Hybridization and capture device, method and apparatus for hybridization and capture of libraries

Abstract

Embodiments of the present disclosure relate to hybridization and capture devices, methods and apparatus for library hybridization and capture. The hybridization and capture device comprises: a reaction table comprising a table surface extending in a first plane, the table surface being provided with a plurality of groups of grooves; and a set of electromagnetic bars configured to: in a non-temperature-sensitive operating phase: perform a blending operation in a non-magnetized and non-heated state, and perform a magnetic transfer operation in a magnetized but non-heated state, the magnetic transfer operation including transferring capture magnetic beads for capturing the library from one of the plurality of sets of slots to another of the plurality of sets of slots; and in a temperature-sensitive operating phase: in a non-magnetic but heating state, performing a blending operation, and in a magnetic and heating state, performing a magnetic transfer operation. In this way, the accuracy and manufacturing difficulty required by the instrument are reduced by using a relatively simple structure of an electromagnetic rod capable of heating and magnetizing and a reaction table for containing a reaction liquid.

Description

Technical Field

[0001] The present application relates to the technical field of clinical medical devices, and in particular to a hybridization and capture device, and a method and apparatus for library hybridization and capture. Background Art

[0002] Methylation sequencing is a high-throughput sequencing technology used to detect DNA methylation status and has important applications in early cancer screening. DNA methylation is a key epigenetic modification that usually occurs on CpG dinucleotides and has a significant impact on the regulation of gene expression. Through methylation sequencing, early tumor-related methylation changes can be detected, thereby providing a highly sensitive and specific early screening method. This technology also has the advantage of being non-invasive and can extract free DNA from blood or urine for testing through liquid biopsy. It is widely applicable to a variety of cancer types and can dynamically monitor tumor progression and treatment effects. Summary of the invention

[0003] In the first aspect of the present disclosure, a hybridization and capture device is provided. The device comprises: a reaction table, comprising a table extending in a first plane, on which a plurality of slots are provided; and a group of electromagnetic rods, configured to: in a non-temperature-sensitive operation phase: perform a mixing operation in a non-magnetic and non-heating state, and perform a magnetic transfer operation in a magnetized but non-heating state, wherein the magnetic transfer operation comprises transferring capture magnetic beads for capturing a library from one group of slots in the plurality of slots to another group of slots; and in a temperature-sensitive operation phase: perform a mixing operation in a non-magnetic but heated state, and perform a magnetic transfer operation in a magnetized and heated state.

[0004] In the second aspect of the present disclosure, a method for library hybridization and capture is provided. The method comprises: using the hybridization and capture device according to the first aspect of the present disclosure, sequentially performing hybridization reaction, magnetic bead washing, magnetic bead resuspension, library capture reaction, hot washing and room temperature washing, wherein multiple groups of grooves in the hybridization and capture device contain corresponding reagents, wherein the non-temperature-sensitive operation stage comprises: magnetic bead washing and room temperature washing, and the temperature-sensitive operation stage comprises: library capture reaction, magnetic bead resuspension and hot washing process.

[0005] In a third aspect of the present disclosure, a device for library hybridization and capture is provided. The device includes at least one processing unit and at least one memory. Here, the at least one memory is coupled to the at least one processing unit and stores instructions for execution by the at least one processing unit. The instructions, when executed by the at least one processing unit, cause the device to execute a method for library hybridization and capture according to the second aspect of the present disclosure.

[0006] It should be understood that the contents described in the summary of the invention are not intended to limit the key or important features of the embodiments of the present disclosure, nor are they intended to limit the scope of the present disclosure. Other features of the present disclosure will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In order to better understand the above and other purposes, features, advantages and functions of the present invention, reference may be made to the preferred embodiments shown in the accompanying drawings. The same reference numerals in the accompanying drawings refer to the same components. It should be understood by those skilled in the art that the accompanying drawings are intended to schematically illustrate the preferred embodiments of the present invention and have no limiting effect on the scope of the present invention, and the components in the drawings are not drawn to scale.

[0008] Figure 1A A schematic diagram showing an example device for library hybridization and capture according to an embodiment of the present disclosure is shown;

[0009] Figure 1B shows a schematic exploded view of an example hybridization and capture device according to an embodiment of the present disclosure;

[0010] Figure 1C shows a schematic diagram of an example hybridization and capture device according to an embodiment of the present disclosure;

[0011] Figures 2A-2D A schematic diagram showing example components of a hybridization and capture device according to an embodiment of the present disclosure;

[0012] Figure 3 A flow chart showing an example method for library hybridization and capture according to an embodiment of the present disclosure is shown;

[0013] Figures 4A-4G A schematic diagram showing various exemplary states of the hybridization and capture device during library hybridization and capture according to an embodiment of the present disclosure; and

[0014] Figure 5 A schematic diagram showing the comparison of core indicators of different miscellaneous capture methods. DETAILED DESCRIPTION

[0015] Various embodiments are now described with reference to the accompanying drawings, wherein throughout the text, similar reference numerals are used to refer to similar elements. In the following description, for the purpose of explanation, many specific details are set forth to facilitate a thorough understanding of one or more embodiments. However, it may be clear in some or all cases that any embodiment described below can be practiced without adopting the specific design details described below. In other examples, known structures and devices are shown in block diagram form to facilitate description of one or more embodiments. A simplified overview of one or more embodiments is given below to provide a basic understanding of the embodiments. This overview is not an exhaustive overview of all contemplated embodiments, is not intended to identify the key or important elements of all embodiments, and is not intended to define the scope of any or all embodiments.

[0016] References to "an embodiment" or "one embodiment" in the framework of this description are intended to indicate that a particular configuration, structure, or characteristic described with respect to the embodiment is included in at least one embodiment. Thus, phrases such as "in an embodiment" or "in an embodiment" that may be present in one or more points of this description do not necessarily refer to the same embodiment. Furthermore, in one or more embodiments, particular configurations, structures, or characteristics may be combined in any appropriate manner.

[0017] In the following disclosure, unless otherwise indicated, when referring to absolute position modifiers (such as terms "front", "back", "top", "bottom", "left", "right", etc.) or relative position modifiers (such as terms "above", "below", "higher", "lower", etc.), or when referring to directional modifiers (such as "horizontal", "vertical", etc.), it refers to the orientation shown in the figure.

[0018] As discussed above, although methylation sequencing has shown great potential in early cancer screening, current technology still faces multiple challenges. First, the cost of whole-genome methylation sequencing is high and difficult to apply to large-scale population screening. Secondly, the sample processing and data analysis process of methylation sequencing is complicated, requiring professional equipment and high-level technical support. In addition, the huge amount of data also poses a huge challenge to bioinformatics analysis. Therefore, targeted methylation sequencing has become a highly regarded alternative. This method captures pre-selected methylated regions through probe hybridization and then performs sequencing. It not only has high specificity and sensitivity, but also significantly reduces the cost and difficulty of data processing.

[0019] However, there are currently few types of equipment that can realize hybridization capture automation, and they mainly rely on expensive pipetting workstations, such as Agilent Bravo, PerkinElmer Sciclone G3, Beckman Coulter Biomek and HamiltonNGS STAR. These workstations cost millions of yuan, and in order to achieve a fully automated hybridization capture process, it is usually necessary to add expensive desktop thermal cycler modules (ODTC) and constant temperature oscillation incubation modules. These modules are used for PCR operations, hot washing and mixing, respectively, and automatic addition and transfer of reagents are achieved through integrated pipetting modules. However, these additional modules not only further increase the cost of the equipment, but also increase the size and complexity of the workstation, resulting in cumbersome operations and limiting the popularity of hybridization capture technology.

[0020] Although the existing hybridization capture equipment based on pipetting workstations can achieve a certain degree of automation, its high price, large size and complex structure greatly limit its wide application in scientific research and clinical practice. Therefore, there is an urgent need for a low-cost, easy-to-use automated hybridization capture solution that can overcome the shortcomings of existing equipment in terms of manufacturing difficulty, temperature control and on-target rate, and provide more efficient technical support for methylation sequencing and capture library preparation.

[0021] In view of this, the disclosed embodiment proposes a hybridization capture automation device based on "magnetic bead transfer". The device includes a reaction table and a group of electromagnetic rods. The surface of the reaction table is provided with a plurality of slots for accommodating reagents. A group of electromagnetic rods corresponding to the plurality of slots can be in a variety of different states. In a non-temperature-sensitive operation stage, a group of electromagnetic rods can perform a mixing operation in a non-magnetized and non-heated state, and perform a bead transfer operation in a magnetized but non-heated state. In a temperature-sensitive operation stage, a group of electromagnetic rods can perform a mixing operation in a non-magnetized but heated state, and perform a bead transfer operation in a magnetized and heated state.

[0022] According to the embodiments of the present disclosure, the structure of the electromagnetic rod capable of heating and magnetization and the reaction table for accommodating the reaction liquid is relatively simple, so all operations of library hybridization and capture can be achieved at a relatively low cost. As a result, the precision and manufacturing difficulty required for the instrument are reduced, so that the manufacturing cost and threshold of the instrument are greatly reduced. In addition, the hybridization capture process has particularly strict requirements on temperature. If the equipment cannot be kept to the capture temperature, the target hit rate will be reduced and unstable. According to the embodiment of the present disclosure, the temperature of the electromagnetic rod is controllable. Such an electromagnet operates the magnetic beads so that the sample and the magnetic beads maintain a constant temperature during the automated hybridization capture, thereby improving the target hit rate and increasing stability. In this way, a temperature-controllable electromagnet can be used to achieve good hybridization and capture functions.

[0023] Example equipment for library hybridization and capture

[0024] Figure 1A FIG. 1 is a schematic diagram of an exemplary apparatus 10 for library hybridization and capture according to an embodiment of the present disclosure. Figure 1A As shown, the device 10 for library hybridization and capture includes a housing 11, a control panel 12 located at the housing 11, and a controller 13 located in the housing 11. The control panel 12 is used to receive instructions from the user and transmit the received instructions to the controller 13. The processor of the controller 13 can be programmed according to the received user instructions and the instructions pre-stored in the non-transitory memory of the controller 13 to control the various components in the device 10 to execute the scheme for library hybridization and capture according to the present disclosure.

[0025] The device 10 also includes a hybridization and capture device 100 located in the housing 11. The hybridization and capture device 100 includes a reaction table 110. The reaction table 110 includes a table body 111. The table body 111 includes a table top 112 extending in a first plane and a bottom surface 114 opposite to the table top 112. A plurality of groups of grooves 113 are provided on the table top 112. The plurality of groups of grooves 113 extend from the table top 112 to the bottom surface 114 in a direction perpendicular to the first plane. Thereafter, a plurality of groups of groove walls 116 of the plurality of groups of grooves 113 continue to extend from the bottom surface 114 in a direction away from the bottom surface 114 and protrude out of the table body 111.

[0026] The reaction station 110 further includes a heating module 115 for heating some of the plurality of groups of grooves. The heating module 115 is disposed on the bottom surface 114 and surrounds the groove wall 116 of some grooves that need to be heated during the library hybridization and capture process according to the embodiment of the present disclosure. The heating module 115 is operated to turn on the heating function and heat the surrounded part of the groove to a predetermined heating temperature.

[0027] The hybridization and capture device 100 further includes a set of electromagnetic rods 120 used in conjunction with the reaction platform 110. Here, the number of electromagnetic rods in the set of electromagnetic rods 120 corresponds to the number of a set of grooves in the plurality of grooves 113 on the reaction platform 110. Figure 1A In the illustrated embodiment, the table 111 is circular, and a plurality of groups of grooves 113 are distributed in groups on the entire table 112 around the axis of the table 111. Here, each group of grooves includes 4 grooves, and a group of electromagnetic rods 120 includes 4 electromagnetic rods. A group of electromagnetic rods 120 can be magnetized to have magnetic force, or demagnetized without magnetic force. In addition, a group of electromagnetic rods 120 can also turn on the heating function to be heated to a predetermined temperature and maintained at the predetermined temperature during the heating function. Correspondingly, after the heating function is turned off, a group of electromagnetic rods 120 will fall back to room temperature.

[0028] The library hybridization and capture process usually includes multiple temperature-sensitive operation stages and multiple temperature-insensitive operation stages. The temperature-insensitive operation stage can, for example, be an operation stage in which the operation has no temperature requirements. In such an operation stage, the effect of temperature on the performance of the reaction is small. Therefore, it can be performed at room temperature. The temperature-insensitive operation stage can, for example, include: magnetic bead washing, room temperature washing, or nucleic acid purification. The temperature-sensitive operation stages can, for example, be an operation stage in which the operation has temperature requirements. In such an operation stage, the effect of temperature on the performance of the reaction is large. Therefore, these operation stages should be performed under the required temperature environment. The temperature-sensitive operation stage can, for example, include: magnetic bead resuspension, library capture reaction, and thermal washing process.

[0029] According to an embodiment of the present disclosure, a group of electromagnetic rods 120 can be in different states, so a group of electromagnetic rods can perform a mixing operation in a non-temperature-sensitive operation stage: in a state of non-magnetization and non-heating, and perform a magnetic transfer operation in a state of magnetization but non-heating. Correspondingly, a group of electromagnetic rods can perform the mixing operation in a temperature-sensitive operation stage: in a state of non-magnetization but heating, and perform the magnetic transfer operation in a state of magnetization and heating. The magnetic transfer operation includes adsorbing magnetic beads in a group of grooves in the multiple groups of grooves 113, and transferring the adsorbed magnetic beads from the first group of grooves to the second group of grooves in the multiple groups of grooves 113. For example, a mixed solution that has undergone an inclusion reaction is contained in the first group of grooves. A group of electromagnetic rods 120 can be operated to adsorb magnetic beads captured with a library in the mixed solution under heating, and transfer the magnetic beads from the first group of grooves used to perform a hybridization reaction to the second group of grooves used for PCR reactions. In addition, the electromagnetic rods 120 can also be operated to perform a stirring operation in the multiple groups of grooves 113. For example, the electromagnetic bar 120 may perform a reciprocating motion in a set of slots to achieve stirring.

[0030] A set of electromagnetic rods 120 can manually perform actions set in the embodiments of the present disclosure. For example, a user can directly manually operate the movement of the electromagnetic rods relative to the reaction table, or can indirectly operate the movement of the electromagnetic rods relative to the reaction table through a mechanical connection component. Figure 1A In the illustrated embodiment, the movement of the electromagnetic rod relative to the reaction platform can be automatically performed by a driving device under the control of the controller 13. To this end, the hybridization and capture device 100 includes a first driving device 140 for driving the reaction platform 110 and a second driving device 130 for driving a set of electromagnetic rods 120.

[0031] like Figure 1AAs shown, the first driving device 140 is supported at the center of the circular table 111. The first driving device 140 is operated to drive the table 111 to rotate around the axis of the table 111. The second driving device 130 is arranged at the top of the housing 11 to suspend a group of electromagnetic rods 120. Four electromagnetic rods are fixed above the reaction table 110 and are operated to drive a group of electromagnetic rods 120 to move in the vertical direction.

[0032] For example, in the process of transferring magnetic beads described above, the controller 13 can control the first drive device 140 to drive the reaction table 110 to rotate until the first group of grooves in the multiple groups of grooves 113 on the reaction table 110 are aligned with a group of electromagnetic rods 120. The controller 13 can control a group of electromagnetic rods 120 to heat and magnetize. Afterwards, the controller 13 can control the second drive device 130 to drive a group of electromagnetic rods 120 to move downward into the first groove to adsorb the magnetic beads in the first groove. The controller 13 controls the second drive device 130 to drive a group of electromagnetic rods 120 to move upward out of the first groove to lift the magnetic beads from the first groove. The controller 13 controls the first drive device 140 again to drive the reaction table 110 to rotate until the second group of grooves in the multiple groups of grooves 113 on the reaction table 110 are aligned with a group of electromagnetic rods 120. Afterwards, the controller 13 controls the second driving device 130 to drive a group of electromagnetic rods 120 to move downward into the second slot, and after a group of electromagnetic rods 120 are in the second slot, controls another group of electromagnetic rods 120 to demagnetize to release the magnetic beads, thereby completing the transfer of the magnetic beads.

[0033] Compared with the hybridization capture automated workstation in the related art that executes the "pipetting-based" process, the hybridization capture automated instrument based on "magnetic bead transfer" disclosed in the present invention reduces the precision and manufacturing difficulty required for the instrument, thereby greatly reducing the manufacturing cost and threshold of the instrument, and the hybridization and capture functions can be achieved through temperature-controllable electromagnets.

[0034] It should be understood that Figure 1A The structure of the device 10 is only exemplary. The relative positions of the housing 11, the control panel 12 and the housing can be set according to design requirements. Figure 1A The driving device shown for driving a set of electromagnetic rods 120 to move relative to the reaction platform 110 is also exemplary. The hybridization and capture device 100 may also include other types of mechanisms suitable for achieving relative movement of the electromagnetic rods and the reaction platform. The present disclosure is not intended to be limited to these.

[0035] The following will refer to Figures 1B to 2D To describe in detail Figure 1A The structure of the acid capture device 100 in FIG. Figure 1B Schematic exploded view of an example hybridization and capture device 100 according to an embodiment of the present disclosure is shown. Figure 1BAs shown, the hybridization and capture device 100 includes a reaction table 110. The reaction table 110 includes a circular table body 111. The table body 111 includes a table top 112 extending in a first plane and a bottom surface 114 opposite to the table top 112. A plurality of groups of grooves 113 are provided on the table top 112. The plurality of groups of grooves 113 extend from the table top 112 to the bottom surface 114 in a direction perpendicular to the first plane. Thereafter, a plurality of groups of groove walls 116 of the plurality of groups of grooves 113 continue to extend from the bottom surface 114 in a direction away from the bottom surface 114 and protrude out of the table body 111. Figure 1B In the embodiment of the present invention, the plurality of groups of grooves 113 are distributed in groups around the axis of the platform body 111 on the entire platform surface 112. Here, each group of grooves includes 4 grooves. It should be understood that the circular reaction platform 110 in this embodiment is only exemplary, and the reaction platform 110 can also be a centrally symmetrical and / or axially symmetrical shape.

[0036] The reaction station 110 further includes a heating module 115 for heating some of the plurality of groups of grooves. The heating module 115 is disposed on the bottom surface 114 and surrounds the groove wall 116 of some grooves that need to be heated during the library hybridization and capture process according to the embodiment of the present disclosure. The heating module 115 is operated to turn on the heating function and heat the surrounded part of the groove to a predetermined heating temperature. The heating module 115 is usually heated in a temperature-sensitive operation stage to perform heating of the groove. Figure 1B In the illustrated embodiment, the heating module 115 includes a variable temperature heating module 117. The variable temperature heating module 117 is configured to maintain a plurality of heating temperatures when turned on. For example, the variable temperature heating module 117 can heat the corresponding reaction tank to a plurality of temperatures required for the PCR reaction during the PCR reaction. The heating module 115 includes a constant temperature heating module 118. The constant temperature heating module 118 is configured to maintain a predetermined heating temperature when turned on.

[0037] The variable temperature heating module 117 and the constant temperature heating module 118 respectively surround different groups of slots. Figure 2A To describe an example configuration of multiple groups of slots 113 and their positional relationship relative to the variable temperature heating module 117 and the constant temperature heating module 118. Figure 2A FIG. 1 is a schematic diagram of an exemplary reaction station 110 of a hybridization and capture device 100 according to an embodiment of the present disclosure. Figure 2A As shown, multiple groups of grooves 113 are provided on the table 112. The multiple groups of grooves 113 include 14 groups of grooves, namely a first group of grooves 205, a second group of grooves 207, a third group of grooves 208, a fourth group of grooves 201, a fifth group of grooves 202, a sixth group of grooves 203, a seventh group of grooves 204, an eighth group of grooves 209, a ninth group of grooves 210, a tenth group of grooves 211, an eleventh group of grooves 206, a twelfth group of grooves 213, a thirteenth group of grooves 212 and a fourteenth group of grooves 214.

[0038] The first group of slots 205 is used for hybridization reaction, and contains a hybridization reaction liquid mixture before the reaction. The hybridization reaction liquid mixture includes a sample, a probe, a blocking solution, and a hybridization reaction liquid. The second group of slots 207 is used to store magnetic beads, and contains magnetic beads and a storage liquid. The third group of slots 208 is used for washing, and contains a magnetic bead washing liquid. The fourth group of slots 201 is used for magnetic bead resuspension, and contains a magnetic bead resuspension liquid before the reaction. The fifth group of slots 202 is used for hot washing, and contains a first hot washing liquid before the reaction. The sixth group of slots 203 and the seventh group of slots 204 are also used for hot washing, and both contain a second hot washing liquid before the reaction. The eighth group of slots 209 is used for room temperature washing, and contains a first washing liquid before the reaction. The ninth group of slots 210 is used for room temperature washing, and contains a second washing liquid before the reaction. The tenth group of slots 211 is used for room temperature washing, and contains a third washing liquid before the reaction. The eleventh group of slots 206 is used for PCR reaction, and contains a PCR reaction liquid before the reaction. The twelfth group of slots 213 is used for eluting magnetic beads and contains an eluent before the reaction. The thirteenth group of slots 212 is used for purification and washing and contains 80% ethanol before the reaction. The fourteenth group of slots 214 is used for storing magnetic rod sleeves, which contain magnetic rod sleeves before the reaction, and after the reaction, the magnetic rods can fall off in the fourteenth group of slots 214.

[0039] In this embodiment, the reagents or reaction solutions used can be the corresponding components in the IDT hybridization capture kit. It should be understood that the groove arrangement of the reaction platform shown is only exemplary. The groove of the reaction platform can be specially designed according to different reaction processes, including the relative position of the groove and the reagents contained. The present disclosure is not intended to limit this.

[0040] Since it is necessary to heat at different temperatures when performing hybridization reaction and PCR reaction, the variable temperature heating module 117 surrounds the first group of grooves 205 for hybridization reaction and the eleventh group of grooves 206 for PCR reaction. In addition, the magnetic bead resuspension and hot washing steps also need to be heated, but the heating temperature can be the same temperature. Therefore, the constant temperature heating module 118 surrounds the fourth group of grooves 201 for magnetic bead resuspension, and the fifth group of grooves 202, the sixth group of grooves 203 and the seventh group of grooves 204 for hot washing. Here, the variable temperature heating module 117 is a fan-shaped module, and a plurality of accommodating cavities are provided on one side of its fan-shaped body facing the bottom surface 114 for accommodating grooves. Since the variable temperature heating module 117 needs to accommodate two groups of grooves, two groups of accommodating cavities are provided. Correspondingly, the constant temperature heating module 118 is also a fan-shaped module, and a plurality of accommodating cavities are provided on one side of its fan-shaped body facing the bottom surface 114 for accommodating grooves. Since the variable constant temperature heating module 118 needs to accommodate four groups of grooves, four groups of accommodating cavities are provided.

[0041] Figure 2BFIG. 2 is a schematic diagram showing an example state 200B of a heating module according to an embodiment of the disclosure. Figure 2B As shown, state 200B includes a heating module 115A whose heating function is not turned on and is at room temperature. The heating module 115A does not turn on the heating function and is at room temperature. The variable temperature heating module 117A and the constant temperature heating module 118A. State 200B also includes a heating module 115B that turns on the heating function and maintains a predetermined heating temperature. The heating module 115B includes a variable temperature heating module 117B and a constant temperature heating module 118B that turn on the heating function and maintain a predetermined heating temperature. It should be understood that although only the state of the heating module being turned on or off as a whole is shown, the switches of the heating functions of the variable temperature heating module 117A and the constant temperature heating module 118A are independent of each other.

[0042] return Figure 1B The hybridization and capture device 100 further includes a cover 150 for closing the plurality of groups of grooves. The cover 150 includes a plurality of groups of holes, namely a group of through holes 152 and a group of pre-existing cavity holes 153. The cover 150 is operated so that a group of through holes 152 or a group of pre-existing cavity holes 153 is aligned with each group of grooves in the plurality of groups of grooves 113. The cover 150 further includes a non-heating portion 151 and a heating portion 154. The number of groups of grooves covered by the heating portion 154 is the same as the number of groups of grooves covered by the heating module 115. Figure 1B In the illustrated embodiment, since the heating modules 115 , namely the variable temperature heating module 117A and the constant temperature heating module 118A, cover a total of 6 groups of grooves, the heating portion 154 also has an area covering the 6 groups of grooves. Figure 2C Schematic diagram showing an example state 200C of a cover according to a disclosed embodiment. Figure 2C As shown, state 200C includes cover 150A with heating portion 154A not turned on and at room temperature. State 200C also includes cover 150B with heating portion 154B turned on and maintained at a predetermined temperature (referred to as a second predetermined temperature).

[0043] During the operation of the hybridization capture device 100 according to the present disclosure, the heating part 153 of the cover 150, the variable temperature heating module 117 and the constant temperature heating module 118 can cooperate with each other to realize the functions required by each process. In some embodiments, the situation where the heating part 153 of the cover 150, the variable temperature heating module 117 and the constant temperature heating module 118 are not heated is mainly used in magnetic bead washing, room temperature washing, and nucleic acid purification processes. The situation where the heating part 153 and the variable temperature heating module 117 of the cover 150 are both heated is mainly used in the hybridization process and the PCR process. The situation where the heating part 153 and the constant temperature heating module 118 of the cover 150 are both heated is mainly used in the magnetic bead resuspension, capture process, and hot washing process.

[0044] return Figure 1B, when the cover 150 rotates relative to the reaction table 110, a group of through holes 152 can be aligned with any group of slots on the reaction table 110. Here, a group of electromagnetic rods 120 can enter the slots of the reaction table 110 through a group of through holes 152, while keeping other slots closed. Similarly, a group of pre-stored cavity holes 153 can also be aligned with any group of slots on the reaction table 110. At this time, a group of electromagnetic rods 120 can move downward to squeeze the pressure door of the pre-stored cavity hole or squeeze the contents in the pre-stored cavity hole and then squeeze the pressure door, and when the pressure received by the pressure door is greater than a predetermined threshold, the pressure door opens. As a result, the pre-stored contents in the pre-stored cavity hole will fall into the slot aligned with the pre-stored cavity hole.

[0045] The hybridization and capture device 100 also includes a set of electromagnetic rods 120 and a set of magnetic rod covers 160 that are sleeved on the outside of the set of electromagnetic rods 120. The magnetic rod covers are usually made of materials that are resistant to chemical corrosion and free of nuclease and protease contamination, such as polypropylene. The magnetic rod covers are usually disposable to avoid cross contamination between different samples. Using a new magnetic rod cover each time can ensure the accuracy and reliability of the experimental results. The magnetic rod cover plays an important role in experimental operations such as nucleic acid extraction, and helps to improve experimental efficiency and the accuracy of results.

[0046] Here, the number of electromagnetic rods in a group of electromagnetic rods 120 corresponds to the number of a group of slots in the plurality of groups of slots 113 on the reaction table 110. A group of electromagnetic rods 120 can be magnetized to have magnetic force, or demagnetized to have no magnetic force. In addition, a group of electromagnetic rods 120 can also turn on the heating function to be heated to a predetermined temperature and maintained at the predetermined temperature during the heating function. Correspondingly, after the heating function is turned off, the group of electromagnetic rods 120 will fall back to room temperature. Figure 2D FIG. 2 is a schematic diagram showing an example state 200D of a set of electromagnetic rods with a magnetic rod sleeve according to a disclosed embodiment. Figure 2D As shown, state 200D includes a group of electromagnetic rods 120A that are at room temperature and not energized with the heating function turned off. State 200D also includes a group of electromagnetic rods 120B that are heated to a predetermined temperature (also referred to as a first predetermined temperature) with the heating function turned on and are not energized. State 200D also includes a group of electromagnetic rods 120C that are energized with the heating function turned off. State 200D also includes a group of electromagnetic rods 120D that are energized with the heating function turned on.

[0047] In some embodiments, in the library hybridization and capture scheme according to the present disclosure, a non-magnetic and non-heated electromagnetic rod can be used before the start of the run and after the end of the run; in the standby state when the electromagnetic rod is not used; and in the magnetic bead and / or reagent and / or sample mixing operations performed during magnetic bead washing, room temperature washing, and nucleic acid purification. The electromagnetic rod in the magnetic state can be used for magnetic separation and magnetic transfer operations performed during magnetic bead washing, room temperature washing, and nucleic acid purification. The electromagnetic rod that is only heated can be used for magnetic bead and / or reagent and / or sample mixing operations performed during magnetic bead resuspension, capture, and hot washing. The electromagnetic rod that is both magnetic and heated can be used for magnetic separation and magnetic transfer operations performed during magnetic bead resuspension, capture, and hot washing.

[0048] Figure 1C A schematic diagram of an example hybridization and capture apparatus 100 is shown in accordance with an embodiment of the present disclosure. Figure 1C Shows Figure 1B Schematic diagram of the various components of the hybridization and capture device 100 in the assembled state. Figure 1C As shown, the variable temperature heating module 117 and the constant temperature heating module 118 are arranged at the bottom of the reaction table 110. The cover 150 can be rotatably arranged on the table surface of the reaction table 110 relative to the reaction table 110. A group of electromagnetic rods 120 with a magnetic rod cover 160 can be rotatably arranged above the cover 150 relative to the reaction table 110. Figure 1C The hybridization and capture device 100 is shown positioned in Figure 1A As shown in the apparatus 10 , the hybridization and capture device 100 can automatically perform the library hybridization and capture method according to the present disclosure.

[0049] In some embodiments, when performing a magnetic transfer operation, the cover 150, the reaction table 110, and a group of electromagnetic rods 120 are operated to align the first group of slots of the reaction table 110, the through holes 152 of the cover 150, and the group of electromagnetic rods 120. A group of electromagnetic rods 120 are operated to enter the first group of slots through the through holes 152. After that, a group of electromagnetic rods 120 are operated to take out the magnetic beads in the first group of slots. After that, the reaction table 110 is operated to align the second group of slots with the through holes 152 of the cover 150 and the group of electromagnetic rods 120. After alignment, a group of electromagnetic rods 120 are operated to enter the second group of slots through the through holes 152. Finally, a group of electromagnetic rods 120 are demagnetized to release the magnetic beads.

[0050] Example Methods for Library Hybridization and Capture

[0051] So far, the apparatus for library hybridization and capture and the structure and operation principle of the hybridization and capture device according to the present disclosure have been introduced. Figures 3 to 4G To describe methods for library hybridization and capture according to the present disclosure. Figure 31 shows a flow chart of an exemplary method 300 for library hybridization and capture according to an embodiment of the present disclosure. The method 300 may also include other steps. The method 300 may, for example, utilize Figure 1B The hybridization and capture device 100 shown is performed manually or by Figure 1A The hybridization and capture of the library is performed using the apparatus 10 shown.

[0052] like Figure 3 As shown, at 302, method 300 includes using a hybridization and capture device to sequentially perform a hybridization reaction, a magnetic bead wash, a magnetic bead resuspension, a library capture reaction, a hot wash, and a room temperature wash. Here, the multiple groups of slots in the hybridization and capture device contain corresponding reagents. The non-temperature-sensitive operation stage includes: magnetic bead wash and room temperature wash, and the temperature-sensitive operation stage includes: library capture reaction, magnetic bead resuspension, and hot wash process.

[0053] For example, using Figure 1A In the embodiment shown in which the apparatus 10 for library hybridization and capture is performed, the apparatus 10 can perform a hybridization reaction using the hybridization and capture device 100. During the hybridization reaction, a hybridization reaction liquid mixture is provided in the first group of grooves 205 among the multiple groups of grooves 113 of the hybridization and capture device 100. The apparatus 10 can seal the first group of grooves 205 using a cover heated to a first predetermined cover temperature, and heat the first group of grooves 205 to a first heating temperature using a variable temperature heating module 117 to perform a hybridization reaction in the first group of grooves 205, thereby obtaining a reacted mixture.

[0054] In some embodiments, the device 10 can perform magnetic bead washing using the hybridization and capture device 100. During the magnetic bead washing process, the device 10 can control a set of electromagnetic rods 120 to perform a mixing operation in a non-magnetic and non-heating state, and to perform a magnetic transfer operation in a magnetized but non-heating state.

[0055] In some embodiments, the device 10 can perform magnetic bead resuspension using the hybridization and capture device 100. During the magnetic bead resuspension process, the device 10 can control a group of electromagnetic rods 120 to perform the mixing operation in a non-magnetic but heated state, and perform the magnetic transfer operation in a magnetized and heated state. The device 10 can also control the cover 150 to close the corresponding group of slots in a heated state. In addition, the device 10 can also control the constant temperature heating module 118 to heat the corresponding slot.

[0056] In some embodiments, the device 10 can perform a library capture reaction using a hybridization and capture device 100. During the library capture reaction, the device 10 can control a group of electromagnetic rods 120 to perform the mixing operation in a non-magnetic but heated state, and perform the magnetic transfer operation in a magnetized and heated state. The device 10 can also control the cover 150 to close the corresponding group of slots in a heated state. In addition, the device 10 can also control the constant temperature heating module 118 and the variable temperature heating module 117 to heat the corresponding slots.

[0057] In some embodiments, the device 10 can perform thermal washing using the hybridization and capture device 100. During the thermal washing process, the device 10 can control a group of electromagnetic rods 120 to perform the mixing operation in a non-magnetic but heated state, and perform the magnetic transfer operation in a magnetized and heated state. The device 10 can also control the cover 150 to close the corresponding group of slots in a heated state. In addition, the device 10 can also control the constant temperature heating module 118 to heat the corresponding slots.

[0058] In some embodiments, the device 10 can perform room temperature washing using the hybridization and capture device 100. During the room temperature washing process, the device 10 can control a set of electromagnetic rods 120 to perform a mixing operation in a non-magnetic and non-heating state, and to perform a magnetic transfer operation in a magnetized but non-heating state.

[0059] exist Figure 3 In the illustrated embodiment, a scheme for performing hybridization capture and PCR amplification based on magnetic bead transfer is provided. Compared with the workstation-based, "pipetting-based" scheme: expensive workstations and complex pipettes, the scheme disclosed in the present invention reduces the precision and manufacturing difficulty required for the instrument, greatly reducing the manufacturing cost and threshold of the instrument, and the hybridization and capture functions can be achieved through temperature-controllable electromagnets.

[0060] Hybridization capture process

[0061] In some embodiments, the hybridization capture process for library hybridization and capture may include: hybridization process; magnetic bead resuspension process; capture process; hot washing process; room temperature washing process; PCR process; purification process. FIG. 4A to FIG. 4G To describe the hybridization capture process.

[0062] 1. Hybridization process

[0063] In some embodiments, the hybridization process may include adding a hybridization reaction solution mixture to a first group of grooves in the plurality of groups of grooves of the hybridization and capture device. Here, the hybridization reaction solution mixture includes a sample, a probe, a blocking solution, and a hybridization reaction solution. The hybridization process may also include closing the first group of grooves with a cover heated to a first predetermined temperature. The hybridization process may also include heating the first group of grooves to a first heating temperature using a variable temperature heating module. The hybridization process may include performing a hybridization reaction in the first group of grooves to obtain a mixture.

[0064] In use Figure 1A In the embodiment of the hybridization process performed by the device 10 for library hybridization and capture shown, the device 10 can rotate the reaction table 110 to align the position of a group of through holes 152 on the cover 150 with the position of the first group of grooves 205. The device 10 adds a hybridization reaction liquid mixture (including samples, probes, blocking liquid, and hybridization reaction liquid) to the first group of grooves 205 through a group of through holes 152 on the cover 150. The device 10 rotates the reaction table 110 to a position corresponding to the magnetic rod sleeve storage position of the reaction table 110, that is, the fourteenth group of grooves 214. At this time, all the grooves on the reaction table 110 are closed by the cover 150. The device 10 starts the variable temperature heating module 117 to the required temperature for the hybridization reaction and maintains it. The device 10 starts the heating function of the cover 150 to heat the heating part 154 to a first predetermined temperature (e.g., 105°C) and maintain it. Thus, a hybridization reaction is performed in the first group of grooves 205.

[0065] Figure 4A FIG. 4 is a schematic diagram showing an example state 400A of a hybridization and capture device in an incubation phase of a hybridization process according to an embodiment of the present disclosure. Figure 4A As shown, a group of through holes 152 of the cover 150 is aligned with the fourteenth group of grooves 214 on the reaction table 110 , so that the heating portion 154 of the cover 150 heated to the first predetermined temperature is aligned with the variable temperature heating module 117 and the constant temperature heating module 118 .

[0066] 2. Magnetic Bead Resuspension Process

[0067] In some embodiments, a magnetic bead washing process is also included before resuspension. The magnetic bead washing process may include transferring the captured magnetic beads in the second group of slots to the third group of slots using a set of electromagnetic rods in a state of magnetization but not heating. The magnetic bead washing process may also include performing a mixing operation using a set of electromagnetic rods in a state of non-magnetization and non-heating to wash the captured magnetic beads. The magnetic bead washing process may also include taking the washed captured magnetic beads out of the third group of slots using a set of electromagnetic rods in a state of magnetization but not heating.

[0068] The magnetic bead resuspension process may include heating the fourth group of slots to a second heating temperature using a constant temperature heating module of the hybridization and capture device. The magnetic bead resuspension process may also include placing the washed captured magnetic beads into the fourth group of slots using a group of electromagnetic rods in a magnetized and heated state. The magnetic bead resuspension process may also include performing a mixing operation using a group of electromagnetic rods in a non-magnetized but heated state to resuspend the captured magnetic beads. The magnetic bead resuspension process may also include taking the captured magnetic beads out of the fourth group of slots for capture using a group of electromagnetic rods in a magnetized and heated state.

[0069] In use Figure 1A In the embodiment of the apparatus 10 for library hybridization and capture shown in the figure, in which the magnetic bead resuspension process is performed, the apparatus 10 uses a set of electromagnetic rods 120 to take out the magnetic rod sleeves in the fourteenth group of slots 214 storing the magnetic rod sleeves of the reaction platform 110 through a set of through holes 152 on the cover 150. (Unless otherwise specified, the magnetic rods below are all magnetic rods with magnetic rod sleeves). Afterwards, the apparatus 10 rotates the reaction platform 110 until the position of the set of through holes 152 on the cover 150 is aligned with the position of the second group of slots 207 containing the magnetic beads and their preservation solution. The apparatus 10 magnetizes the set of electromagnetic rods 120, so that the set of electromagnetic rods 120 enters the second group of slots 207 containing the nucleic acid capture magnetic beads (i.e., the first group of magnetic beads) and their preservation solution through the set of through holes 152, and adsorbs the nucleic acid capture magnetic beads. The apparatus 10 removes the set of electromagnetic rods 120 adsorbed with the nucleic acid capture magnetic beads from the second group of slots 207.

[0070] The device 10 rotates the reaction table 110 until the position of a group of through holes 152 on the cover 150 corresponds to the position of the third group of grooves 208 containing the magnetic bead washing solution. The device 10 allows a group of electromagnetic rods 120 adsorbed with nucleic acid capture magnetic beads to enter the third group of grooves 208 containing the magnetic bead washing solution through a group of through holes 152. The device 10 demagnetizes a group of electromagnetic rods 120 to release the adsorbed nucleic acid capture magnetic beads into the third group of grooves 208. The device 10 controls a group of electromagnetic rods 120 to perform reciprocating motion (such as rotation, slapping) to mix the magnetic beads. The device 10 controls a group of electromagnetic rods 120 to re-magnetize and adsorb the nucleic acid capture magnetic beads in the third group of grooves 208. The device 10 removes the magnetic rods attached with nucleic acid capture magnetic beads from the groove. The device 10 starts the constant temperature heating module 118 to keep the constant temperature heating module 118 at the required temperature for capture (i.e., the second heating temperature). The device 10 activates the heating function of the set of electromagnetic rods 120 to maintain the set of electromagnetic rods 120 at the required temperature (ie, the first predetermined temperature) for capturing. The device 10 also activates the heating function of the cover 150, thereby completing the preparation stage of the magnetic bead resuspension process.

[0071] The device 10 rotates the reaction table 110 until the position of a group of through holes 152 on the cover 150 corresponds to the position of the fifth group of grooves 201 containing the magnetic bead resuspension liquid. The device 10 allows a group of electromagnetic rods 120 adsorbed with nucleic acid capture magnetic beads (i.e., the first group of magnetic beads) to enter the fifth group of grooves 201 containing the magnetic bead resuspension liquid through a group of through holes 152. The device 10 demagnetizes a group of electromagnetic rods 120 to release the adsorbed nucleic acid capture magnetic beads.

[0072] Figure 4B FIG. 4 is a schematic diagram showing an exemplary state 400B of a hybridization and capture device in a magnetic bead resuspension process according to an embodiment of the present disclosure. Figure 4B As shown, a group of through holes 152 on the cover 150 is aligned with the fifth group of slots 201. The constant temperature heating module 118 and the heating portion 154 of the cover 150 start the heating function to ensure the temperature of the magnetic beads in the fifth group of slots 201 for resuspension. At the same time, the reagents in other slots may evaporate under the heating of the constant temperature heating module 118. After the steam encounters the heating portion 154, it will not condense due to the high temperature of the heating portion 154.

[0073] Afterwards, the device 10 controls the set of electromagnetic rods 120 to reciprocate (such as rotate, beat) to mix the magnetic beads. The device 10 re-magnetizes the set of electromagnetic rods 120 and adsorbs the nucleic acid capture magnetic beads in the tank, and finally removes the set of electromagnetic rods 120 attached with the nucleic acid capture magnetic beads from the tank.

[0074] 3. Capture Process

[0075] In some embodiments, the capture process may include placing the captured magnetic beads resuspended by the magnetic beads into the mixed solution after hybridization reaction in the first group of slots using a set of electromagnetic rods in a magnetized and heated state. The capture process may include performing a mixing operation in the first group of slots using a set of electromagnetic rods in a non-magnetized but heated state. The capture process may include sealing the first group of slots with a cover heated to a second predetermined cover temperature so that the captured magnetic beads are incubated in the mixed solution.

[0076] In use Figure 1AIn the embodiment of the apparatus 10 for library hybridization and capture shown, the apparatus 1 maintains the temperature of the constant temperature heating module 118, the variable temperature heating module 117 and the set of electromagnetic rods 120 at the required temperature for capture, and keeps the heating function of the cover 150 turned on. The apparatus 10 rotates the reaction table 110 until the position of a set of through holes 152 on the cover 150 corresponds to the position of the first set of grooves 205 containing the blocking and hybridization mixture. The apparatus 10 allows the set of electromagnetic rods 120 adsorbed with nucleic acid capture magnetic beads to enter the groove through a set of through holes 152, and the apparatus 10 demagnetizes the set of electromagnetic rods 120 to release the adsorbed nucleic acid capture magnetic beads. The apparatus 10 causes the set of electromagnetic rods 120 to reciprocate (such as rotate, tap) to mix the magnetic beads. The apparatus 10 incubates the nucleic acid capture magnetic beads in the first set of grooves 205 containing the blocking and hybridization mixture for a period of time (for example, 12 hours), and occasionally causes the set of electromagnetic rods 120 to perform a stirring operation. Figure 4C FIG. 4 is a schematic diagram showing an exemplary state 400C of a hybridization and capture device in a capture process according to an embodiment of the present disclosure. Figure 4C As shown, a group of electromagnetic rods 120 adsorbed with nucleic acid capture magnetic beads enter the first group of grooves 205 through a group of through holes 152 for incubation. At this time, a group of electromagnetic rods 120, variable temperature heating module 117, constant temperature heating module 118, and heating unit 154 are all in a heating state.

[0077] 4. Hot washing process

[0078] In some embodiments, the thermal washing process may include heating the fifth group of slots, the sixth group of slots, and the seventh group of slots to a third heating temperature using a constant temperature heating module of the hybridization and capture device. The thermal washing process may also include taking out the captured magnetic beads with the library from the first group of slots using a group of electromagnetic rods in a magnetized and heated state. The thermal washing process may also include performing thermal washing operations in the fifth group of slots, the sixth group of slots, and the seventh group of slots in sequence. The thermal washing operation includes: placing the captured magnetic beads into a corresponding group of slots using a group of electromagnetic rods in a magnetized and heated state; performing a mixing operation in a corresponding group of slots using a group of electromagnetic rods in a non-magnetized but heated state; and taking out the captured magnetic beads from a corresponding group of slots using a group of electromagnetic rods in a magnetized and heated state.

[0079] In use Figure 1AIn the embodiment of the apparatus 10 for library hybridization and capture shown in the figure, which performs a thermal washing process, the apparatus 10 turns off the heating function of the variable temperature heating module 117, maintains the temperature of the constant temperature heating module 118 and the magnetic rod at the temperature required for capture, and maintains the heating function of the cover 150. The apparatus 10 repeats the following steps in the fifth group of slots 202 containing the first thermal washing solution, the sixth group of slots 203 containing the second thermal washing solution, and the seventh group of slots 204 containing the second thermal washing solution: a group of electromagnetic rods 120 are energized, enter a group of slots containing nucleic acid capture magnetic beads through a group of through holes 152, and adsorb the nucleic acid capture magnetic beads; the group of electromagnetic rods 120 adsorbing the nucleic acid capture magnetic beads are removed from the group of slots; the reaction table 110 is rotated to a group of through holes 152 on the cover 150. The position of one group of slots corresponds to the position of another group of slots; a group of electromagnetic rods 120 adsorbed with nucleic acid capture magnetic beads are allowed to enter another group of slots through a group of through holes 152; a group of electromagnetic rods 120 are demagnetized to release the adsorbed nucleic acid capture magnetic beads into the group of slots; a group of electromagnetic rods 120 are controlled to perform reciprocating motion (such as rotation, slapping) to mix the magnetic beads; a group of electromagnetic rods 120 are controlled to be re-magnetized and adsorb the nucleic acid capture magnetic beads in the group of slots; and the magnetic rods attached with the nucleic acid capture magnetic beads are removed from the slots.

[0080] Figure 4D FIG. 4 is a schematic diagram showing an exemplary state 400D of a hybridization and capture device in a hot wash process according to an embodiment of the present disclosure. Figure 4D As shown, a group of electromagnetic rods 120 places magnetic beads into a group of grooves containing hot washing liquid through a group of through holes 152 to perform hot washing. Here, a group of electromagnetic rods 120, variable temperature heating module 117, constant temperature heating module 118, and heating unit 154 are all in a heating state.

[0081] 5. Room temperature washing process

[0082] In some embodiments, the room temperature washing process may include keeping the eighth group of slots, the ninth group of slots, and the tenth group of slots at room temperature. The room temperature washing process may include sequentially performing room temperature washing operations on the captured magnetic beads that have been thermally washed in the eighth group of slots, the ninth group of slots, and the tenth group of slots. The room temperature washing operation includes: placing the captured magnetic beads into the corresponding group of slots using a group of electromagnetic rods in a state of being magnetized but not heated; performing a mixing operation in the corresponding group of slots using a group of electromagnetic rods in a state of being non-magnetized and non-heated; and taking the captured magnetic beads out of the corresponding group of slots using a group of electromagnetic rods in a state of being magnetized but not heated.

[0083] In use Figure 1AIn the embodiment shown in which the device 10 for library hybridization and capture performs a room temperature washing process, the device 10 turns off the heating functions of the variable temperature heating module 117 , the constant temperature heating module 118 , the magnetic rod, and the cover 150 . The device 10 repeats the following steps in the eighth group of slots 209 containing the first washing solution, the ninth group of slots 210 containing the second washing solution, and the tenth group of slots 211 containing the third washing solution: a group of electromagnetic rods 120 are magnetized, enter a group of slots containing nucleic acid capture magnetic beads through a group of through holes 152, and adsorb the nucleic acid capture magnetic beads; the group of electromagnetic rods 120 adsorbed with nucleic acid capture magnetic beads are removed from the group of slots; the reaction table 110 is rotated until the position of a group of through holes 152 on the cover 150 corresponds to the position of another group of slots; the group of electromagnetic rods 120 adsorbed with nucleic acid capture magnetic beads enter another group of slots through a group of through holes 152; the group of electromagnetic rods 120 are demagnetized to release the adsorbed nucleic acid capture magnetic beads into the group of slots; the group of electromagnetic rods 120 are controlled to reciprocate (such as rotate, beat) to mix the magnetic beads; the group of electromagnetic rods 120 are controlled to be magnetized again, and adsorb the nucleic acid capture magnetic beads in the group of slots; the magnetic rods attached with nucleic acid capture magnetic beads are removed from the slots.

[0084] Figure 4E FIG. 4 is a schematic diagram showing an exemplary state 400E of a hybridization and capture device in a room temperature washing process according to an embodiment of the present disclosure. Figure 4E As shown, a group of electromagnetic rods 120 places the magnetic beads into a group of grooves containing room temperature washing liquid through a group of through holes 152 to perform room temperature washing. Here, a group of electromagnetic rods 120, variable temperature heating module 117, constant temperature heating module 118, and heating unit 154 are not in a heating state.

[0085] 6.PCR Process

[0086] In some embodiments, the PCR process may include placing the captured magnetic beads with the library captured into the eleventh group of slots using the group of electromagnetic rods in a magnetized and heated state. The PCR process may also include performing the PCR reaction by adjusting the temperature of the eleventh group of slots using a variable temperature heating module of the hybridization and capture device.

[0087] In use Figure 1AIn the embodiment of the device 10 for library hybridization and capture shown, the device 10 rotates the reaction table 110 until the position of a group of through holes 152 on the cover 150 corresponds to the position of the eleventh group of grooves 206 containing the PCR reaction solution. The device 10 allows a group of electromagnetic waves 120 adsorbed with nucleic acid capture magnetic beads to enter the eleventh group of grooves 206 containing the PCR reaction solution through a group of through holes 152, and the device 10 demagnetizes a group of electromagnetic rods 120 to release the adsorbed nucleic acid capture magnetic beads. The device 10 causes a group of electromagnetic rods 120 to reciprocate (such as rotating and beating) to mix the magnetic beads. The device 10 rotates the reaction table 110 until the position of a group of through holes 152 on the cover 150 is aligned with the fourteenth group of grooves 214 of the reaction table 110, so that the cover 150 covers the eleventh group of grooves 206. The device 10 starts the variable temperature heating module 117 to perform a PCR reaction, and starts the heating function of the cover 150. Figure 4F FIG. 4 is a schematic diagram showing an exemplary state 400F of a hybridization and capture device in a PCR process according to an embodiment of the present disclosure. Figure 4F As shown, a group of through holes 152 of the cover 150 is aligned with the fourteenth group of slots 214 on the reaction station 110, so that the heated heating portion 154 of the cover 150 is aligned with the variable temperature heating module 117 and the constant temperature heating module 118 to perform a PCR reaction.

[0088] 7. Purification process

[0089] In some embodiments, the purification process may include aligning a set of pre-stored cavities containing purification magnetic beads of the cover of the hybridization and capture device with an eleventh set of slots containing a reaction solution reacted by PCR. The purification process may also include squeezing the purification magnetic beads using a set of electromagnetic rods so that the purification magnetic beads push open the pressure gates of a set of pre-stored cavities and enter the eleventh set of slots. The purification process may also include performing a mixing operation using a set of electromagnetic rods in a non-magnetic and non-heated state to incubate the purification magnetic beads. The purification process may also include transferring the capture magnetic beads and the purification magnetic beads from the eleventh set of slots to the thirteenth set of slots using a set of electromagnetic rods in a magnetized but non-heated state to perform a washing operation. The purification process may also include transferring the capture magnetic beads and the purification magnetic beads from the thirteenth set of slots to the twelfth set of slots using a set of electromagnetic rods in a magnetized but non-heated state so that the capture magnetic beads and the purification magnetic beads are incubated in the eluent. The purification process may also include removing the magnetic beads from the twelfth set of slots using a set of electromagnetic rods in a magnetized but non-heated state to obtain the captured library.

[0090] In use Figure 1AIn the embodiment of the apparatus 10 for library hybridization and capture shown, the apparatus 10 rotates the reaction table 110 so that a group of pre-stored cavities 153 on the cover 150 correspond to the position of the eleventh group of slots 206 containing the PCR reaction solution on the reaction table 110. The apparatus 10 controls a group of electromagnetic rods 120 to squeeze a group of pre-stored cavities 153 so that the purified magnetic beads (i.e., the second group of magnetic beads) in a group of pre-stored cavities 153 enter the eleventh group of slots 206 containing the PCR reaction solution. The apparatus 10 rotates the reaction table 110 until the position of a group of through holes 152 on the cover 150 corresponds to the position of the eleventh group of slots 206 containing the PCR reaction solution. The apparatus 10 controls a group of electromagnetic rods 120 to enter the slot 6 containing the PCR reaction solution through a group of through holes 152, and the magnetic rods cause a group of electromagnetic rods 120 to reciprocate (such as rotate, tap) to mix the magnetic beads so that they are allowed to stand and incubate. In the washing step in the thirteenth group of slots 212 containing 80% ethanol, the device transfers the magnetic beads from the thirteenth group of slots 212 to the twelfth group of slots 213 containing the eluent, and performs static incubation after demagnetization and mixing. The device 10 controls a group of electromagnetic rods 120 to re-magnetize to adsorb the nucleic acid capture magnetic beads in the slot, and removes the magnetic rods adsorbed with the magnetic beads from the slot. The device 10 rotates the reaction table 110 until the position of a group of through holes 152 on the cover 150 is aligned with the position of the fourteenth group of slots 214 of the reaction table 110. The device 10 controls a group of electromagnetic rods 120 so that the magnetic rods on a group of electromagnetic rods 120 are sleeved under the stopper of the slot and fall into the fourteenth group of slots 214. Finally, the hybridization-captured library is in the third slot 213 containing the eluent.

[0091] Figure 4G FIG. 4 is a schematic diagram showing an exemplary state 400G of a hybridization and capture device in a purification process according to an embodiment of the present disclosure. Figure 4G As shown, a group of electromagnetic rods 120 places the magnetic beads into a third group of slots containing an eluent through a group of through holes 152 to perform elution. Here, a group of electromagnetic rods 120, a variable temperature heating module 117, a constant temperature heating module 118, and a heating unit 154 are not in a heating state.

[0092] The entire process of hybridization capture is automated by the apparatus and method for library hybridization and capture disclosed herein. During the entire process, the magnetic beads are operated by using temperature-controllable electromagnets, so that the sample and the magnetic beads are kept at a constant temperature during the automated hybridization capture, thereby improving the on-target rate and increasing stability.

[0093] Experiment and result analysis

[0094] Figure 5A schematic diagram showing the comparison of core indicators of different hybrid capture methods is shown. In this embodiment, a hybrid capture experiment was performed. Experimental samples: NA12878 cell line interrupted and then methylated, pre-library after library construction, the pre-library input amount was 600ng. Hybridization capture reagents used in the experiment: IDT's xGen hybridization capture kit and commercial probes provided by IDT. It should be noted that IDT's kit is only a feasible example hybrid capture kit, and other types of hybrid capture kits can also be used according to the scheme of the present disclosure.

[0095] 1. Experimental groups:

[0096] Control group: manual operation was performed according to the official procedure of IDT's xGen hybrid capture kit;

[0097] Experimental group 1: Hamilton NGS STAR workstation and xGen hybrid capture kit were used for capture process. Hamilton NGS STAR workstation is an automated workstation recommended by IDT that can be used with xGen hybrid capture kit.

[0098] Experimental Group 2: Hybridization capture was performed using the hybridization and capture device according to the present disclosure.

[0099] 2. Experimental conditions:

[0100] The temperature for hybridization, capture, hot washing, and magnetic bead resuspension was 65°C;

[0101] The hybridization time was 12 h;

[0102] The number of PCR cycles was 11.

[0103] 3. Experimental preparation:

[0104] After adding the sample to the Blocker Master Mix of the IDT kit, vacuum drying was performed;

[0105] The vacuum-dried sample was resuspended in the Hybridization Master Mix of the IDT kit.

[0106] 4. Experimental Procedure

[0107] Control group:

[0108] Manual operation was performed according to the official procedure of IDT's xGen hybrid capture kit.

[0109] Experimental Group 1:

[0110] Perform hybridization on an external PCR instrument;

[0111] Automated capture was performed on a Hamilton NGS STAR workstation.

[0112] Experimental Group 2:

[0113] The components of the IDT hybrid capture kit are placed in the corresponding slots of the hybridization and capture device according to the present disclosure, and the volume is the volume specified in the official instructions of the IDT kit. The correspondence between the reagents of the hybridization and capture device according to the present disclosure and the reagents in the IDTxGen kit is shown in the following table

[0114] Table 1 Reagent comparison table

[0115]

[0116]

[0117] The details of the operation when performing hybridization and capture reactions using the hybridization and capture device according to the present disclosure are as follows:

[0118] The operating temperature of the constant temperature heating module 118 is 65°C;

[0119] During the hybridization process and the PCR process, the operating temperature of the heating part of the cover 150 is 105° C.; during the hot washing and capturing process, the operating temperature of the heating part of the cover 150 is 70° C.;

[0120] The temperature of the heating state of the temperature-controllable electromagnetic rod is 65°C and the magnetic separation time is 30s;

[0121] During the capture process, the samples were mixed once every 10 min using a temperature-controlled electromagnetic rod for 20 s.

[0122] 5. Experimental Results

[0123] Take the same amount of final libraries from the control group, experimental group 1, and experimental group 2 for sequencing, and analyze the sequencing results. Here, the target ratio of the targeted region after deduplication (target_ratio_dedup) is selected to evaluate the target ratio of the genome targeted region after deduplication. Target_ratio_dedup reflects the specificity of the capture process, that is, the efficiency of capturing target molecules in a specific area. This indicator reflects how much proportion of DNA fragments are actually wanted to be captured and analyzed in the specific area of ​​interest during genome sequencing.

[0124] like Figure 5As shown, chart 510 shows the results of the parameter target_ratio_de-weighting of the control group, experimental group 1, and experimental group 2. It can be seen from chart 510 that the target_ratio_de-weighting of experimental group 2 according to the present disclosure is higher than that of the control group and experimental group 1. This is because the temperature-controllable electromagnetic rod raises the temperature to the required temperature during the process of magnetic bead resuspension, hot washing, capture, etc., avoiding non-specific capture caused by cooling of the magnetic beads in these processes. In the control group and experimental group 1, since the magnetic frame and the gun tip are not heated, the reagent temperature will be reduced during the process of magnetic bead resuspension, hot washing, capture, etc., resulting in non-specific capture, thereby resulting in a reduction in target_ratio_de-weighting.

[0125] It can be further seen from Chart 510 that the volatility of the 2-point target_ratio_deduplication values ​​of the experimental group according to the present disclosure is better than that of the control group. This is because the fluctuation of the reagent temperature during the magnetic bead resuspension, hot washing, capture, etc. is positively correlated with the operation time. During the manual operation, it is difficult to ensure that the operation time of each sample is consistent, which leads to greater volatility of the target_ratio_deduplication during the manual operation.

[0126] For hybrid capture, the yield of the final library depends not only on the recovery rate of the target gene, but also on the recovery rate of non-target genes. However, it is hoped that as many target genes as possible are recovered and as few non-target genes are recovered. Therefore, it is difficult to evaluate the performance of hybrid capture from the yield of the final library. Here, the ratio of unique sequences in the targeted region (target_unique_ratio), an important indicator for evaluating the complexity of the library, is also selected to evaluate the recovery rate of the target gene. The ratio of unique sequences in the targeted region indicates the ratio of unique reads in the targeted region of the genome. In simple terms, it is the proportion of DNA fragments that can be uniquely read in a specific target region of a genome. This ratio indicates how many DNA fragments are unique, rather than repeated, in these target regions. The higher this ratio is, the more types of DNA fragments are captured, representing a higher diversity and complexity of the sample.

[0127] Icon 520 shows the results of the ratio of unique sequences in the targeted regions of the control group, experimental group 1, and experimental group 2. It can be seen from icon 520 that the ratio of unique sequences in the targeted regions of experimental group 2 according to the present disclosure is higher than that of the control group and experimental group 1. This is because in order to avoid the ratio of unique sequences in the targeted region being reduced due to the long magnetic absorption time at room temperature during the processes of magnetic bead resuspension, hot washing, and capture, the control group can only quickly perform magnetic separation operations, resulting in the loss of some magnetic beads, thereby reducing the ratio of unique sequences in the targeted region. For experimental group 1, on the one hand, since the washing solution contains high concentrations of salt and surfactants, some magnetic beads are washed away together with the supernatant during the magnetic separation process of discarding the supernatant, resulting in sample loss; on the other hand, since some samples will be hung on the gun tip when the gun tip is mixed and the liquid is transferred, and in order to avoid the temperature of the sample on the gun tip from decreasing and affecting the ratio of unique sequences in the targeted region, the gun tip needs to be discarded after each reaction, resulting in the loss of the sample on the gun tip. According to the experimental group 2 disclosed in the present invention, since the temperature of the temperature-controllable electromagnetic rod increases the temperature to the required temperature during the process of magnetic bead resuspension, hot washing, and capture, on the one hand, the magnetic separation time can be extended, so that the magnetic beads are fully transferred, thereby avoiding sample loss; on the other hand, since the magnetic rod cover is maintained at the temperature required for capture and hot washing during the experiment, there is no need to replace the magnetic rod cover during the experiment, thereby reducing sample loss. In summary, the device described in the present invention not only has a higher degree of automation, but also has a higher target hit rate and library complexity through the specially designed temperature-controllable electromagnetic rod.

[0128] The embodiments of the present disclosure have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the various implementations disclosed herein.

Claims

1. A hybridization and capture device (100), comprising: A reaction table (110) comprises a table top (112) extending in a first plane, and a plurality of grooves (113) are formed on the table top; and A set of electromagnetic rods (120) is configured as follows: During non-temperature-sensitive operating phases: In a state where magnetization is not applied and heating is not performed, a mixing operation is performed, and in a state where magnetization is applied but heating is not performed, a magnetic transfer operation is performed, wherein the magnetic transfer operation comprises transferring capture magnetic beads for capturing the library from one group of grooves in the plurality of groups of grooves (113) to another group of grooves; as well as During temperature-sensitive operating phases: The mixing operation is performed in a non-magnetic but heated state, and the magnetic transfer operation is performed in a magnetic and heated state.

2. The hybridization and capture device (100) according to claim 1, wherein the non-temperature sensitive operation phase comprises: Magnetic bead washing, room temperature washing, nucleic acid purification, and the temperature-sensitive operation stages include: magnetic bead resuspension, library capture reaction, and hot washing process.

3. The hybridization and capture device (100) according to claim 1, wherein the reaction station (110) further comprises: The platform (111) comprises a bottom surface (114) opposite to the platform surface (112), and the multiple groups of groove walls (116) of the multiple groups of grooves (113) extend from the bottom surface (114) in a direction away from the platform surface (112); as well as A heating module (115) is arranged on the bottom surface (114) and surrounds the groove walls (116) of a portion of the grooves in the plurality of groups of grooves (113). The heating module (115) is configured to heat the portion of the grooves to a predetermined heating temperature when turned on.

4. The hybridization and capture device (100) according to claim 3, wherein the heating module (115) comprises: A variable temperature heating module (117), configured to maintain a heating temperature among a plurality of heating temperatures when turned on; as well as The constant temperature heating module (118) is configured to maintain a heating temperature when turned on.

5. The hybridization and capture device (100) according to claim 4, wherein the constant temperature heating module (118) is configured to heat the grooves in the multiple groups of grooves (113) for containing reagents required for the temperature-sensitive operation stage during the temperature-sensitive operation stage.

6. The hybridization and capture device (100) according to claim 4, wherein the variable temperature heating module (117) is configured to heat the corresponding grooves in the multiple groups of grooves (113) to the multiple heating temperatures in hybridization reactions and polymerase chain reactions.

7. The hybridization and capture device (100) according to claim 3 further includes a cover (150) covering the table top, the cover (150) including multiple groups of holes (152, 153), and the cover (150) is operated so that a group of holes in the multiple groups of holes (152, 153) is aligned with each group of grooves in the multiple groups of grooves (113) in sequence.

8. The hybridization and capture device (100) according to claim 7, wherein the cover (150) further comprises a heating portion (154), and the number of groups of grooves covered by the heating portion (154) is the same as the number of groups of grooves covered by the heating module (115).

9. The hybridization and capture device (100) according to claim 8, wherein the heating section (154) is configured to heat during the temperature-sensitive operation phase, and during hybridization reactions and polymerase chain reactions.

10. The hybridization and capture device (100) according to claim 7, wherein the plurality of groups of holes (152, 153) include a group of through holes (152) and a group of pre-stored cavity holes (153) suitable for accommodating reagents, The set of pre-existing cavity holes (153) includes a pressure gate configured to open in response to a pressure greater than a pressure threshold.

11. The hybridization and capture device (100) according to claim 10, wherein the group of electromagnetic rods (120) is configured to move toward the group of pre-stored cavity holes (153) when the group of electromagnetic rods (120) is aligned with the group of pre-stored cavity holes (153) and a group of grooves in the multiple groups of grooves (113), so that the pressure door of the group of pre-stored cavity holes (153) opens, so that the purified magnetic beads stored in the group of pre-stored cavity holes (153) fall into the aligned group of grooves.

12. The hybridization and capture device (100) according to claim 1, wherein the set of electromagnetic rods is configured to be heated to a predetermined temperature in the range of 60°C-70°C.

13. The hybridization and capture device (100) according to claim 12, wherein the predetermined temperature is in the range of 63°C-65°C.

14. The hybridization and capture device (100) according to claim 1, wherein the reaction table (110) is of a centrally symmetrical and / or axially symmetrical shape, and the plurality of groups of grooves (113) are evenly distributed on the table surface (112) around the axis of the reaction table (110).

15. A method (300) for library hybridization and capture, comprising: Using the hybridization and capture device (100) according to any one of claims 1 to 14, hybridization reaction, magnetic bead washing, magnetic bead resuspension, library capture reaction, hot washing and room temperature washing are performed in sequence, wherein the multiple groups of grooves (113) of the hybridization and capture device (100) contain corresponding reagents, The non-temperature-sensitive operation phase includes: the magnetic bead washing and the room temperature washing, and the temperature-sensitive operation phase includes: the library capture reaction, the magnetic bead resuspension and the hot washing process.

16. The method (300) of claim 1, wherein performing the hybridization reaction comprises: Adding a hybridization reaction solution mixture into a first group of grooves (205) among the multiple groups of grooves (113) of the hybridization and capture device (100), wherein the hybridization reaction solution mixture includes a sample, a probe, a blocking solution and a hybridization reaction solution; closing the first set of slots (205) with the cover heated to a first predetermined cover temperature; Using the variable temperature heating module (117) to heat the first group of grooves (205) to a first heating temperature; as well as A hybridization reaction is performed in the first group of grooves (205) to obtain a reacted mixed solution.

17. The method (300) according to claim 15, wherein the plurality of grooves (113) of the hybridization and capture device (100) comprises a second group of grooves (207) containing capture magnetic beads and a third group of grooves (208) containing capture magnetic bead washing solution, and The magnetic bead washing comprises: Transferring the captured magnetic beads in the second group of grooves (207) to the third group of grooves (208) by using the group of electromagnetic rods (120) in a state of being magnetized but not heated; Performing a mixing operation using the set of electromagnetic rods (120) in a non-magnetic and non-heated state to wash the captured magnetic beads; as well as The washed capture magnetic beads are taken out from the third set of grooves (208) using the set of electromagnetic rods (120) in a state of being magnetized but not heated.

18. The method (300) according to claim 15, wherein the plurality of grooves (113) of the hybridization and capture device (100) further comprises a fourth groove (201) containing a magnetic bead resuspension solution, and The magnetic bead resuspension step comprises: Using the constant temperature heating module (118) of the hybridization and capture device (100) to heat the fourth group of grooves (201) to a second heating temperature; Using the set of electromagnetic rods (120) in a magnetized and heated state, the washed capture magnetic beads are placed into the fourth set of slots (201); Performing a mixing operation using the set of electromagnetic rods (120) in a non-magnetic but heated state to re-suspend the captured magnetic beads; as well as The capture magnetic beads are taken out from the fourth group of grooves (201) for capture by using the group of electromagnetic rods (120) in a magnetized and heated state.

19. The method (300) of claim 15, wherein performing the library capture reaction comprises: Using the set of electromagnetic rods (120) in a magnetized and heated state, the captured magnetic beads that have been resuspended by the magnetic beads are placed in the mixed solution that has undergone the hybridization reaction in the first set of grooves (205); Performing a mixing operation in the first group of slots (205) using the group of electromagnetic rods (120) in a non-magnetic but heated state; The first set of grooves (205) is sealed using the cover heated to a second predetermined cover temperature to allow the capture magnetic beads to be incubated in the mixed solution.

20. The method (300) according to claim 15, wherein the plurality of groups of slots (113) of the hybridization and capture device (100) further comprises a fifth group of slots (202) containing a first hot washing solution, a sixth group of slots (203) containing a second hot washing solution, and a seventh group of slots (204), and The hot washing step further comprises: Using the constant temperature heating module (118) of the hybridization and capture device (100) to heat the fifth group of grooves (202), the sixth group of grooves (203) and the seventh group of grooves (204) to a third heating temperature; Using the set of electromagnetic rods (120) in a magnetized and heated state, the captured magnetic beads having captured the library are taken out from the first set of grooves (205); A hot washing operation is performed in the fifth group of slots (202), the sixth group of slots (203) and the seventh group of slots (204) respectively, wherein the hot washing operation comprises: Using the set of electromagnetic rods (120) in a magnetized and heated state, the captured magnetic beads are placed in a corresponding set of grooves; Using the group of electromagnetic rods (120) in a non-magnetic but heated state to perform a mixing operation in a corresponding group of slots; and The captured magnetic beads are taken out from a corresponding group of grooves by using the group of electromagnetic rods (120) in a magnetized and heated state.

21. The method (300) according to claim 15, wherein the plurality of groups of grooves (113) of the hybridization and capture device (100) include an eighth group of grooves (209) containing a first washing solution, a ninth group of grooves (210) containing a second washing solution, and a tenth group of grooves (211) containing a third washing solution, and The room temperature washing also includes: Maintaining the eighth group of grooves (209), the ninth group of grooves (210), and the tenth group of grooves (211) at room temperature; as well as The captured magnetic beads that have been subjected to thermal washing are sequentially subjected to room temperature washing operations in the eighth group of slots (209), the ninth group of slots (210), and the tenth group of slots (211), wherein the room temperature washing operations include: Using the group of electromagnetic rods (120) in a state of being magnetized but not heated, the captured magnetic beads are placed in a corresponding group of grooves; Performing a mixing operation in a corresponding group of slots using the group of electromagnetic rods (120) in a non-magnetic and non-heated state; and The captured magnetic beads are taken out from a corresponding group of grooves using the group of electromagnetic rods (120) in a state of being magnetized but not heated.

22. The method (300) according to claim 15, wherein the plurality of groups of grooves (113) of the hybridization and capture device (100) include an eleventh group of grooves (206) containing polymerase chain reaction (PCR) reaction solution; The method further comprises performing a PCR reaction, comprising: Using the set of electromagnetic rods (120) in a magnetized and heated state, the captured magnetic beads having the captured library are placed into the eleventh set of grooves (206); The PCR reaction is performed by adjusting the temperature of the eleventh group of wells (206) using the variable temperature heating module (117) of the hybridization and capture device (100).

23. The method (300) according to claim 15, wherein the plurality of groups of grooves (113) of the hybridization and capture device (100) include a twelfth group of grooves (213) containing an eluent and a thirteenth group of grooves (212) containing ethanol, and, The method further comprises performing nucleic acid purification, comprising: Aligning a group of pre-stored cavities (153) containing purified magnetic beads on the cover (150) of the hybridization and capture device (100) with an eleventh group of grooves (206) containing a reaction solution subjected to a PCR reaction; as well as Using the group of electromagnetic rods (120) to squeeze the purified magnetic beads, so that the purified magnetic beads push open the pressure gates of the group of pre-stored cavities (153) and enter the eleventh group of slots (206); Performing a mixing operation using the set of electromagnetic rods (120) in a non-magnetic and non-heated state to incubate the purified magnetic beads; Using the group of electromagnetic rods (120) in a state of being magnetized but not heated, the capture magnetic beads and the purification magnetic beads are transferred from the eleventh group of grooves (206) to the thirteenth group of grooves (212) to perform a washing operation; Using the group of electromagnetic rods (120) in a magnetically-permeable but non-heated state, the capture magnetic beads and the purification magnetic beads are transferred from the thirteenth group of slots (212) to the twelfth group of slots (213), so that the capture magnetic beads and the purification magnetic beads are incubated in the eluent; as well as The magnetic beads are removed from the twelfth group of grooves (213) by using the group of electromagnetic rods (120) in a state of being magnetized but not heated, so as to obtain the captured library.

24. An apparatus (10) for library hybridization and capture, comprising: at least one processing unit; At least one memory, the at least one memory is coupled to the at least one processing unit and stores instructions for execution by the at least one processing unit, the instructions, when executed by the at least one processing unit, causing the device (10) to perform a method (300) for library hybridization and capture according to any one of claims 15 to 23.

Images