Reagent Exchange in the Machine

Abstract

The method includes flowing a capture reagent through a reagent management system and a flow cell of the instrument. The flow cell has a first polynucleotide positioned therein. The capture reagent adds a first base to a base sequence. The base sequence includes a second polynucleotide complementary to the first polynucleotide. After the first base is added to the second polynucleotide, an image of an identification signal emitted from the first base is captured. A cleavage reagent is flowed through the reagent management system and the flow cell to remove a first terminator from the first base to allow a subsequent base in the base sequence to be added to the second polynucleotide. A buffer reagent is flowed through the reagent management system and the flow cell in multiple cycles in successive forward and reverse flow directions.

Description

Cross - reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 863,444, filed on June 19, 2019, entitled Reagent Exchange In An Instrument. The entire disclosure of the above - mentioned application is incorporated herein by reference. BACKGROUND OF THE INVENTION

[0002] An instrument utilized in the execution of a method for determining the order of nucleotides in a polynucleotide may include a reagent management system (RMS) in fluid communication with a flow cell. The RMS can store, select, and sequentially flow a plurality of reagents through the flow channels of the flow cell.

[0003] Such a method may include multiple cycles of an array of selected reagents that are routed (or sent) through the flow channels of the flow cell to perform various controlled chemical reactions on a cluster of cloned first polynucleotides positioned in the flow channels of the flow cell. By the chemical reaction, tagged nucleotides can form base pairs having the base sequence of the first polynucleotide, one base pair at a time. When paired, the tag is excited and emits an identification signal that identifies the base pair. Each tagged polynucleotide is added in the flow cell to a growing complementary second polynucleotide strand that is a double - strand with the first polynucleotide.

[0004] Thus, the complementary second polynucleotide strand can be constructed one base at a time to determine the base sequence in the cluster of the first polynucleotides. The greater the number of known nucleotides in the complementary second polynucleotide strand that can be constructed (i.e., the longer the length of the second polynucleotide strand), the more data can be provided regarding the order of nucleotides in the cluster of the first polynucleotides, and the faster the overall analysis will be.

[0005] However, if the reagent exchange during the flow of the reagent is insufficient, then cross-contamination between the reagents can occur. Therefore, there is a need for an apparatus and method for determining the order of nucleotides in a polynucleotide that improves the reagent exchange between the flows of the reagents. SUMMARY OF THE INVENTION

[0006] The present disclosure provides an advantage by providing an apparatus operable to carry out a method for determining the order of nucleotides in a polynucleotide in accordance with one or more aspects of the present disclosure. The method includes a wash phase, and when flowing a buffer reagent (or buffer reagent) through a reagent management system and a flow cell of the apparatus, the total sweep volume of the buffer reagent is greater than the total fluid consumption of the buffer reagent. Further, the ratio of the total sweep volume to the total fluid consumption may be 6, 10, or greater. This method enhances reagent exchange more than other methods, and the total sweep volume relative to the total fluid consumption is 1. Thus, compared to such other methods, pre-phasing is reduced and the length of the polynucleotide that can grow is increased.

[0007] Methods according to one or more aspects of the present disclosure include flowing an incorporation regent through a reagent management system and a flow cell of an instrument. The flow cell has a first polynucleotide positioned therein. The incorporation regent adds a first base (or the first base) to a nucleotide sequence. The nucleotide sequence includes a second polynucleotide complementary to the first polynucleotide. After the first base is added to the second polynucleotide, an image of the discrimination signal resulting from the first base is captured. A cleavage reagent is flowed through the reagent management system and the flow cell to remove a first terminator from the first base, enabling subsequent bases in the nucleotide sequence to be added to the second polynucleotide. A buffer reagent is flowed through the reagent management system and the flow cell in a plurality of cycles of successive forward and reverse flow directions to wash the cleavage reagent from the reagent management system and the flow cell.

[0008] In some illustrations of the method, the plurality of cycles includes 4 or more cycles.

[0009] In some illustrations of the method, the plurality of cycles includes 12 or more cycles.

[0010] In some illustrations of the method, the reagent management system and the flow cell are positioned within a cartridge of the instrument, and the cartridge is removably insertable into the instrument.

[0011] In some illustrations of the method, the incorporation regent, the cleavage reagent, and the buffer reagent are stored in reagent wells of the reagent management system, and the reagent wells are configured to store a finite total amount of each reagent.

[0012] In some exemplary methods, flowing the buffer reagent includes flowing a first forward fluid volume (or forward flow volume) of the buffer reagent in the forward flow direction (or forward flow direction) of each cycle of a plurality of cycles; and flowing a second reverse fluid volume (or reverse flow volume) of the buffer reagent in the reverse flow direction (or reverse flow direction) of each cycle of the plurality of cycles, wherein the first forward fluid volume is greater than the second reverse fluid volume.

[0013] In some exemplary methods, the forward fluid volume is about 3% or more greater than the reverse fluid volume.

[0014] In some exemplary methods, flowing the buffer reagent includes an incubation period of about 10 seconds or more after the last cycle of a plurality of cycles, during which there is substantially no flow of the buffer reagent through the reagent management system and the flow cell; and flowing a third forward fluid volume of the buffer reagent in the forward flow direction after the incubation period.

[0015] In some exemplary methods, during the flowing of the buffer reagent, the total sweep volume of the buffer reagent is substantially given by the formula ((1 st FFV + 2 nd RFV)*N) + 3 rd determined by FFV. Where 1 st FFV is the first forward fluid volume, 2 nd RFV is the second reverse fluid volume, N is the total number of cycles in the plurality of cycles, and 3 rd FFV is the third forward fluid volume is.

[0016] In some exemplary methods, during the flowing of the buffer reagent, the total fluid consumption of the buffer reagent is substantially given by the formula ((1 st FFV - 2 nd(RFV) × N) + 3 rd It is determined by FFV.

[0017] In some illustrations of the method, the ratio of the total sweep volume of the buffer reagent to the total fluid consumption of the buffer reagent is equal to about 6, or greater than about 6.

[0018] In some illustrations of the method, the forward flow and the reverse flow are separated by a delay time of about 1 second or less for each cycle of a plurality of cycles in the forward and reverse directions.

[0019] In some illustrations of the method, the forward flow and the reverse flow are separated by a delay time of about 200 milliseconds or less for each cycle of a plurality of cycles in the forward and reverse directions.

[0020] In some illustrations of the method, each cycle of a plurality of cycles in the forward and reverse directions is separated by a delay time of about 200 milliseconds or less.

[0021] Any of the illustrations of the method may be implemented in any suitable combination to achieve the advantages described herein.

[0022] Devices according to one or more aspects of the present disclosure include an operable reagent management system positioned within the device. The reagent management system includes a plurality of reagent wells. The reagent wells have at least a first reagent well, a second reagent well, and a third reagent well, and are configured to contain a capture reagent, a cleavage reagent, and a buffer reagent positioned therein, respectively. The reagent management system is operable to select a flow of reagent from one of the plurality of reagent wells. The flow cell is operable to be positioned within the device. The flow cell includes a flow channel in fluid communication with the reagent management system. The flow channel is operable to route the flow of reagent over a first polynucleotide positioned in the flow channel. The device is operable as follows: The reagent management system and the capture reagent are flowed through the flow cell, and a first base is added to the base sequence. The base sequence comprises a second polynucleotide complementary to the first polynucleotide; After the first base is added to the second polynucleotide, an image of the discrimination signal generated from the first base is captured; The reagent management system and the cleavage reagent are flowed through the flow cell, and the first terminator is removed from the first base to enable the addition of subsequent bases in the base sequence to the second polynucleotide; and In a plurality of cycles of consecutive forward and reverse flow directions, the buffer reagent is flowed through the reagent management system and the flow cell, and the cleavage reagent is flushed out from the reagent management system and the flow cell.

[0023] In some examples, the instrument comprises a cartridge operable to include the reagent management system and the flow cell. Here, the capture reagent, the cleavage reagent, and the buffer reagent are stored in the reagent wells of the reagent management system, and the reagent wells are configured to store a finite total amount of the nuclear reagent.

[0024] In some examples, the instrument is operable to flow the buffer reagent through the reagent management system and the flow cell in a plurality of cycles of 4 or more consecutive forward and reverse flow directions.

[0025] In some examples, the instrument is operable to flow the buffer reagent as follows: A first volume of the buffer reagent is flowed in the forward flow direction of each cycle of the plurality of cycles; A second volume of the buffer reagent is flowed in the reverse flow direction of each cycle of the plurality of cycles, and the first volume is larger than the second volume; After the last cycle of the plurality of cycles, an incubation period with substantially no flow of the buffer reagent through the reagent management system and the flow cell is imposed for about 10 seconds or more; and After the incubation period, a third forward flow volume of the buffer reagent is flowed in the forward flow direction.

[0026] In some examples, the device is operable to flow a buffer reagent as follows: The total sweep volume of the buffer reagent is substantially the formula ((1 st FFV + 2 nd RFV) * N) + 3 rd determined by FFV, where: 1 st FFV is the first forward fluid volume, 2 nd RFV is the second reverse fluid volume, N is the total number of cycles in a plurality of cycles, and 3 rd FFV is the third forward fluid volume is. The total fluid consumption of the buffer reagent is substantially the formula ((1 st FFV - 2 nd RFV) * N) + 3 rd determined by FFV; and the ratio of the total sweep volume of the buffer reagent to the total fluid consumption of the buffer reagent is equal to about 6, or greater than about 6.

[0027] In some examples, the device is operable to flow a buffer reagent such that the forward flow and the reverse flow are separated by a delay time of about 200 milliseconds or less for each cycle of a plurality of cycles in the forward and reverse directions.

[0028] Any example of the device can be implemented in any suitable combination to achieve the advantages described herein.

[0029] It should be recognized that all combinations of the foregoing aspects, including the methods and devices, and further concepts described in more detail below (provided that such concepts are not mutually inconsistent) are considered to be part of the subject matter of the invention disclosed herein.

[0030] These and other objects, features, and advantages of the present disclosure will become apparent from the following detailed description of various aspects of the present disclosure, taken in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0031] The present disclosure will be more fully understood from the following detailed description, taken in conjunction with the accompanying drawings.

[0032]

Figure 1

Figure 2

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Figure 7

Figure 8

Best Mode for Carrying Out the Invention

[0033] Here, to provide a thorough understanding of the methods, systems, and device structures, functions, manufacture, and use principles disclosed herein, specific examples are described. One or more examples are shown in the accompanying drawings. Those skilled in the art will understand that the methods, systems, and devices specifically described herein and shown in the accompanying drawings are non-limiting examples, and that the scope of the present disclosure is defined only by the claims. Features illustrated or described in connection with one example may be combined with features of other examples. Such modifications and variations are intended to be included within the scope of the present disclosure.

[0034] "Substantially," "approximately," "about," "relatively," or other similar terms that may be used throughout the present disclosure, including in the claims, are used to describe or account for small variations, such as those due to changes in a reference or parameter in a process. Such small variations include zero variations from a reference or parameter as well. For example, these may refer to less than ±10% or equal to ±10%, less than ±5% or equal to ±5%, less than ±2% or equal to ±2%, less than ±1% or equal to ±1%, less than ±0.5% or equal to ±0.5%, less than ±0.2% or equal to ±0.2%, less than ±0.1% or equal to ±0.1%, less than ±0.05% or equal to ±0.05%, etc.

[0035] Referring to FIG. 1, a simplified example of the chemical structure of nucleotide 100 according to the aspects disclosed herein is shown. Nucleotide 100 is composed of the following three subunit molecules: a nitrogenous base 102 (also known as a nucleobase or base), a five-carbon sugar 104 (such as ribose or deoxyribose), and at least one phosphate group 106. A plurality of nucleotides bonded together are known as a polynucleotide. The first polynucleotide 108 and the second polynucleotide 110, most commonly seen in FIG. 2, are examples of such polynucleotides.

[0036] The nitrogenous base 102 of nucleotide 100 may be one of the following five types: adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). Each type of base 102 pairs (i.e., chemically bonds) with only one other type of base and forms complementary base pairs in the complementary double-stranded polynucleotides of two polynucleotides, such as the complementary double-stranded polynucleotide 112 shown in FIG. 2.

[0037] As used herein, complementary refers to forming a pair between the first base of a first nucleotide and a specific second base of a second nucleotide that can chemically bond to it. The second base (and its associated nucleotide) is considered to be complementary to the first base (and its associated nucleotide) herein. For example, adenine-thymine (A-T) and guanine-cytosine (G-C) are complementary base pairs that can connect the complementary double polynucleotide strands of deoxyribonucleic acid (DNA). Also, adenine-uracil (A-U) and guanine-cytosine (G-C) are complementary base pairs that can connect the complementary double polynucleotide strands of ribonucleic acid (RNA).

[0038] Referring to FIG. 2, a simplified example of a first polynucleotide 108 and a growing complementary second polynucleotide 110 according to the aspects described herein is shown. The first polynucleotide 108 and the second polynucleotide 110 are both connected to form a double-stranded polynucleotide 112.

[0039] The first polynucleotide 108 is chemically fixed (or anchored) 114 to the floor 116 of the flow channel 224 of the flow cell 202 (best seen in FIG. 7). The first polynucleotide 108 includes nucleotides 118-1 to 118-N, where N represents the total number of nucleotides 118 in the first polynucleotide 108 and also indicates the length of the first polynucleotide 108. The specific type of nitrogenous base associated with each nucleotide 118 is identified by the circled letters A, G, C, or T, which represent the types of bases adenine, guanine, cytosine, and thymine, respectively. The first polynucleotide 108 can be one of many clusters of identical cloned (or cloned) first polynucleotides 108 fixed to the floor 116 of the flow channel 224.

[0040] The second polynucleotide 110 includes at least nucleotides 120-1 to 120-6. Further, the specific type of nitrogenous base associated with each nucleotide 120 is identified by the circled letters A, G, C, or T. Each nucleotide 120 includes a chemical tag (such as a fluorescent tag) that, when excited (e.g., by excitation light), will emit an identification signal (such as fluorescence) that identifies the specific base of the nucleotide 120.

[0041] The bases of the nucleotides 120 can only bind to the complementary bases of the nucleotides 118 to form a series of complementary base pairs 122-1 to 122-6. The base pairs 122 and the nucleotides 118, 120 associated with them form the double-stranded polynucleotide 112.

[0042] In the implementation of the method to determine the order of nucleotides 118-1 to 118-N in the first polynucleotide 108, devices such as device 200 utilized in the implementation of the method may include a reagent management system 204 in fluid communication with a flow cell 202 (most clearly seen in FIG. 7). The reagent management system can store, select, and sequentially flow a plurality of reagents through the flow channel 224 of the flow cell 202. The reagent management system and / or the flow cell may or may not be disposed in a removable and insertable device cartridge 230 (most clearly seen in FIG. 7).

[0043] As described in more detail herein, to perform various controlled chemical reactions with the first polynucleotide 108, such methods include a plurality of cycles of an array of selected reagents 208-218 (most clearly seen in FIG. 7) that are routed through the flow channel 224 of the flow cell 202. At least one reagent known as an incorporation reagent that flows through the first polynucleotide 108 includes nucleotide 120. Each nucleotide 120 in the incorporation reagent includes a chemical tag (such as a fluorescent tag) that emits an identification signal (such as fluorescence) that identifies a particular base of the nucleotide 120 when excited (e.g., by excitation light). Further, the base of each nucleotide 120 in the incorporation reagent includes a separable terminator that prevents other bases from binding to it. By the chemical reaction, nucleotide 120 can have the base sequence 118 of the first polynucleotide 108 and form a plurality of base pairs 122, one base pair 122 at a time.

[0044] However, if the reagent exchange between reagent flows is not sufficient, then cross - contamination between reagents can occur. That is, if the first reagent is not removed and is not exchanged with the subsequent second reagent flow of the method, then the first reagent can contaminate the second reagent. This can cause a phenomenon known as prephasing, where multiple tagged bases are inadvertently added to the complementary second polynucleotide strand that grows in each cycle. Prephasing reduces the accuracy of the method for determining the nucleotide sequence. Also, prephasing is cumulative and can repeat in each cycle of the method, further reducing the accuracy in each cycle. Additionally, prephasing can reduce the number of bases that can be constructed with the complementary second polynucleotide (i.e., reduce the length), and the order of these bases can be determined within an acceptable degree of accuracy. Reagent exchange can be particularly difficult in regions of poor fluid flow conditions, such as the range or corners (or angles, corner) of a flow cell with non - uniform channels.

[0045] In addition to an increase in prephasing, inappropriate reagent exchange can contribute to other phenomena that can reduce the accuracy of various methods for determining the nucleotide sequence. For example, inappropriate reagent exchange can lead to an increase in phasing, where tagged bases are not added to the growing complementary second nucleotide. Also, inappropriate reagent exchange can potentially lead to a decrease in the proportion of clusters passing through the filter, which is an indicator of the purity of the signal from each cluster.

[0046] Furthermore, when the RMS is placed within the cartridge of the instrument, efficient reagent exchange becomes even more important because the reagent storage capacity of the reagent wells can be limited by the spatial constraints of the cartridge.

[0047] In the example shown in FIG. 2, the method allows the sequentially joining of tagged nucleotides 120-1 through 120-5 to nucleotides 118-1 through 118-5 to form complementary base pairs 122-1 through 122-5. Nucleotide 120-6 is the next in the sequence and attempts to bind (or pair) with nucleotide 118-6 to form a complementary base pair.

[0048] When paired, the chemical tag of nucleotide 120-6 is excited to emit an identification signal that identifies base pair 122-6. Once identified, the terminator bound to the base of nucleotide 120-6 is removed, thereby allowing subsequent bases to bind. The tagged nucleotide 120-6 is added to the growing complementary second polynucleotide strand 110, which is a double strand having the first nucleotide strand 108.

[0049] Thus, the complementary second nucleotide strand 110 can build one base at a time to determine the subsequent bases 118 in the cluster of the first polynucleotide 108. The greater the number of nucleotides 120 in the complementary second polynucleotide strand 110 that can be built (i.e., the longer the length of the second polynucleotide strand), the more data can be provided regarding the order of the nucleotides in the cluster of the first polynucleotide 108, and the overall analysis is faster.

[0050] Referring to FIG. 3, an example of a simplified flowchart of a method 130 for determining the order of nucleotides in a polynucleotide according to the aspects described herein is shown. Method 130 includes four basic phases, which are an uptake phase 132, an imaging phase 134, a cleavage phase 136, and a washing phase 138.

[0051] During capture step 132, the capture reagent is flowed through the reagent management system 204 and the flow cell 202 of the instrument 200 (best seen in FIG. 7). The flow cell 202 has a first polynucleotide 108 (best seen in FIG. 2) positioned therein. The capture reagent adds a first base (associated with nucleotide 120-6) to a base sequence (associated with nucleotides 120-1 to 120-5). The base sequences (and their associated nucleotides 120-1 to 120-6) include a second polynucleotide 110 that is complementary to the first polynucleotide 108.

[0052] More specifically, the capture reagent flows over a cluster of cloned first polynucleotides 108 that are pre-fixed to the flow channel 224 of the flow cell 202. The capture reagent includes known nucleotides 120, where the bases of these nucleotides 120 sequentially pair with the bases of the first polynucleotide 108 to form a growing second polynucleotide 110 that is complementary to the first polynucleotide 108. The first polynucleotide 108 and the second polynucleotide 110 form a double-stranded polynucleotide such as a DNA fragment. The types of bases of the first polynucleotide 108 and the second polynucleotide 110 can be any one of adenine (A), guanine (G), cytosine (C), thymine (T), or uracil (U).

[0053] Also, the nucleotides 120 in the capture reagent include a tag (in this example, a fluorescent tag) that enables the identification of the type of base with an excitation signal (in this example, excitation light). The nucleotides 120 also include a cleavable terminator (or blocking group) known in the art, which prevents more than one base of the second polynucleotide from pairing with the bases of the first polynucleotide 108 at once.

[0054] During the imaging phase 14, after the first base of nucleotide 120-6 is added to the second polynucleotide 110, an image of the discrimination signal resulting from the first base of nucleotide 120-6 is captured.

[0055] More specifically, the fluorescent tag of nucleotide 120 associated with the second polynucleotide 110 is excited by the excitation light and emits characteristic fluorescence. Next, an image of the fluorescence is taken to identify the type of base of nucleotide 120-6 newly added to the sequence of nucleotides 120-1 to 120-6 that form the growing second polynucleotide 110. Since the second polynucleotide 110 is complementary to the first polynucleotide 108, the order of nucleotide 118 in the first polynucleotide 108 can be determined. The longer the second polynucleotide 110 can be grown, the more of the order of nucleotide 118 in the first polynucleotide 108 can be determined.

[0056] During the cleavage phase 136, the cleavage reagent is flowed through the reagent management system 204 and the flow cell 202 to remove the terminator from nucleotide 120-6. This enables subsequent nucleotides (not shown) in the sequence of nucleotides 120-1 to 120-6 to be added to the second polynucleotide 110.

[0057] During the washing phase 138, the buffer reagent (or washing reagent) is flowed through the reagent management system 204 and the flow cell 202 in a plurality of cycles of continuous forward and reverse flow directions to wash away the cleavage reagent from the reagent management system and the flow cell. The buffer reagent can be a mixture of chemical species used to remove unintended chemical species from the flow cell and the reagent management system. The unintended chemical species can be, for example, active reagents remaining from the previous step (such as excess cleavage reagent) or various impurities obtained as by-products from the previous step. The buffer reagent can be, for example, buffered saline to which lipoic acid is added.

[0058] As used herein, the forward flow refers to the direction of the flow of reagent from the reagent well 232 of the reagent management system 204 to the flow cell 202 (best seen in FIG. 7). As used herein, the reverse flow refers to the direction of the flow of reagent from the flow cell 202 to the reagent well 232.

[0059] In the example of FIG. 3, a plurality of cycles are shown as 12 repeated cycles of the forward flow direction 140 and the subsequent reverse flow direction 142 of the buffer reagent. However, the buffer reagent can be cycled any number of times in the forward flow direction 140 and then the subsequent reverse flow direction 142. For example, the plurality of cycles can be 2, 4, 6, 12 or more cycles.

[0060] The plurality of cycles of the continuous forward flow direction 140 and reverse flow direction 142 during the wash phase 138 allow for a more complete removal (i.e., reagent exchange) of the cleavage reagent introduced into the system during the cleavage phase 136 than previous methods that utilized the same finite volume of buffer reagent. This is because in previous methods, the reagent flowed only in the forward direction during the wash phase. Therefore, in previous methods, the same buffer reagent passed through the reagent management system and the flow cell only once, and there was a possibility that the cleavage reagent was not sufficiently removed during the wash phase. Under these conditions, during a subsequent capture phase, the subsequent capture reagent can be contaminated with the remaining cleavage reagent. The remaining cleavage reagent can then prematurely remove the terminator from the nucleotide during the subsequent capture phase. This can potentially allow for an unwanted amount of prephasing, where more than one base binds to the growing second polynucleotide at once.

[0061] If prephasing allows accumulation every new cycle, then significant uncertainty in the order of bases in the polynucleotide can occur. The uncertainty can increase over each cycle and the accuracy of the data obtained can decrease as the polynucleotide chain grows. Thus, the total length of the second polynucleotide chain that can be grown can be limited by prephasing so that the order of the bases can be determined with a reasonable degree of accuracy.

[0062] In comparison, the multiple cycles in the forward direction 140 and reverse direction 142 of the present disclosure allow the same buffer reagent to pass through the reagent management system and the flow cell several times for more complete washing. In one example, by using multiple consecutive forward and reverse direction cycles as described herein, more efficient use of a finite volume of buffer reagent can be provided, particularly when it is not desirable to increase the volume of the buffer reagent, for example, due to space and / or cost constraints. As a result, less buffer reagent may be required to achieve a given level of reagent exchange.

[0063] As used herein, consecutive forward direction 140 and reverse direction 142 refer to the forward and reverse directions that occur adjacent to each other without intervening steps. The absence of intervening steps means that there is no substantial incubation period (or delay time) with no flow between the forward and reverse flow of the reagent.

[0064] Thus, the flow in the forward direction 140 and the flow in the reverse direction 142 should be separated by a delay time of about 1 second or less, about 200 milliseconds or less, or about 100 milliseconds or less for each cycle of the multiple cycles in the forward and reverse directions. Further, each cycle of the multiple cycles in the forward and reverse directions should be separated by a delay time of about 1 second or less, about 200 milliseconds or less, or about 100 milliseconds or less.

[0065] In one example, to prevent contamination of the reagent well 232 in the reagent management system 204 (best seen in FIG. 7), the volume of buffer reagent flowing in the forward direction 140 should be greater than the volume of buffer reagent flowing in the reverse direction. In other words, to prevent contamination of the reagent well during the wash phase: A first forward fluid volume (1 st FEV) of buffer reagent is flowed in the forward direction for each cycle of a plurality of cycles; and A second reverse fluid volume (2 nd RFV) of buffer reagent is flowed in the reverse direction for each cycle of a plurality of cycles, where the first forward fluid volume is greater than the second reverse fluid volume. More specifically, the first forward fluid volume (1 st FEV) may be about 3% or more greater than the second reverse fluid volume (2 nd RFV).

[0066] After a plurality of forward directions 140 and reverse directions 142 occur, the wash phase 138 proceeds to an incubation period 144. The incubation period 144 is a period during which there is substantially no flow of buffer reagent through the reagent management system 204 and the flow cell 202. The incubation period 144 may have a duration of about 10 seconds or more after the last cycle of the plurality of cycles 140, 142 occurs.

[0067] After the incubation period 144, the wash phase 138 proceeds to a third forward flow 146. That is, a third forward fluid volume (3 rd FFV) of buffer reagent is flowed in the forward direction after the incubation period 144. The incubation period 144 and the third forward flow 146 may help to diffuse the cleavage reagent from cracks that may occur at the edges of the reagent management system or the flow cell.

[0068] After completion of the wash phase 138, the method then returns to the capture phase 132 at 148 and begins the process of adding the next nucleotide 120 in the nucleotide sequence to the second polynucleotide 110.

[0069] In some existing methods, since the buffer reagent flows only in the forward direction during the washing phase, the total amount of buffer reagent consumed during the washing phase (total fluid consumption) is essentially equal to the total amount of buffer reagent volume swept through the reagent management system and the flow cell during the washing phase (total swept volume). In contrast, in the method of the present disclosure, the total swept volume can be many times larger than the total fluid consumption during the washing phase.

[0070] More specifically, during the period when the buffer reagent is flowing (washing phase), the total swept volume of the buffer reagent is given by the formula ((1 st FFV + 2 nd RFV)*N) + 3 rd and is substantially determined by FFV, where: 1 st FFV is the first forward fluid volume, 2 nd RFV is the second reverse fluid volume, N is the total number of cycles in a plurality of cycles, and 3 rd FFV is the third forward fluid volume. Furthermore, during the period when the buffer reagent is flowing (washing phase), the total fluid consumption of the buffer reagent is substantially determined by the following formula: ((1 st FFV - 2 nd RFV)*N) + 3 rd FFV The term "substantially determined" as used in the formulas of the foregoing type is used to account for and explain measurement errors that may occur when determining parameters such as 1 st FFV, 2 nd RFV, and 3 rd FFV.

[0071] Since the buffer reagent of the same volume can pass through the reagent management system and the flow cell several times, the ratio of the total swept volume of the buffer reagent to the total fluid consumption of the buffer reagent can be substantially greater than 1. More specifically, the ratio of the total swept volume of the buffer reagent to the total fluid consumption of the buffer reagent can be equal to or greater than about 6, about 10, about 12 or more.

[0072] In a cartridge based instrument system, the reagent management system and / or the flow cell can be positioned within the cartridge. The cartridge can be removably insertable into the instrument, i.e., the cartridge can be inserted into and removed from the instrument.

[0073] Thus, in a cartridge based instrument system, a large ratio of the total swept volume to the total fluid consumption (2, 6, 10, 12 or greater) can be particularly advantageous. This is because the capture reagent, cleavage reagent and buffer reagent can be stored in the reagent wells of the reagent management system, and the reagent management system can be part of the cartridge. Thus, the reagent wells of the reagent management system can be configured to store a finite total volume of each reagent to fit the cartridge. The large ratio of the total swept volume to the total fluid consumption allows the same finite volume of reagent to sweep through the reagent management system and the flow cell several times before being consumed, thus enhancing reagent exchange.

[0074] Referring to FIG. 4, an example of a flowchart of another method 150 for determining the nucleotide sequence in a polynucleotide according to the aspects disclosed herein is shown. Method 150 includes a capture phase 152, an imaging phase 154, a cleavage phase 156, and a wash phase 158.

[0075] During the capture phase 152, the capture reagent 160 is flowed through the reagent management system 204 and the flow cell 202 of the instrument 200 (best seen in FIG. 7). The capture reagent may be a mixture of chemicals that incorporate fluorescently labeled nucleotides into the DNA strand.

[0076] The flow cell 202 has a first polynucleotide 108 positioned therein (best seen in FIG. 2). The capture reagent adds a first base (associated with nucleotide 120-6) to a base sequence (associated with nucleotides 120-1 to 120-5). The base sequences (and the associated nucleotides 120-1 to 120-6) form a second polynucleotide 110 that is complementary to the first polynucleotide 108.

[0077] Also, the nucleotide 120 in the capture reagent also includes a tag (in this example, a fluorescent tag) that enables the identification of the base type using an excitation signal (in this example, excitation light). The nucleotide 120 also includes a cleavage terminator (or blocking group) known in the art, which prevents more than one base of the second polynucleotide 110 from pairing with the base of the first polynucleotide 108 at a time.

[0078] Next, during the capture phase 152, the wash reagent 162 is used to remove any excess capture reagent 160. The wash reagent can be a mixture of chemicals utilized to remove active reagents from the flow cell.

[0079] This method proceeds to the imaging phase 154, where the scan reagent 164 is used to protect the surface of the flow cell from optical damage before the image is taken. The scan reagent 164 can be a mixture of chemicals that stabilize the nucleotide strand during the detection process.

[0080] Next, during the imaging phase 154, after the first base of nucleotide 120-6 is added to the second polynucleotide 110, an image of the discrimination signal resulting from the first base of nucleotide 120-6 is captured 166.

[0081] More specifically, the fluorescent tag of nucleotide 120 associated with the second polynucleotide 110 is excited by the excitation light and emits characteristic fluorescence. Then, an image of the fluorescence is taken, and in the sequence of nucleotides 120-1 to 120-6 that form the growing second polynucleotide 110, discrimination information regarding the type of base of the newly added nucleotide 120-6 is provided.

[0082] Next, during the imaging phase 154, the additional tagging reagent 168 is used to add a second tag to the newly added nucleotide 120-6. The tagging reagent 168 can be a mixture of chemical substances utilized to deliver a fluorescent tag to the nucleotide.

[0083] Next, during the imaging phase 154, the washing reagent 170 is used to remove the excess tagging reagent 168. The washing reagent 170 can be a mixture of chemical substances utilized to remove the active reagent from the flow cell.

[0084] Next, during the imaging phase 154, the second scanning reagent 172 is applied to protect the surface of the flow cell from optical damage before the second image is taken. The second scanning reagent can be a mixture of chemical substances that stabilize the nucleotide chain during the detection process.

[0085] Next, during the imaging phase 154, a second image of the second discrimination signal resulting from the first base of nucleotide 120-6 is captured 174. More specifically, the second fluorescent tag of nucleotide 120 associated with the second polynucleotide 110 is excited by the excitation light and emits characteristic second fluorescence. Then, an image of the second fluorescence is taken, providing further discrimination information regarding the type of base of the newly added nucleotide 120-6.

[0086] The method proceeds to the cleavage phase 156, and the wash reagent 176 is used to remove any excess second scan reagent 172. The wash reagent can be a mixture of chemicals utilized to remove the active reagent from the flow cell.

[0087] Next, during the cleavage phase 156, the cleavage reagent 178 is flowed through the reagent management system 204 and the flow cell 202 to remove the terminator from the nucleotide 120-6. This enables the addition of subsequent nucleotides (not shown) in the sequence of nucleotides 120-1 to 120-6 to the second polynucleotide 110.

[0088] The method proceeds to the wash phase 158, and the first buffer reagent is flowed through the reagent management system 204 and the flow cell 202 in a plurality of cycles of consecutive forward and reverse flow directions to wash the cleavage reagent 178 from the reagent management system and the flow cell. The first buffer reagent can be, for example, buffered saline to which lipoic acid is added.

[0089] More specifically, a first forward fluid volume (1 st FFV) of 22 microliters (μL) of the first buffer reagent is flowed in the forward flow direction 180 to initiate each cycle of the plurality of cycles. Next, a second reverse fluid volume (2 nd RFV) of 19 microliters (μL) of the first buffer reagent is flowed in the reverse flow direction 182 to terminate each cycle of the plurality of cycles. In this specific example of FIG. 4, there are a total of 12 cycles of forward and reverse flow.

[0090] Next, during the wash phase 158, an incubation period 184 with substantially no flow of the first buffer reagent through the reagent management system 204 and the flow cell 202 is imposed on the system. The incubation period 184 in this particular example has a duration of about 30 seconds, although other incubation periods may be used.

[0091] After incubation period 184, wash phase 158 proceeds to the third forward flow 186. That is, the third forward volume (3 rd FFV) of 12 microliters (μL) of the first buffer reagent is flowed in the forward direction after incubation period 184.

[0092] The first buffer reagent is utilized in wash phase 158 to substantially remove cleavage reagent 178 from the system. However, certain chemical substances in the first buffer reagent that are effective in the removal of cleavage reagent 178 may also interfere with the capture reagent in the next cycle. One such example of such a chemical substance may be lipoic acid.

[0093] Accordingly, during wash phase 158, the forward flow of the second buffer reagent 188 is flowed through the reagent management system and the flow cell to remove the first buffer reagent. The second buffer reagent may be physiological saline without lipoic acid. The second buffer reagent is not important in the removal of the cleavage reagent, but does not interfere with the capture reagent 160 in the next cycle.

[0094] After completion of wash phase 158, the method then returns to capture phase 152 at 190 and initiates the process of adding the next nucleotide 120 in the nucleotide sequence to the second polynucleotide 110.

[0095] As previously described herein, during the wash phase, the total sweep volume of the first buffer reagent is given by the formula ((1 st FFV + 2 nd RFV) * N) + 3 rd FFV is substantially determined by, where: 1 st FFV is the first forward volume and is equal to 22 μL, 2 nd RFV is the second reverse volume and is equal to 19 μL, N is the total number of cycles in a plurality of cycles and is equal to 12, and 3 rd FFV is the third forward volume and is equal to 12 μL. Therefore, in the example of FIG. 4, the total sweep volume is equal to the following: (22 + 19)*12 + 12 = 504 μL

[0096] Furthermore, during the wash phase, the total fluid consumption of the first buffer reagent is substantially determined by the following formula: (1 st FFV + 2 nd RFV)*N + 3 rd FFV Furthermore, in the example of FIG. 4, the total fluid consumption is equal to the following: (22 - 19)*12 + 12 = 48 μL

[0097] Furthermore, in the example of FIG. 4, the ratio of the total sweep volume to the total fluid consumption is equal to 504 / 48 = 10.5. For comparison, the ratio of the total sweep volume to the total fluid consumption of the previous system is essentially 1. This is because the previous system utilizes only the forward flow of the buffer reagent that is effective for removing the cleavage reagent.

[0098] FIGS. 5 and 6 show results obtained from non - limiting examples.

[0099] Referring to FIG. 5, Table 190 showing examples of various ratios of the total sweep volume to the total fluid consumption of the first buffer reagent obtained during the exemplary wash phase 158 of method 150 is shown. In rows 190 - 1 through 190 - 7, the total fluid consumption was held constant at 48 μL. Also, in rows 190 - 2 through 190 - 7, the first forward fluid volume (1 st FFV), the second reverse fluid volume (2 nd RFV), the number of cycles (N), and the third forward fluid volume (3 rd FFV) were varied such that the total sweep volume increased from 100 μL to 1200 μL while the total fluid consumption was held constant at 48 μL. As can be seen, the ratio of the total sweep volume to the total fluid consumption varied from 2.08 (row 190 - 2) to 25 (190 - 7).

[0100] Row 190-1 above represents a comparative example of the previous method and there were no cycles of backflow and forward flow of the buffer reagent. Instead, row 190-1 had only forward flow during the wash phase, and thus its total sweep volume was equal to its total fluid consumption.

[0101] Referring to FIG. 6, graphs 192, 194 of the ratio of total sweep volume to total fluid consumption plotted from the data obtained in the working example of FIG. 5 according to the embodiments disclosed herein against the prephasing gradient are shown. Graph 194 was plotted at ambient temperature (e.g., ambient temperature within the range of 25-35 °C). Graph 192 was plotted at a temperature 8 °C higher than the ambient temperature (e.g., a temperature within the range of 33-43 °C).

[0102] As shown in graphs 192 and 194, the prephasing gradient is expressed as the percentage (%) of molecules in a cluster of polynucleotides in a flow cell advancing 1 base pair per cycle. The prephasing gradient is a measure of the rate of prephasing per cycle.

[0103] The lower the prephasing value, the better the quality of the data at a given polynucleotide length during the processing of the method herein. Further, the lower the prephasing value, the longer the polynucleotide that can be grown.

[0104] As can be determined from graphs 192 and 194, the optimal range of the ratio of total sweep volume to total fluid consumption at which the lowest prephasing gradient can be achieved is equal to or greater than about 6. Also, an alternative optimal range that can be determined from graphs 192, 194 of the plotted data of the example of FIG. 5 is from about 6 to about 12.

[0105] The optimal range is related to a low pre-phasing gradient. In most cases in the embodiment of FIG. 6, the optimal range is related to a pre-phasing gradient ratio that is less than 2.0%. Such a low ratio of pre-phasing gradient allows the complementary second polynucleotide 110 to construct a base length longer than that previously obtained with a finite amount of reagent within an acceptable accuracy, as provided by the surprising results obtained from the embodiments of FIGS. 5 and 6. In the embodiments of FIGS. 5 and 6, the cartridge-based instrument system was able to accurately construct the second complementary polynucleotide 110 up to a length of 200 bases, or at a higher percentage of time (e.g., 90% or more of the time). Thus, the base pairs of the first polynucleotide 108 and the second polynucleotide 110 could also be accurately constructed up to a length of 200 base pairs or more at the same high percentage of time.

[0106] Referring to FIG. 7, an example of a schematic diagram of an instrument 200 according to the aspects described herein is shown. In this particular example, the instrument includes a cartridge 230 engaged therewith. The cartridge 230 includes a flow cell 202 in fluid communication with a reagent management system (RMS) 204. In this particular example, the RMS 204 is in fluid communication with the flow cell 202 via an intermediate connection 206. The cartridge 230 may be removably insertable into the instrument 200. This means that the cartridge can be inserted into and removed from the instrument.

[0107] Although the example of FIG. 7 shows a cartridge-based instrument 200 having an RMS 204 and a flow cell 202 included in the cartridge 230, other instruments 200 may not include such a cartridge-based system. Rather, in some instruments 200, the components of the RMS 204 may be integrally and firmly attached within the instrument 200, and only the flow cell 202 may be removable from the instrument 200. Further, the flow cell 202 may or may not be removable from the cartridge 230.

[0108] The RMS includes a plurality of reagent wells 232. Each reagent well 232 is operable to contain a plurality of reagents 208-218 positioned therein. The RMS 204 is operable to select a reagent flow 234 from one of the plurality of reagents 208-218.

[0109] Also, the RMS is operable to direct the reagent flow 234 in both forward and reverse directions. As used herein, the forward flow refers to the direction of the reagent flow 234 from the reagent well 232 to the flow cell 202. As used herein, the reverse flow refers to the direction of the reagent flow 234 from the flow cell 202 to the reagent well 232.

[0110] The reagents 208-218 may be any several types or combinations of reagents, depending on the type and order of the chemical reactions carried out in the flow cell. For example, the reagents 208-218 may be of the following types: The reagents 208 and 209 may be different formulations (or preparations) of the capture reagent. The reagents 210 and 211 may be different formulations of the scan reagent. The reagent 212 may be a cleavage reagent. The reagents 214 and 216 may be different formulations of the buffer reagent. The reagent 218 may be air.

[0111] The connection 206 includes a first channel 236 that is in fluid communication with the RMS 204 via the RMS outlet port 256. The first channel 236 is operable to route the reagent flow 234 to the flow channel 224 of the flow cell 202 via the inlet port 220 of the flow cell 202. Also, the connection 206 includes a second channel 238 that is in fluid communication with the flow channel 224 via the outlet port 222 of the flow cell 202. The second channel 238 is operable to route the reagent flow 234 that returns from the flow cell 202 to the RMS 204 via the RMS inlet port 258 after the reagent flow 234 has passed through the flow channel 224.

[0112] The flow cell 202 of the machine 200 includes a flow channel 224 that is in fluid communication with a first channel 236 via an inlet port 220 and in fluid communication with a second channel 238 via an outlet port 222. The flow channel 224 is operable to effect various chemical reactions between various reagent flows 234 from a plurality of reagents 208-218 and a polynucleotide such as the polynucleotide 108 of FIG. 2 positioned in the flow cell 224.

[0113] The example of FIG. 7 illustrates a flow cell 202 having a single inlet port 220 and a single outlet port 222, although other configurations of the flow cell may be utilized. For example, the flow cell 202 may include a plurality of inlet ports 220 for receiving reagent flows from a plurality of channels of the connection 206. Also, by way of example, the flow cell may include a plurality of outlet ports 222 for sending reagent flows to a plurality of channels of the connection 206.

[0114] The example of FIG. 7 shows a flow cell 202 in fluid communication with the RMS 204 via an intermediate connection 206, although the flow cell 202 may alternatively be directly connected to the RMS 204 without having the connection 206 therebetween. That is, the RMS outlet port 256 may be directly connected to the flow cell inlet port 220, and the RMS inlet port 258 may be directly connected to the flow cell outlet port 222.

[0115] In this example, the RMS 204 includes a rotary valve 242 for selecting the reagents 208-218. The rotary valve 242 has an internal rotary valve body 244. The valve body 244 includes a central port 246 and a rotatable port 248 that are connected by a rotary channel 250. The valve body 244 pivots around the central port 246 and moves the rotatable port 248.

[0116] A plurality of reagent wells 232 containing reagents 208 - 218 may be arranged around the periphery of the rotary valve 242 or may be arranged away from the rotary valve 242. Each reagent well 232 is in fluid communication with a corresponding well channel 252. Each well channel 252 includes a well channel port 254 that can align with a rotatable port 248 of the rotary valve 242 to receive the reagent flow 234 from any given reagent well 232.

[0117] When the rotatable port 248 aligns with one of the well channel ports 254, by establishing a flow path for the reagent flow 234, the reagent flow 234 can flow from the selected well 232 through the well channel 252, through the rotary valve 242, through the common line 255, and out of the RMS outlet port 256. Then, the reagent flow 234 passes through the first channel 236, enters the inlet port 220 of the flow cell 202, continues through the flow channel 224, and the selected reagent among the plurality of reagents 208 - 218 can react with the polynucleotide 108.

[0118] Unreacted reagents and / or reaction by - products may flow out from the outlet port 222 of the flow cell 202 through the second channel 238. Then, the reagent flow 234 may re - enter the RMS 204 through the RMS inlet port 258.

[0119] The RMS inlet port 258 of the RMS 204 is in fluid communication with a first pinch valve. The first pinch valve 260 is in fluid communication with a second pinch valve 262. The first pinch valve 260 and the second pinch valve 262 include an elastic central portion that can be actuated mechanically or pneumatically to pinch off or release the reagent flow 234 passing through the pinch valves 260, 262. Further, although pinch valves 260, 262 are illustrated in this example, other types of valves may be utilized to perform the same function. For example, valves 260, 262 may be rotary valves.

[0120] Also, a mounted pump 264 (such as a syringe pump or the like) is also arranged on the RMS 204. Although the mounted pump 264 may be other types of pumps, it is referred to as the syringe pump 264 in this specification. The syringe pump 264 is connected in a tee formation between the first pinch valve 260 and the second pinch valve 262. Both of the pinch valves 260 and 262 are opened and closed by the device 200 to engage or disengage the syringe pump 264 with the flow cell 202 and / or the waste tank 270.

[0121] The syringe pump 264 includes a reciprocating plunger 266 arranged in a cylinder 268 having a cylinder bore (or cylinder hole, cylinder bore) 270. The plunger 266 is received within the cylinder bore to form a seal of the plunger - cylinder bore. The plunger 266 is driven by the device 200 to reciprocate within the cylinder bore 270, and pumps and transports the reagents 208 - 218 in the downstream direction from the reagent well 232 to the waste tank 272.

[0122] In addition to the device 200 being operable to pump and transport the reagent flow 234 in the downstream direction from the reagent well 232 to the flow cell 202 and to the pump 264, the instrument 200 may be operable to pump and transport the reagent flow 234 in the reverse direction. That is, the reagents 208 - 218 may be pumped and transported in the reverse flow direction from the pump 264 to the flow cell 202 and to the reagent well 232. Therefore, the device is operable to flow the buffer reagents 214, 216 through the RMS 204 and the flow cell 202 in a plurality of consecutive downstream and reverse flow cycles, for example, according to the method 130 of FIG. 3 and 150 of FIG. 4.

[0123] Also, when the chemical reaction caused by the reagents 208 - 218 is induced to affect the detectable characteristics of the polynucleotide 108, the device 200 includes a detection module 226 operable to detect photons of light or other forms of detectable characteristics.

[0124] The implementation form shown in FIG. 7 is an implementation form of the device 200 that uses a rotary valve 242 to route various reagents 208-218 to the flow cell 202 via a common line 255, but other devices 200 do not have to use the rotary valve 242. For example, the well channels 252 from each reagent well 232 may extend directly to one of a plurality of separate RMS outlet ports 256.

[0125] In that case, in order to control the reagent flow 234 from each reagent well 232, the well channels 252 may each include a valve (not shown). Further, the first channels 236 may be a plurality of first channels for receiving the corresponding reagent flows 234 from the corresponding RMS outlet ports 256. Further, the inlet port 220 of the flow cell 202 may be a plurality of inlet ports 220 for receiving various reagent flows 234 from each of the plurality of first channels 236.

[0126] Referring to FIG. 8, an example of a schematic block diagram of the device 200 of FIG. 7 is shown. The device 200 includes a docking station 274 for receiving the cartridge 230. Various electrical and mechanical assemblies within the device 200 interact with the cartridge 230 in order to manipulate the cartridge during the microfluidic analysis operation of the various chemical reactions performed in the flow cell 202.

[0127] The device 200 includes, among other things, one or more processors 276 that will execute program instructions stored in the memory 278 to perform the microfluidic analysis operation. The processor communicates electronically with a rotary valve drive assembly 280, a syringe pump drive assembly 282, a pinch valve drive assembly 284, a detection module 226, and a temperature adjustment assembly 306.

[0128] The user interface 286 is provided for the user to control and monitor the operation of the device 200. The communication interface 288 can transmit data and other information between the device 200 and a remote computer, network, etc.

[0129] The rotary valve drive assembly 280 includes a drive shaft 290 that is mechanically coupled to a rotary valve interface bracket 292. The rotary valve interface bracket 292 is selectively and mechanically coupled to the rotary valve 242 of the cartridge 230. The rotary valve drive assembly 280 includes a rotary motor 294 and, in some implementations, a translational motor 296. The translational motor 296 can move the drive shaft 290 in a translational direction between an engaged state and a disengaged state with the rotary valve 242. The rotary motor 294 manages the rotation of the rotary valve body 244 of the rotary valve 242.

[0130] Also, the rotary valve drive assembly 280 includes a position encoder 298 that monitors the position of the drive shaft 290. The encoder 298 provides position data to the processor 276.

[0131] The syringe pump drive assembly 282 includes a syringe pump motor 300 coupled to an extendable shaft 302. The shaft 302 is driven by the syringe pump motor 300 between an extended position and a retracted position, causing the plunger 366 to reciprocate within the cylinder bore 270 of the cylinder 268 of the syringe pump 264.

[0132] The pinch valve drive assembly 284 includes a set of two pneumatically driven pinch valve drive motors 304. The two pinch valve drive motors 304 are mechanically coupled to the corresponding one of the first pinch valve 260 and the second pinch valve 262. The pinch valve drive motor 304 may pneumatically open and close the first pinch valve 260 and / or the second pinch valve 262 by utilizing pneumatic pressure to pinch off or release the elastic central portion of the first pinch valve 260 and / or the second pinch valve 262. Alternatively, the pinch valve drive motor 304 may be electrically driven.

[0133] The detection module 226 may include all of the cameras and / or detection sensors appropriate and / or necessary to enable the detection of emitted photons related to the polynucleotide 108 in the flow cell 202, or other forms of detectable characteristics. Then, device circuitry (not shown) within the instrument 200 may process and transmit data signals resulting from these detected emissions. The data signals may then be analyzed to reveal characteristics of the polynucleotide 108.

[0134] Also, a temperature regulation assembly 306 (or other environmental control device) may be included within the instrument 200. The temperature regulation assembly 306 may be utilized to provide temperature control of the flow cell 202 during various chemical reactions. More specifically, both heating and cooling of the flow cell 202 are provided by the temperature regulation assembly 306, thereby enabling thermal cycling of the flow cell 202. The environmental control device may control or adjust parameters other than temperature (e.g., pressure).

[0135] It should be understood that all combinations of the foregoing concepts and the concepts described hereinafter, as more particularly described herein (subject to the condition that such concepts are not mutually inconsistent), are considered to be part of the subject matter of the invention disclosed herein. In particular, all combinations of the claimed subject matter at the end of this disclosure are considered to be part of the subject matter of the invention disclosed herein.

[0136] Although the foregoing disclosure has been described with reference to specific exemplifications, it should be understood that many variations can be made within the spirit and scope of the concepts of the invention described. Accordingly, it is intended that this disclosure not be limited to the exemplifications described, but rather have the full scope defined by the language of the claims that follow. Preferred embodiments of the present invention are as follows. [1] Flowing an uptake reagent through a reagent management system of a device and a flow cell, the flow cell having a first polynucleotide positioned therein, the uptake reagent adding a first base to a base sequence, the base sequence comprising a second polynucleotide complementary to the first polynucleotide; Capturing an image of an identification signal resulting from the first base after the first base has been added to the second polynucleotide; Flowing a cleavage reagent through the reagent management system and the flow cell to remove a first terminator from the first base to enable addition of subsequent bases in the base sequence to the second polynucleotide; and Flowing a buffer reagent through the reagent management system and the flow cell in a plurality of successive forward and reverse cycles comprising a method. [2] The method according to [1], wherein the plurality of cycles comprises 4 or more cycles. [3] The method according to [1], wherein the plurality of cycles comprises 12 or more cycles. [4] The method according to any one of [1] to [3], wherein the reagent management system and the flow cell are positioned within a cartridge of the device, the cartridge being removably insertable into the device. [5] The method according to any one of [1] to [4], wherein the uptake reagent, the cleavage reagent and the buffer reagent are stored in reagent wells of the reagent management system, the reagent wells being operable to store a finite total volume of each reagent. [6] Flowing the buffer reagent comprises flowing a first forward fluid volume of the buffer reagent in the forward direction of each cycle of the plurality of cycles; and flowing a second reverse fluid volume of the buffer reagent in the reverse direction of each cycle of the plurality of cycles wherein the first forward fluid volume is greater than the second reverse fluid volume, according to any one of [1] to [5]. [7] The method according to [6], wherein the forward fluid volume is greater than the reverse fluid volume by about 3% or more. [8] Flowing the buffer reagent comprises an incubation period of about 10 seconds or more after the last cycle of the plurality of cycles, during which there is substantially no flow of the buffer reagent through the reagent management system and the flow cell; and after the incubation period, flowing a third forward fluid volume of the buffer reagent in the forward direction comprising a method according to any one of [1] to [7]. [9] While flowing the buffer reagent, the total sweep volume of the buffer reagent is given by the formula ((1 st FFV + 2 nd RFV)*N) + 3 rd Substantially determined by FFV, where 1 st FFV is the first-order fluid volume, 2 nd RFV is the second reverse fluid volume, N is the total number of cycles in a plurality of cycles, and 3 rd FFV is the third-order fluid volume is the method described in [8].

[10] While flowing the buffer reagent, the total fluid consumption of the buffer reagent is given by the formula ((1 st FFV - 2 nd RFV)*N) + 3 rd substantially determined by FFV, the method described in [9].

[11] The ratio of the total sweep volume of the buffer reagent to the total fluid consumption of the buffer reagent is equal to or greater than about 6, the method described in

[10] .

[12] The flow in the forward direction and the flow in the reverse direction are separated by a delay time of about 1 second or less for each cycle of a plurality of cycles in the forward and reverse directions, the method according to any one of [1] to

[11] .

[13] The flow in the forward direction and the flow in the reverse direction are separated by a delay time of about 200 milliseconds or less for each cycle of a plurality of cycles in the forward and reverse directions, the method according to any one of [1] to

[11] .

[14] Each cycle of a plurality of cycles in the forward and reverse directions is separated by a delay time of about 200 milliseconds or less, the method according to any one of [1] to

[13] .

[15] An apparatus comprising a reagent management system positioned within the apparatus; and a flow cell positioned within the apparatus comprising The reagent management system comprises a plurality of reagent wells, the reagent wells having at least a first reagent well, a second reagent well, and a third reagent well, the first reagent well, the second reagent well, and the third reagent well being operable to contain a capture reagent, a cleavage reagent, and a buffer reagent respectively positioned therein, and the reagent management system is for selecting the flow of reagent from one of the plurality of reagent wells, The flow cell comprises a flow channel in fluid communication with the reagent management system, the flow channel being for routing the flow of reagent over a first polynucleotide positioned in the flow channel, The apparatus flows a capture reagent through the reagent management system and the flow cell to add a first base to a base sequence comprising a second polynucleotide complementary to the first polynucleotide; after the first base is added to the second polynucleotide, captures an image of the discrimination signal resulting from the first base; To remove the first terminator from the first base so that subsequent bases in the base sequence can be added to the second polynucleotide, a cleavage reagent is flowed through the reagent management system and the flow cell; and In a plurality of consecutive forward and reverse cycles, a buffer reagent is flowed through the reagent management system and the flow cell, An apparatus that is operable.

[16] The apparatus according to

[15] , wherein the apparatus is operable to flow a buffer reagent through the reagent management system and the flow cell in a plurality of 4 or more consecutive forward and reverse cycles.

[17] The apparatus according to

[15] , wherein the apparatus is operable to flow a buffer reagent through the reagent management system and the flow cell in a plurality of 12 or more consecutive forward and reverse cycles.

[18] The apparatus according to any one of

[15] to

[17] , wherein the apparatus is operable to flow a buffer reagent such that the forward flow and the reverse flow are separated by a delay time of about 200 milliseconds or less for each cycle of the plurality of forward and reverse cycles.

[19] The apparatus according to any one of

[15] to

[18] , wherein the reagent management system is removably insertable into the apparatus.

[20] The apparatus according to any one of

[15] to

[19] , wherein the flow cell is removably insertable into the apparatus.

[21] The apparatus is A cartridge for housing the reagent management system and the flow cell And comprises The capture reagent, the cleavage reagent, and the buffer reagent are stored in the reagent wells of the reagent management system, and the reagent wells store a finite total volume of each reagent. The apparatus according to

[15] to

[20] .

[22] The apparatus according to

[21] , wherein the cartridge is removably insertable into the apparatus.

[23] The apparatus is A first volume of the buffer reagent is flowed in the forward direction for each cycle of the plurality of cycles; A second volume of the buffer reagent is flowed in the reverse direction for each cycle of the plurality of cycles The apparatus is operable to flow a buffer reagent such that The first volume is larger than the second volume. The apparatus according to any one of

[15] to

[22] .

[24] The apparatus according to

[23] , wherein the forward volume is about 3% or more larger than the reverse volume.

[25] The apparatus is After the last cycle of the plurality of cycles, an incubation period of about 10 seconds or more is imposed during which there is substantially no flow of the buffer reagent through the reagent management system and the flow cell; and After the incubation period, a third forward volume of the buffer reagent is flowed in the forward direction The device as described in

[23] or

[24] , which is operable to flow a buffer reagent.

[26] The device is such that the total sweep volume of the buffer reagent is substantially determined by the formula ((1 st FFV + 2 nd RFV) * N) + 3 rd FFV, where 1 st FFV is the first forward fluid volume, 2 nd RFV is the second reverse fluid volume, N is the total number of cycles in a plurality of cycles, and 3 rd FFV is the third forward fluid volume; the total fluid consumption of the buffer reagent is substantially determined by the formula ((1 st FFV - 2 nd RFV) * N) + 3 rd FFV; and such that the ratio of the total sweep volume of the buffer reagent to the total fluid consumption of the buffer reagent is equal to or greater than about 6, the device as described in

[25] , which is operable to flow a buffer reagent.

Claims

1. A reagent management system for a machine and flowing an intake reagent through a flow cell, the flow cell having a first polynucleotide positioned within the flow cell, the intake reagent adding a first base to a nucleotide sequence, the nucleotide sequence comprising a second polynucleotide complementary to the first polynucleotide; After the first base is added to the second polynucleotide, capturing an image of an identification signal resulting from the first base; Flowing a cleavage reagent through the reagent management system and the flow cell to remove a first terminator from the first base to enable addition of subsequent bases in the nucleotide sequence to the second polynucleotide; and Flowing a buffer reagent through the reagent management system and the flow cell in a plurality of successive forward and reverse cycles comprising: Flowing the buffer reagent comprises flowing a first forward fluid volume of the buffer reagent in the forward direction of each cycle of the plurality of cycles; and Flowing a second reverse fluid volume of the buffer reagent in the reverse direction of each cycle of the plurality of cycles comprising: A method for determining the order of nucleotides in a polynucleotide, wherein the first forward fluid volume is greater than the second reverse fluid volume.

2. The method according to claim 1, wherein the plurality of cycles includes 4 or more cycles.

3. The method according to claim 1, wherein the plurality of cycles includes 12 or more cycles.

4. The method according to any one of claims 1 to 3, wherein the reagent management system and the flow cell are positioned within a cartridge of the machine, and the cartridge is removably insertable into the machine.

5. The method according to any one of claims 1 to 4, wherein the intake reagent, the cleavage reagent, and the buffer reagent are stored in reagent wells of the reagent management system, and the reagent wells are operable to store a finite total volume of each reagent.

6. The method according to any one of claims 1 to 5, wherein the forward fluid volume is at least 3% greater than the reverse fluid volume.

7. Flowing the buffer reagent includes an incubation period of at least 10 seconds after the last cycle of the plurality of cycles, during which there is substantially no flow of the buffer reagent through the reagent management system and the flow cell; and After the incubation period, flowing a third forward fluid volume of the buffer reagent in the forward direction The method according to any one of claims 1 to 6.

8. While flowing the buffer reagent, the total sweep volume of the buffer reagent is given by the formula ((1 ｓｔ FFV + 2 ｎｄ RFV) * N) + 3 ｒｄ is substantially determined by FFV, where 1 ｓｔ FFV is the first-order fluid volume, 2 ｎｄ RFV is the second inverse fluid volume, where N is the total number of all cycles in the plurality of cycles, and 3 ｒｄ FFV is the third-order fluid volume The method according to claim 7.

9. While flowing the buffer reagent, the total fluid consumption of the buffer reagent is given by the formula ((1 ｓｔ FFV - 2 ｎｄ RFV) * N) + 3 ｒｄ The method according to claim 8, substantially determined by FFV.

10. The method according to claim 9, wherein the ratio of the total swept volume of the buffer reagent to the total fluid consumption of the buffer reagent is equal to or greater than 6.

11. The method according to any one of claims 1 to 10, wherein the flow in the forward direction and the flow in the reverse direction are separated by a delay time of 1 second or less for each cycle of a plurality of cycles in the forward direction and the reverse direction.

12. The method according to any one of claims 1 to 10, wherein the flow in the forward direction and the flow in the reverse direction are separated by a delay time of 200 milliseconds or less for each cycle of a plurality of cycles in the forward direction and the reverse direction.

13. The method according to any one of claims 1 to 12, wherein each cycle of a plurality of cycles in the forward direction and the reverse direction is separated by a delay time of 200 milliseconds or less.

14. An apparatus comprising: a reagent management system positioned within the apparatus; and a flow cell positioned within the apparatus wherein: the reagent management system comprises a plurality of reagent wells, the reagent wells comprising at least a first reagent well, a second reagent well, and a third reagent well, the first reagent well, the second reagent well, and the third reagent well being operable to contain a capture reagent, a cleavage reagent, and a buffer reagent respectively positioned therein, the reagent management system being for selecting a flow of reagent from one of the plurality of reagent wells; the flow cell comprises a flow channel in fluid communication with the reagent management system, the flow channel being for routing a flow of reagent over a first polynucleotide positioned in the flow channel; the apparatus is operable to: flow a capture reagent through the reagent management system and the flow cell to add a first base to a base sequence comprising a second polynucleotide complementary to the first polynucleotide; capture an image of an identification signal resulting from the first base after the first base has been added to the second polynucleotide; flow a cleavage reagent through the reagent management system and the flow cell to remove a first terminator from the first base so as to enable addition of subsequent bases in the base sequence to the second polynucleotide; and flow a buffer reagent through the reagent management system and the flow cell in a plurality of successive forward and reverse cycles, wherein a first volume of the buffer reagent is flowed in the forward direction for each cycle of the plurality of cycles. Operable to flow the buffer reagent such that a second volume of the buffer reagent flows in a reverse flow direction for each of a plurality of cycles and is operable; A device in which a first forward fluid volume is greater than a second reverse fluid volume. **Claim 15** The device according to claim 14, operable to flow a buffer reagent through a reagent management system and a flow cell in a plurality of four or more cycles of successive forward and reverse flow directions. **Claim 16** The device according to claim 14, operable to flow a buffer reagent through a reagent management system and a flow cell in a plurality of twelve or more cycles of successive forward and reverse flow directions. **Claim 17** The device according to any one of claims 14 to 16, operable to flow the buffer reagent such that for each of a plurality of cycles in a forward flow direction and a reverse flow direction, the forward flow and the reverse flow are separated by a delay time of 200 milliseconds or less. **Claim 18** The device according to any one of claims 14 to 17, wherein the reagent management system is removably insertable into the device. **Claim 19** The device according to any one of claims 14 to 18, wherein the flow cell is removably insertable into the device. **Claim 20** The device has a cartridge for housing a reagent management system and a flow cell, and is configured such that an intake reagent, a cleavage reagent, and a buffer reagent are stored in reagent wells of the reagent management system, and the reagent wells store a finite total volume of each reagent. The device according to any one of claims 14 to 19. **Claim 21** The device according to claim 20, wherein the cartridge is removably insertable into the device. **Claim 22** The device according to any one of claims 14 to 21, wherein the forward fluid volume is at least 3% greater than the reverse fluid volume. **Claim 23** The device is operable such that after a last cycle of a plurality of cycles, an incubation period of at least 10 seconds is imposed during which there is substantially no flow of the buffer reagent through the reagent management system and the flow cell; and after the incubation period, the buffer reagent is flowed such that a third forward fluid volume of the buffer reagent flows in the forward flow direction. The device according to any one of claims 14 to 22. **Claim 24** The device The total sweep volume of the buffer reagent is given by the formula ((1 ｓｔ FFV + 2 ｎｄ RFV) * N) + 3 ｒｄ is substantially determined by the FFV, where 1 ｓｔ FFV is the first-order fluid volume, 2 ｎｄ RFV is the second inverse fluid volume, where N is the total number of cycles in a plurality of cycles, and 3 ｒｄ FFV is the third-order fluid volume; The total fluid consumption of the buffer reagent is given by the formula ((1 ｓｔ FFV - 2 ｎｄ RFV) * N) + 3 ｒｄ is substantially determined by FFV; and the ratio of the total sweep volume of the buffer reagent to the total fluid consumption of the buffer reagent is equal to or greater than 6. The apparatus according to claim 23, which is operable to flow a buffer reagent.

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