, as previously described herein, reagents and solutions from containers 1040A-1040E can be controllably delivered to sensor device 150 via reagent values 1043, which are controlled through system controller 1010. As such, a selected reagent or solution can be sequentially directed to sensor device 150 via fluidic multiplexer device inlet channel 1036 of fluidic multiplexer device 1030. During the course of a sequencing experiment, various deoxynucleotide triphosphates (dNTPs) can be sequentially flowed over sensor device 150 in a defined order. In response to nucleotide addition, the pH within the local environment of microwell 310 can change. Such a change in pH can be detected by ion sensitive field effect transistors (ISFET) sensors, which are a type of chemFET sensor, such as chemFET . As such, a change in pH can be used to generate an output signal indicating the incorporation of a dNTP complementary to the target polynucleotide of the amplified bead 410.

[0066] Regarding preparation of a single-template bead, as illustrated in method 500 of FIG. 17, a plurality of bead supports 504 can be placed in a solution along with a plurality of polynucleotides 502 (target or template polynucleotides). The plurality of bead supports 504 can be activated or otherwise prepared to bind with the polynucleotides 502. For example, the bead supports 504 can include an oligonucleotide (capture primer) complementary to a portion of a polynucleotide of the plurality of polynucleotides 502. In another example, the bead supports 504 can be modified with target polynucleotides 502 using techniques such as biotin-streptavidin binding.

[0067] With respect to seeding the particles to arrive at a preparation of single-template beads, the a target polynucleotide can be subjected to polymerase chain reaction (PCR) amplification or recombinase polymerase amplification (RPA). In an example, the particles 504 include a capture primer complementary to a portion ofthe template polynucleotide 502. The template polynucleotide can hybridize to the capture primer. The capture primer can be extended to form single-template bead 506 that includes a single target polynucleotide attached thereto. Other beads may remain unattached to a target nucleic acid, such as untemplated bead 508 of FIG. 17 and other template polynucleotide can be free floating in solution, as depicted in FIG. 17. As will be discussed in more herein, the preparation of monoclonal single template bead 506 is done to minimize formation of polyclonal beads having more than one target polynucleotide.

[0068] In method 500 of FIG. 17, single-template bead 506 including a target polynucleotide can be attached to a magnetic bead 510 to form a bead assembly 512. In particular, the magnetic bead 510 can be attached to single-template bead 506 by a double stranded polynucleotide linkage. In an example, a further probe including a linker moiety can hybridize to a portion of the target polynucleotide on single-template bead 506. The linker moiety can attached to a complementary linker moiety on the magnetic bead 510. In another example, the template polynucleotide is used to form the target nucleic acid attached to single-template beads 506 can include a linker moiety that attaches to the magnetic bead 510. In another example, the template polynucleotide complementary to target polynucleotide attached to single-template bead 506 can be generated from a primer that is modified with a linker that attaches to the magnetic bead 510. The linker moiety attached to the polynucleotide and the linker moiety attached to the magnetic bead can be complementary to and attach to each other. In an example, the linker moieties have affinity and can include: an avidin moiety and a biotin moiety; or other biorecognition pairs as previously described herein for FIG. 16.

[0069] As depicted in FIG. 17, bead assembly 512 can be purified by separating elements, such as untemplated bead 508 and free target polynucleotides 502 from bead assembly 12. In FIG. 17, bead assembly 12 can be immobilized with application of a magnetic field to a reaction container in which method 500 is being prepared, such as, for example, but not limited by, a test tube, microfuge tube or microtiter plate and the like. Accordingly, bead assembly 12 is retained on the wall of the reaction container, while elements such untemplated bead 508 and free target polynucleotides 502 remain in the supernatant. The supernatant can be removed while retaining bead assembly 12, and the immobilized bead assembly can be washed thereby forming an enriched and purified population of a bead assembly. To prepare a purified solution of single-template beads, the immobilized bead assembly can be further subjected to conditions under which single-template bead 506 is released from the magnetic bead 510, for example via mechanical agitation by vortexing or sonication. As such, a purified preparation of single-template bead can be recovered from the supernatant.

[0070] As depicted in FIG. 18, purified sample 610 of single-template beads 606 can be loaded into sensor device 150, which can be sensor device 100 of FIG. 1 or sensor device 200 of FIG. 2, that can include microwell 310, as previously described herein. For example, purified sample 610 of single-template bead 606 can be loaded into device 150 using incubation loading. The target polynucleotide of single-template bead 606 can be amplified, referred to herein as templating, while in the well 310, to provide amplified or template bead 614, depicted with multiple copies 616 of the target polynucleotides. In particular, amplified bead 614 has a monoclonal population of target polynucleotides. Such an amplification reaction can be performed using polymerase chain reaction (PCR) amplification, recombination polymerase amplification (RPA), isothermal amplification or a combination thereof. An enzyme such as a polymerase is present, bound to, or is in close proximity to the hydrophilic particle or hydrogel particle. In the example of FIG. 18, a polymerase is present in solution or in the microwell to facilitate duplication of the polynucleotide. The polymerase enzyme used can be a naturally-occurring polymerase, recombinant polymerase, mutant polymerase, variant polymerase, fusion or otherwise engineered polymerase, chemically modified polymerase, synthetic polymerase, or analog, derivative, fragment or subunit thereof.

[0071] While multiple copies of polynucleotides 616 of amplified bead 614 are illustrated as being on a surface, the polynucleotides can extend within the amplified bead 614. Hydrogel and hydrophilic particles having a low concentration of polymer relative to water can include polynucleotide segments on the interior of and throughout amplified bead 614 or polynucleotides can reside in pores and other openings. In particular, amplified bead 614 can permit diffusion of enzymes, nucleotides, primers and reaction products used to monitor the reaction. A high number of polynucleotides per particle produces a better signal.

[0072] Sequencing can be performed on sensor device 150 loaded with amplified beads 614 of FIG. 18. For example, sequencing system 1000 of FIG. 12 can be used as previously described for amplified bead 410 of FIG. 16 to sequence amplified beads 614 of FIG. 18.

EXAMPLE:

[0073] Loading was performed using incubation loading with a GX5™ multilane chip, such as described for sensor device 200 of FIG. 2, which is used in conjunction with Genexus™ chip coupler (A40269). Sequencing was performed using a Genexus™ instrument, such as described for sequencing system 2500 of FIG. 13B and FIG. 13C. A standard Genexus™ singlelane library run was set up according to the user guide with Genexus™ Control Library from the Ion Torrent Genexus™ Control kit (A40267). The Genexus™ Control Library from the Ion Torrent Genexus™ Control kit can be used, for example, to confirm the function of a Genexus instrument, to assess sample performance, as well as for use in troubleshooting.

[0074] Prior to starting the run, Genexus™ strip 3 of Genexus™ Templating Strips for GX5™ (A40263) was manually modified. The magnetic loading beads were first removed from well 7 (counting left from right starting at 1) with a pipette. Then, well 7 was rinsed three times with 600 pL of nuclease-free water to completely remove all magnetic bead residue. 208 pL of nuclease-free water was deposited into the empty well and the modified Strip 3 was loaded onto the Genexus instrument as prompted. The Genexus instrument was operated with the deck doors open to enable the user to change the loading method from magnetic loading to incubation loading. This was accomplished by removing the magnetic separation plate and replacing it with an empty dummy plate just before the contents of well 2 in strip 3 were added to the loading solution in well A9 of the magnetic separation plate. At the same time, 6 pL of an aqueous solution comprised of 0.2 M Tris-HCI, pH 8.0, 1 M KCI, 0.23 M MgCl2, and 0.10% Triton®X-100 was added to the loading solution in well A9 of the original magnetic separation plate. Just prior to the loading solution being injected into the chip lane, the dummy plate was removed and replaced with the original magnetic separation plate containing the loading solution. The rest of the loading module was allowed to commence, with the loading solution incubating inside the chip for 54 minutes at 35 °C. Templating, sequencing, and analysis was allowed to commence identical to a typical Genexus sequencing run.

[0075] Table 6 shows the comparison between the performance qualification specifications for the Genexus™ Control Library and the incubation loading run. As can be discerned from inspection of Table 6, the incubation loading sequencing run met or exceeded the performance qualification specifications for AQ20 mean read length, average uniformity of base coverage, raw read accuracy, and total reads.

Table 6: Genexus™ Control Library specifications v. GX5 incubation loading run

[0076] As such, according to the present disclosure, in a first example, a method for preparation of a sensor device for analysis comprises introducing a loading solution over the sensor device, said sensor device including a microwell array formed over a sensor array, wherein the loading solution contains a number of beads providing a ratio of at least 2 beads per microwell, then selecting incubation conditions suitable for loading between about 65% to about 95% of microwells of the microwell array with the loading solution, and loading the sensor device by incubation of the loading solution with the sensor device using the selected incubation conditions.

[0077] A second example includes the subject matter of the first example, and further includes incubation of the sensor device with the loading solution is done for between about 10 minutes to about 1 hour.

[0078] A third example includes the subject matter of any of examples 1 or 2, and further includes incubation of the sensor device with the loading solution is in a temperature range between about 4°C to about 60°C.

[0079] A forth example includes the subject matter of any of examples 1-3, and further includes that a ratio of a bead diameter to a cross-sectional diameter of a microwell opening is between about 60% to about 120%.

[0080] A fifth example includes the subject matter of any of examples 1-4, and further includes that a bead diameter is not less than about 65% of the microwell height and not more than about 120% of a cross-sectional area of a microwell opening.

[0081] A sixth example includes the subject matter of any of examples 1-6, and further includes performing a sequencing assay on the sensor device loaded with a sample of beads.

[0082] A seventh example includes the subject matter of example 6, and further specifies that the sample of beads comprises a sample of hydrophilic polymer beads.

[0083] An eighth example includes the subject matter of example 7, and further specifies that the hydrophilic polymer bead comprises a hydrogel bead.

[0084] A ninth example includes the subject matter of example 6, and further specifies that the sample of beads loaded on the sensor device comprises a sample of single-copy tern plated beads.

[0085] A tenth example includes the subject matter of example 6, and further specifies that the sample of beads loaded on the sensor device comprises a sample of amplified beads.

[0086] An eleventh example includes the subject matter of example 6, wherein sequencing comprises controllably flowing a sequence of deoxynucleotide triphosphate (dNTP) reagent over the device. [0087] A twelfth example includes the subject matter of example 11, wherein flowing the sequence of deoxynucleotide triphosphate (dNTP) reagent over the device is performed in a defined order.

[0088] A thirteenth example includes the subject matter of example 11, and further includes generating an output signal indicating an incorporation of a dNTP complementary to a target polynucleotide on a template bead.

[0089] A fourteenth example includes the subject matter of any of example 1-13, and further specifies that the sensor device comprises a chemically-sensitive field effect transistor (chemFET) sensor device.

[0090] A fifteenth example includes the subject matter of example 14, and further specifies that the chemFET sensor device is an ion-selective field effect transistor (ISFET) sensor device.

[0091] A sixteenth example includes the subject matter of example 15, and further specifies that the ISFET sensor device is selective for hydrogen ion.

[0092] A seventeenth example includes the subject matter of example 16, and further specifies that the sensor device comprises at least 10⁷-10⁹ sensors.

[0093] An eighteenth example includes the subject matter of any of examples 1-17, and further specifies that the device is a multilane sensor device; the method and further including, introducing the loading solution over a selected lane of the multilane device, and loading the multilane sensor device by incubation of the loading solution with the selected lane of the multilane device.

[0094] A nineteenth example includes the subject matter of example 18, and further includes performing a sequencing assay on the sensor device loaded with a sample of beads.

[0095] A twentieth example includes the subject matter of example 19, and further specifies that the sample of beads comprises a sample of single-copy template beads.

[0096] A twenty first example includes the subject matter of example 18, and further specifies that sequencing comprises controllably flowing a sequence of deoxynucleotide triphosphate (dNTP) reagent over the lane of the multilane device.

[0097] While various examples of the present disclosure have been shown and described herein, numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the various examples described herein may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for preparation of a sensor device for analysis comprising: introducing a loading solution over the sensor device, said sensor device including a microwell array formed over a sensor array, wherein the loading solution contains a number of beads providing a ratio of at least 2 beads per microwell; selecting incubation conditions suitable for loading between about 65% to about 95% of microwells of the microwell array with the loading solution; and loading the sensor device by incubation of the loading solution with the sensor device using the selected incubation conditions.

2. The method of claim 1, wherein incubation of the sensor device with the loading solution is done for between about 10 minutes to about 1 hour.

3. The method of claims 1 or 2, wherein incubation of the sensor device with the loading solution is in a temperature range between about 4°C to about 60°C.

4. The method of any one of claims 1-3, wherein a ratio of a bead diameter to a cross- sectional diameter of a microwell opening is between about 60% to about 120%.

5. The method of any one of claims 1-4, wherein a bead diameter is not less than about 65% of the microwell height and not more than about 120% of a cross-sectional area of a microwell opening.

6. The method of any one of claims 1-6, further comprising performing a sequencing assay on the sensor device loaded with a sample of beads.

7. The method of claim 6, wherein the sample of beads comprises a sample of hydrophilic polymer beads.

8. The method of claim 7, wherein the hydrophilic polymer bead comprises a hydrogel bead.

9. The method of claim 6, wherein the sample of beads loaded on the sensor device comprises a sample of single-copy templated beads.

10. The method of claim 6, wherein the sample of beads loaded on the sensor device comprises a sample of amplified beads.

11. The method of claim 6, wherein sequencing comprises controllably flowing a sequence of deoxynucleotide triphosphate (dNTP) reagent over the device.

12. The method of claim 11, wherein flowing the sequence of deoxynucleotide triphosphate (dNTP) reagent over the device is performed in a defined order.

13. The method of claim 11, further comprising generating an output signal indicating an incorporation of a dNTP complementary to a target polynucleotide on a template bead.

14. The method of any one of claims 1-13, wherein the sensor device comprises a chemically-sensitive field effect transistor (chemFET) sensor device.

15. The method of claim 14, wherein the chemFET sensor device is an ion-selective field effect transistor (ISFET) sensor device.

16. The method of claim 15, wherein the ISFET sensor device is selective for hydrogen ion.

17. The method of claim 16, wherein the sensor device comprises at least 10⁷-10⁹ sensors.

18. The method of any one of claims 1-17, wherein the device is a multilane sensor device; the method further comprising: introducing the loading solution over a selected lane of the multilane device; and loading the multilane sensor device by incubation of the loading solution with the selected lane of the multilane device.

19. The method of claim 18, further comprising performing a sequencing assay on the sensor device loaded with a sample of beads.

20. The method of claim 19, wherein the sample of beads comprises a sample of singlecopy template beads.

21. The method of claim 18, wherein sequencing comprises controllably flowing a sequence of deoxynucleotide triphosphate (dNTP) reagent over the lane of the multilane device.