THREE-DEGREE-OF-FREEDOM LIBRARY PREPARATION CASSETTE AND METHOD USING THE SAME

Abstract

The present invention relates to a three-degree-of-freedom library preparation cassette and a method using the same. Two-degree-of-freedom movement capability is provided for mutual positioning between a pipette and reagent wells, within a limited small volume, by combining rotation of a mixing reagent tray and relative horizontal movement between the pipette and the mixing reagent tray, thus greatly increasing the number of reagent wells that can be provided, thereby increasing the functions and applications of the whole system; and the device is simple in structure, low in cost, and suitable for disposable use.

Claims

1. A three-degree-of-freedom library preparation cassette, comprising a pipette and a mixing reagent tray arranged within the sealed cassette, wherein the pipette is at least vertically movably arranged within the sealed cassette; the mixing reagent tray is a circular tray and is rotatable clockwise or counterclockwise at least about a geometric center thereof as an axis; the mixing reagent tray is provided with a plurality of reagent wells, which are configured to at least include two different well pitches; a downward projection relative straight-line distance between the pipette and the geometric center of the mixing reagent tray is changeable by horizontal movement of the pipette or the mixing reagent tray, so that the pipette corresponds vertically to any reagent well on the mixing reagent tray as selected and performs a liquid drawing or liquid injection operation on the reagent well; and the well pitches comprise a straight-line distance from the geometric center of the mixing reagent tray to a geometric center of the reagent well, and/or a straight-line distance from the geometric center of the mixing reagent tray to a position on the reagent well corresponding to the liquid drawing or liquid injection operation by the pipette.

2. The cassette according to claim 1, wherein the pipette moves horizontally from the geometric center of the mixing reagent tray to an edge of the mixing reagent tray along any straight line passing through the geometric center of the mixing reagent tray.

3. The cassette according to claim 2, wherein the mixing reagent tray is provided with a plurality of reagent wells arranged annularly successively in concentric circles with three different well pitches from inside to outside, forming three groups consisting of a plurality of reagent wells arranged successively along the concentric circles of different well pitches, respectively, namely, an inner circle reagent well group, a middle circle reagent well group and an outer circle reagent well group.

4. The cassette according to claim 3, wherein the inner circle reagent well group comprises at least one magnetic bead reagent well, which is detachable from the mixing reagent tray; the middle circle reagent well group comprises a plurality of library construction reagent wells; and the outer circle reagent well group at least comprises a plurality of reaction reagent wells, a plurality of library capture reagent wells, one cleaning reagent well, and one waste reagent well.

5. The cassette according to claim 4, wherein the magnetic bead reagent well, the library construction reagent wells, reaction reagent well, the library capture reagent wells, the cleaning reagent well and the waste reagent well have different volumes and/or shapes.

6. The cassette according to claim 4, wherein at least one of the plurality of reaction reagent wells is detachable from the mixing reagent tray.

7. The cassette according to claim 5, wherein the sum of downward projected areas of the magnetic bead reagent well, the library construction reagent wells, the reaction reagent wells, the library capture reagent wells, the cleaning reagent well and the waste reagent well occupies 90% or more of a downward projected area of the mixing reagent tray.

8. The cassette according to claim 2, wherein the mixing reagent tray is further integrally formed with a driven gear, and an external driving device drives, through gear engaged connection, the driven gear to rotate and cause the mixing reagent tray to rotate.

9. The cassette according to claim 8, wherein the driven gear does not occupy a top reagent well region of the mixing reagent tray.

10. The cassette according to claim 9, wherein the mixing reagent tray is further provided with a limit strip, which is controlled to lock/release the rotation of the mixing reagent tray.

11. The cassette according to claim 2, wherein the pipette is connected to a rotating shaft and a screw, respectively, ends of which extend out of the sealed cassette; the screw rotates and drives the pipette to move horizontally; and the rotating shaft rotates and causes a vertical driving gear provided on the rotating shaft to rotate and drive a pipette head component of the pipette to move vertically.

12. The cassette according to claim 11, wherein the rotating shaft is in clearance fit with the vertical driving gear provided on the rotating shaft, and the vertical driving gear is capable of freely moving axially along the rotating shaft; and the rotating shaft and the vertical driving gear are in circumferential limit connection having at least one tangential surface to transmit torque.

13. The cassette according to claim 11, wherein the ends of the rotating shaft and the screw extending out of the sealed cassette are each provided with both an inner spline and an outer spline; the external driving device transmits torque to the rotating shaft and the screw by combined action of an outer spline and an inner spline corresponding to the inner spline and the outer spline.

14. The cassette according to claim 1, wherein the cassette further comprises a mixing reagent tray base, a pipette pump assembly and a cassette housing; the mixing reagent tray base comprises a bottom seat and a top cover; the mixing reagent tray is placed within the mixing reagent tray base and is provided with vertical movement limits and horizontal plane movement limits by the bottom seat and the top cover; the pipette pump assembly provides a liquid drawing or liquid injection driving force for the pipette; and the cassette housing is independently provided with a hot lid capable of heating and thermally insulating specified reagent wells and a sampling through hole capable of opening/sealing and closing specified reagent wells.

15. A method of using a three-degree-of-freedom library preparation cassette, comprising the following steps: S1, cassette installation: installing a mixing reagent tray containing required reagents sealed therein, into a cassette housing by means of a mixing reagent tray base to form a complete cassette; S2, library preparation: connecting the complete cassette installed with the mixing reagent tray to an external driving device, and using the external driving device to operate the mixing reagent tray to rotate, and enable horizontal movement of the pipette and vertical movement of the pipette, to accomplish a library preparation process; and S3, reaction product drawing and waste disposal: disconnecting the used cassette from the external external driving device, and drawing reaction products from reaction reagent wells, and properly disposing the remaining part as a waste.

16. The method according to claim 15, wherein step S1 comprises mounting a detached separately stored magnetic bead reagent well loaded with magnetic beads onto the mixing reagent tray.

17. The method according to claim 15, wherein step S1 comprises locking the mixing reagent tray in an initial position of preparation by using a limit strip.

18. The method according to claim 17, wherein step S2 comprises releasing the mixing reagent tray to rotate by pushing the limit strip by the external driving device.

19. The method according to claim 15, wherein step S2 comprises heating and thermally insulating the reaction reagent wells provided in the mixing reagent tray by using a hot lid.

20. The method according to claim 15, wherein step S3 comprises drawing the preparation products from the reaction reagent wells through a sampling through hole.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 is a schematic diagram of a first embodiment of a three-degree-of-freedom library preparation cassette of the present invention.

[0042] FIG. 2 is an exploded diagram of the first embodiment of the three-degree-of-freedom library preparation cassette of the present invention.

[0043] FIG. 3 is a schematic diagram of an embodiment of a pipette of the present invention.

[0044] FIG. 4 is a top-view diagram of the first embodiment of a mixing reagent tray of the present invention.

[0045] FIG. 5 is a schematic diagram of an embodiment of assembly of the mixing reagent tray and a mixing reagent tray base of the present invention.

[0046] FIG. 6 is a schematic diagram of an embodiment of assembly of a three-degree-of-freedom library preparation cassette and an external driving device of the present invention.

[0047] FIG. 7 is a schematic diagram of distribution of reagent wells in a second embodiment of a mixing reagent tray of the present invention.

[0048] Reference numerals: 1—pipette, 11—rotating shaft, 12—screw, 2—mixing reagent tray, 21—reagent well, 211—magnetic bead reagent well, 212—library construction reagent well, 213—reaction reagent well, 214—cleaning reagent well, 215—waste reagent well, 216—library capture reagent well, 22—driven gear, 23—limit strip, 3—mixing reagent tray base, 31—bottom seat, 32—top cover, 4—pipette pump assembly, 5—cassette housing, 51—hot lid, 52—sampling through hole, 6—external driving device.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0049] For clearer understanding of the present invention, it will be described in detail in conjunction with the accompanying drawings and embodiments.

[0050] As shown in FIG. 1, which is an overall schematic diagram of the appearance of a first embodiment of a three-degree-of-freedom library preparation cassette of the present invention, it can be seen that most of main components of the cassette are arranged within a sealed cassette housing 5, and parts exposed outside the cassette housing 5 mainly include a rotating shaft 11 and a screw 12 for connecting an external driving device 6, and a driven gear 22, i.e., the cassette as shown in Embodiment 1 has three drive locations independent from each other and thus has the ability to provide three-degree-of-freedom driving. Also visible from the outside of the cassette housing 5 are a hot lid 51 for heating and thermally insulating some specified wells of reagent wells 21 and a sampling through hole 52 for drawing liquid from some specified wells of the reagent wells 21; the sampling through hole 52 is mainly used for drawing prepared library reagents, so the sampling through hole 52 needs to be kept sealed and closed during library preparation and is opened only when samples need to be put before library preparation and when the prepared library reagents, that is the preparation products, need to be carried out after preparation.

[0051] As shown in FIG. 2, which is an exploded view of the first embodiment, the structure of the interior of the cassette in the first embodiment can be seen from FIG. 2. A mixing reagent tray 2 with a number of reagent wells 21 is detachably mounted on the bottom of the cassette housing 5 by being arranged in a mixing reagent tray base 3; a pipette 1 is connected with the rotating shaft 11 and the screw 12 successively, and a pipette pump assembly 4 connected is used to provide a driving force for liquid drawing and injection operations. At a lower end of the pipette 1 is a pipette head component for drawing and injecting liquid. By rotation of the screw 12, the pipette 1 can be controlled to move forward and backward along the screw, and in conjunction with rotation of the mixing reagent tray 2, it enables vertical alignment of the pipette head component of the pipette 1 with any reagent well 21, and then by rotation of the rotating shaft 11 to drive the pipette head component of the pipette 1 to descent vertically, the pipette head component can puncture a thin film on the reagent well 21, extend into the aligned reagent well 21 and come into contact with a reagent stored in the reagent well 21 to perform a drawing operation. A similar operation process is applied to a reagent liquid injection operation.

[0052] As shown in FIG. 3, which is a schematic structural diagram of an embodiment of the pipette 1, it mainly explains how the pipette 1 described in the present invention is driven by a combination of the rotating shaft 11 and the screw 12 to achieve a horizontal and vertical two-degrees-of-freedom movement capability. The screw 12 responsible for driving the pipette 1 to move horizontally forward and backward is configured as a driving structure of a higher priority, and positive and reverse rotation of the screw 12 can drive the pipette 1 to move horizontally forward and backward overall; the rotating shaft 11 responsible for driving the pipette 1 to move vertically is a driving structure of a lower priority than the screw 12, and positive and reverse rotation of the rotating shaft 11 only drives the pipette head component of the pipette 1 to move vertically up and down; to ensure pipetting precision, the pipette head component of the pipette 1 first descends to the bottom of the well where pipetting is to be performed, the bottom of the well being used as an initial position, then moves up a certain distance, and thereby draws and/or releases the liquid; furthermore, torque transmission of the rotating shaft 11 is implemented by circumferential limit connection including at least one tangential surface, so that the torque transmission of the rotating shaft 11 does not affect the horizontal movement of the pipette 1, and simultaneous horizontal and vertical movements of the pipette 1 can be achieved.

[0053] Another focus of the present invention is a design of reagent wells with different well pitches to increase the number of reagent wells 21 that can be accommodated in the mixing reagent tray 2. In traditional library preparation cassette design, reagent wells are mostly arranged in a straight line or in a concentric circle with the same well pitch, which can reduce the structural complexity of pipetting components in the cassette, but also greatly limits the number of reagent wells that can be arranged, because in the case the volume of the cassette is controlled, in order to ensure effective drawing and injection of reagents, the area occupied by the reagent wells cannot be reduced indefinitely. As shown in FIG. 4, which is a schematic diagram of a preferred embodiment of a mixing reagent tray 2 that can be used in the cassette of the present invention, it achieves several times the number of reagent wells 21 in the same volume in the prior art by classifying and arranging the reagent wells 21 with different functions into three concentric circles with different well pitches, and uses a design of a driven gear 22 integrally formed in an outer circle. In this embodiment, an inner circle of the mixing reagent tray 2 is provided with four magnetic bead reagent wells 211, and the four magnetic bead reagent wells 211 can all be loaded with the same magnetic bead reagent to provide a sufficient amount of reagent, and may also be loaded with different magnetic bead reagents respectively as needed so that a single cassette can support the use of multiple types of magnetic bead reagents at the same time, and the section of the magnetic bead reagent wells 211 is designed to be detachable from the mixing reagent tray 2 so that the magnetic bead reagent can be stored separately; from inside to outside, a middle circle of the mixing reagent tray 2 is provided with up to sixteen library construction reagent wells 212 independent from each other, which can be loaded with the same or different reagents as needed, thus greatly increasing functions that can be achieved by the cassette described in the present invention; an outer circle part is independently provided with a total of six reaction reagent wells, every two of which are arranged in a group, respectively, for implementing the main reaction process, wherein one or more groups of reaction reagent wells can be detachable from the mixing reagent tray and can be replaced with other functional modules (e.g. QC modules) as needed, to further increase the use functions of the cassette; the outer circle of the reagent tray 2 is further provided with thirteen library capture reagent wells 216 independent from each other, which are loaded with various reagents used in library capture, thus greatly increasing the functions that can be achieved by the cassette described in the present invention; the remaining part of the outer circle is provided with a plurality of irregular reagent wells 21, including cleaning reagent wells 214 and waste reagent wells 215 indispensable for experiments, the cleaning reagent wells 214 and waste reagent wells 215 making full use of the remaining space on the mixing reagent tray 2, ultimately achieving the effect that more than 90% of the area of the mixing reagent tray 2 is effectively used.

[0054] As shown in FIG. 5, which is a schematic diagram of an embodiment of assembly of the mixing reagent tray and the mixing reagent tray base of the present invention, using the mixing reagent tray base 3 can limit vertical and horizontal movements of the mixing reagent tray 2 by a bottom seat 31 and a top cover 32, and enables the mixing reagent tray 2 to be detachably mounted to the cassette housing 5. Furthermore, to facilitate the use of the cassette, the mixing reagent tray 2 is also provided with a limit strip 23 for rotational limit. In the embodiment shown in FIG. 5, the limit strip 23 is a mechanical limit structure, which is inserted into the mixing reagent tray base 3 so that its end limit tooth comes into matched contact with the driven gear 22 to limit rotation of the mixing reagent tray 2. This can simplify the operation process during initialization of the cassette, and facilitates initialization positioning.

[0055] The connection between the cassette as a whole and the external driving device 6 can be as shown in FIG. 6. The rotating shaft 11 and the screw 12 each transmit torque through an inner spline and an outer spline protruding from the cassette housing 5, and the external driving device 6 is provided with corresponding outer splines and inner splines, so that the torque is shared by using matched connections of the two sets of inner and outer splines in the torque transmission process, and the problem of precision reduction caused by device abrasion can be effectively avoided; and the mixing reagent tray 2 is driven by engaged connection of the driven gear 22 and a driving gear on the external driving device 6 to achieve three-degree-of-freedom driving capability of the cassette as a whole. In this configuration, main driving structures are arranged on the external driving device 6 and do not need direct contact with the reagents, and can be used repeatedly; and the cassette part has a simple structure and a low cost, and is suitable for disposable use to avoid reagent contamination.

[0056] To further illustrate the three-degree-of-freedom library preparation cassette described in the present invention, a typical library construction process is described using a second embodiment of the mixing reagent tray 2 with distribution of the reagent wells 21 as shown in FIG. 7. It is to be noted that all numerical symbols marked in FIG. 7 are independent numbers of different reagent wells 21 thereof, i.e., the mixing reagent tray 2 has 47 mutually independent reagent wells 21 numbered from 1 to 47, as shown in FIG. 7. To avoid confusion, in the following description of the library construction process, specific reagent wells 21 are denoted in the form of w+number, e.g. w1, w6 or w30. Reagent well numbers and corresponding reagent well names/types are provided in Table 1 below.

TABLE-US-00001 TABLE 1 Reagent well number Reagent well name/type  w1 Reaction well 1  w2 Reaction well 2  w3 Waste liquid well  w5 Magnetic bead washing reagent  w6 Magnetic bead mixing reagent w10 Captured magnetic bead washing reagent 1 w11 w12 Magnetic bead washing reagent w19 Captured magnetic bead washing reagent 2 w20 Captured magnetic bead washing reagent 2 w21 Captured magnetic bead washing reagent 1 w22 Captured magnetic bead washing reagent 1 w23 Captured magnetic bead washing reagent 3 w25 Captured magnetic bead washing reagent 4 W26.sup.  Library elution reagent w27 Magnetic bead washing reagent w28 End-filling reagent w30 Connecting reagent 1 w31 Connecting reagent 2 w32 PCR amplification reagent 1 w33 PCR amplification primer 1 w37 Blocking reagent w38 Capture Reagent w40 Capture reagent 2 w42 PCR amplification reagent 2 w43 PCR amplification primer 2 w45 magnetic bead w46 Captured magnetic bead w47 magnetic bead

[0057] Library preparation using the mixing reagent tray 2 as shown in FIG. 7 includes the following steps: [0058] a DNA is transferred to w1; [0059] an end-filling reagent is transferred from w28 to w1; [0060] mixing is performed; [0061] a bottom module is rotated; [0062] a PCR module covers the tube bottom; [0063] a hot lid is lowered to cover w1 and w2; [0064] reaction is performed; [0065] reaction is completed; [0066] the bottom module leaves w1; [0067] the hot lid leaves w1 and w2; [0068] a connecting reagent 1 is transferred from w30 to w1; [0069] a connecting reagent 2 is transferred from w31 to w1; [0070] mixing is performed sufficiently; [0071] a heating module is raised; [0072] heated reaction is performed; [0073] reaction is completed; [0074] the heating module is removed; [0075] magnetic beads are mixed; [0076] magnetic beads are transferred from w45 or w47 to w1; [0077] mixing is performed sufficiently; [0078] standing is performed for certain time; [0079] the bottom is rotated, and a magnetic module covers the underside of w1; [0080] standing is performed for certain time; [0081] a supernatant is removed; [0082] a washing reagent is transferred from any of w5, w12 and w27 to w1; [0083] standing is performed for certain time; [0084] a supernatant is removed; [0085] a washing reagent is transferred from any of w5, w12 and w27 to w1; [0086] standing is performed for certain time; [0087] a supernatant is removed; [0088] the magnetic module leaves w1; [0089] a PCR amplification reagent 1 is transferred from w32 to w1; [0090] a PCR amplification primer 1 is transferred from w33 to w1; [0091] mixing is performed sufficiently; [0092] the PCR module covers w1; [0093] the hot lid covers w1 and w2; [0094] PCR reaction is performed; [0095] the bottom PCR module is removed; [0096] the hot lid is removed; [0097] a magnetic bead mixing reagent is transferred from w6 to w1; [0098] mixing is performed sufficiently and evenly; [0099] standing is performed for certain time; [0100] the bottom is rotated, and the magnetic module covers the underside of w1; [0101] standing is performed for certain time; [0102] a supernatant is removed; [0103] a washing reagent is transferred from any of w5, w12 and w27 to w1; [0104] standing is performed for certain time; [0105] a supernatant is removed; [0106] a washing reagent is transferred from any of w5, w12 and w27 to w1; [0107] standing is performed for certain time; [0108] a supernatant is removed; [0109] the magnetic module leaves w1; [0110] an eluent is transferred to w1; [0111] mixing is performed sufficiently and evenly; [0112] standing is performed for certain time; [0113] the bottom is rotated, and the magnetic module covers the underside of w1; [0114] standing is performed for certain time; [0115] a supernatant is drawn from w1 into w2; [0116] library construction is completed; [0117] a cleaning solution is drawn to clean w1; [0118] all liquid in w1 is transferred away; [0119] a blocking solution is added from w37 to w2; [0120] magnetic beads are mixed uniformly; [0121] magnetic beads of a fixed volume are drawn from w45 or w47 into w2; [0122] mixing is performed sufficiently and evenly; [0123] standing is performed for certain time; [0124] the bottom is rotated, and the magnetic module covers the underside of w2; [0125] standing is performed for certain time; [0126] a supernatant is removed; [0127] a washing reagent is transferred from any of w5, w12 and w27 to w2; [0128] standing is performed for certain time; [0129] a supernatant is removed; [0130] a washing reagent is transferred from any of w5, w12 and w27 to w2; [0131] standing is performed for certain time; [0132] a supernatant is removed; [0133] the magnetic module leaves w2; [0134] a capture reagent is transferred from w38 to w2; [0135] mixing is performed sufficiently and evenly; [0136] standing is performed for certain time; [0137] the bottom is rotated, and the magnetic module covers the underside of w2; [0138] standing is performed for certain time; [0139] a supernatant is transferred from w2 to w1; [0140] a cleaning solution is drawn to clean w2; [0141] all liquid in w2 is transferred away; [0142] a bottom heating module is raised to cover the underside of w1 and w2; [0143] the hot lid is lowered to cover w1 and w2; [0144] heated reaction is performed; [0145] a side magnetic heating module covers w10 and w11; [0146] a bottom magnetic heating module is raised to cover the underside of w19 and w20; [0147] captured magnetic beads are cleaned; [0148] the captured magnetic beads are mixed; [0149] the capture beads are transferred from w46 to w11; [0150] standing is performed for certain time; [0151] all supernatant in w11 is removed; [0152] a captured magnetic bead cleaning reagent is transferred to w11; [0153] standing is performed for certain time; [0154] all supernatant in w11 is removed; [0155] a captured magnetic bead cleaning reagent is transferred to w11; [0156] standing is performed for certain time; [0157] all supernatant in w11 is removed; a capture reagent 2 is transferred from w40 to w11; [0158] the hot lid is opened; [0159] all the reagents in w11 are transferred to w2; [0160] mixing is performed sufficiently and evenly; [0161] the hot lid is closed; [0162] standing is performed for certain time; [0163] the hot lid is opened; [0164] a capture washing reagent 1 is drawn from w21 or w22 into w2; [0165] the reagents in w2 are mixed sufficiently and evenly; [0166] the bottom module is replaced with a side magnetic module; [0167] w10 and w11 are interchanged, and w19 and w20 are interchanged, synchronously, and the temperature is maintained; [0168] standing is performed for certain time; [0169] a supernatant is drawn and removed from w2; [0170] a capture washing reagent 2 is drawn from w19 or w20 into w2; [0171] the bottom module is replaced with the heating module; [0172] w10 and w11 are interchanged, and w19 and w20 are interchanged, synchronously, and the temperature is maintained; [0173] mixing is performed sufficiently and evenly; [0174] standing is performed for certain time; [0175] the bottom module is replaced with the side magnetic module; [0176] w10 and w11 are interchanged, and w19 and w20 are interchanged, synchronously, and the temperature is maintained; [0177] a supernatant is drawn and removed from w2; [0178] a capture washing reagent 2 is drawn from w19 or w20 into w2; [0179] the bottom module is replaced with the heating module; [0180] w10 and w11 are interchanged, and w19 and w20 are interchanged, synchronously, and the temperature is maintained; [0181] mixing is performed sufficiently and evenly; [0182] standing is performed for certain time; [0183] the bottom module is replaced with the side magnetic module; [0184] a supernatant is drawn and removed from w2; [0185] all bottom modules are removed; [0186] a magnetic bead washing reagent (room temperature) is drawn from w5 into w2; [0187] mixing is performed sufficiently and evenly; [0188] the side magnetic module covers w2; [0189] standing is performed for certain time; [0190] a supernatant is drawn and removed; [0191] a capture washing reagent 3 is drawn from w23 into w2; [0192] the bottom module is removed; [0193] mixing is performed sufficiently and evenly; [0194] the side magnetic module covers w2; [0195] standing is performed for certain time; [0196] a supernatant is drawn and removed; [0197] a capture washing reagent 4 is drawn from w25 into w2; [0198] the bottom module is removed; [0199] mixing is performed sufficiently and evenly; [0200] the side magnetic module covers w2; [0201] standing is performed for certain time; [0202] a supernatant is drawn and removed; [0203] the bottom module is removed; [0204] a capture PCR amplification reagent 2 is drawn from w42 into w2; [0205] a capture PCR amplification primer 2 is drawn from w43 into w2; [0206] mixing is performed sufficiently and evenly; [0207] the bottom PCR module covers w1 and w2; [0208] the hot lid is lowered to cover w1 and w2; [0209] PCR reaction is performed; [0210] PCR reaction is completed and the hot lid is removed; [0211] the PCR module is removed; [0212] magnetic beads are mixed; [0213] magnetic beads of a certain volume are added from w45 or w47 into w2; [0214] mixing is performed sufficiently and evenly; [0215] standing is performed for certain time; [0216] the side magnetic module covers w2; [0217] standing is performed for certain time; [0218] a supernatant is drawn and removed; [0219] a magnetic bead washing reagent 2 is drawn into w2; [0220] standing is performed for certain time; [0221] a supernatant is drawn and removed; [0222] a magnetic bead washing reagent 2 is drawn into w2; [0223] standing is performed for certain time; [0224] a supernatant is drawn and removed; [0225] a library elution reagent is drawn from w26 into w2; [0226] the bottom module is removed; [0227] mixing is performed sufficiently and evenly; [0228] the side magnetic module is raised; [0229] standing is performed for certain time; [0230] a supernatant is drawn into w1; [0231] the bottom module is removed; [0232] the hot lid covers w1 and w2 to prevent volatilization; and [0233] capture library construction is complete.

[0234] The use of the three-degree-of-freedom library preparation cassette described in the present invention can also simplify amplification operation steps in the prior art. Again with reference to the distribution of reagent wells 21 as shown in FIG. 7, reagent well names/types as shown in Table 2 can preferably be used in amplification operations.

TABLE-US-00002 TABLE 2 Reagent well number Reagent well name/type w1 Reaction well 1 w2 Reaction well 2 w3 PCR1 solution chamber w4 Primer chamber w5 Washing solution 1 chamber w6 Eluent chamber w7 PCR2 reaction solution chamber w8 Washing solution 2 chamber w9 Waste liquid chamber w10  Recovery chamber w44  Magnetic bead chamber

[0235] Specific amplification operation steps include: [0236] the position of the cassette is initialized, and it is confirmed that each well is at a correct position; [0237] the cassette is rotated to move to a predetermined position; [0238] a pipetting device is moved to a specified position; [0239] a bottom disc is rotated so that W3 is under a suction pipette; [0240] a transfer pipette punctures a foil seal on w3; [0241] the transfer pipette further goes down into W3 and identifies the bottom position; [0242] the transfer pipette is raised to a predetermined height position; [0243] the disc is rotated so that w1 is at a specified position; [0244] the transfer pipette transfers liquid into w1; [0245] the transfer pipette returns to a predetermined height; [0246] the transfer pipette punctures a foil seal on w4; [0247] the transfer pipette further goes down into w4 and identifies the bottom position; [0248] a certain amount of liquid is drawn; [0249] the transfer pipette is raised to a predetermined height position; [0250] the disc is rotated so that w1 is at a specified position; [0251] the transfer pipette transfers the liquid into w1; [0252] the transfer pipette evenly mixes the liquid in w1 by repeated operations of suction and release; [0253] the transfer pipette returns to a predetermined height; [0254] the hot lid is pressed down to cover the position of w1; [0255] a base PCR unit is raised to cover the underside of w1; [0256] the base unit starts an entire PCR cyclic process; [0257] after the process is completed, the hot lid leaves w1; [0258] a bottom module leaves w1; [0259] the transfer pipette is moved to w44; [0260] a certain volume of magnetic beads is drawn; [0261] the transfer pipette is raised to a predetermined height position; [0262] the transfer pipette is moved to a fixed position above w1; [0263] a fixed volume of magnetic beads is added into w1; [0264] the transfer pipette evenly mixes the liquid in w1 by up and down suction and release; [0265] the transfer pipette leaves w1 to a specified height position; [0266] standing is performed for certain time; [0267] a module with an annular magnetic bracket at the bottom is moved to cover the underside of w1; [0268] standing is performed for certain time; [0269] the transfer pipette enters w1 and draws a certain amount of liquid; [0270] the transfer pipette leaves w1 to a specified height position; [0271] the bottom is rotated so that w9 is placed under the transfer pipette; [0272] the liquid is transferred to w9; [0273] the transfer pipette is moved to a fixed height; [0274] the bottom disc is rotated so that w5 is under the transfer pipette; [0275] the transfer pipette sucks a certain volume of washing solution; [0276] the transfer pipette is moved to a fixed height; [0277] the disc is rotated so that w1 is under the transfer pipette; [0278] the transfer pipette transfers the liquid into w1; [0279] the transfer pipette is moved to a fixed height; [0280] standing is performed for certain time; [0281] the transfer pipette is moved into w1 and draws a certain amount of liquid; [0282] the transfer pipette is moved to a fixed height; [0283] the bottom is rotated so that w9 is placed under the transfer pipette; [0284] the liquid is transferred to w9; [0285] the transfer pipette is moved to a fixed height; [0286] the bottom disc is rotated such that w5 is under the transfer pipette; [0287] the transfer pipette sucks a certain volume of washing solution; [0288] the transfer pipette is moved to a fixed height; [0289] the disc is rotated so that w1 is under the transfer pipette; [0290] the transfer pipette transfers the liquid into w1; [0291] the transfer pipette is moved to a fixed height; [0292] standing is performed for certain time; [0293] the transfer pipette is moved into w1 and draws a certain amount of liquid; [0294] the transfer pipette is moved to a fixed height; [0295] the bottom is rotated so that w9 is placed under the transfer pipette; [0296] the liquids is transferred to w9; [0297] the transfer pipette is moved to a fixed height; [0298] the bottom disc is rotated so that w7 is under the transfer pipette; [0299] the transfer pipette draws a certain volume of reagent; [0300] the transfer pipette is moved to a fixed height; [0301] the bottom module leaves w1; [0302] the bottom disc is rotated so that the position of w1 is under the transfer pipette; [0303] the transfer pipette releases the liquid into w1; [0304] the transfer pipette evenly mixes the liquid in w1 by suction and release; [0305] the transfer pipette is moved to a fixed height; [0306] standing is performed for certain time; [0307] a bottom magnetic module is raised to cover the underside of w1; [0308] standing is performed for certain time; [0309] the transfer pipette sucks a certain volume of liquid; [0310] the transfer pipette is moved to a fixed height; [0311] the bottom disc is rotated so that w2 is under the transfer pipette; [0312] the transfer pipette releases the liquid into w2; [0313] the bottom magnetic module is detached from w2; [0314] the bottom module covers the position of w2; [0315] the hot lid is lowered to cover w2; [0316] reaction is performed with the module; [0317] after the process is complete, the hot lid leaves w2; [0318] the bottom module leaves w2; [0319] the transfer pipette is moved to w44; [0320] a certain volume of magnetic beads is drawn; [0321] a transfer pipette spring rises such that the tip of the transfer pipette leaves w44 to a predetermined height position; [0322] the transfer pipette is moved to a fixed position above w2; [0323] a fixed volume of magnetic beads is added into w2; [0324] the transfer pipette evenly mixes the liquid in w2 by up and down suction and release; [0325] the transfer pipette leaves w2 to a specified height position; [0326] standing is performed for certain time; [0327] the module with the annular magnetic bracket at the bottom is moved to cover the underside of w2; [0328] standing is performed for certain time; [0329] the transfer pipette enters w2 and draws a certain amount of liquid; [0330] the transfer pipette leaves w2 to a specified height position; [0331] the bottom is rotated so that w9 is placed under the transfer pipette; [0332] the liquid is transferred to w9; [0333] the transfer pipette is moved to a fixed height; [0334] the bottom disc is rotated so that w8 is under the transfer pipette; [0335] the transfer pipette sucks a certain volume of washing solution; [0336] the transfer pipette is moved to a fixed height; [0337] the disc is rotated so that w2 is under the transfer pipette; [0338] the transfer pipette transfers the liquid into w2; [0339] the transfer pipette is moved to a fixed height; [0340] standing is performed for certain time; [0341] the transfer pipette is moved into w2 and draws a certain amount of liquid; [0342] the transfer pipette is moved to a fixed height; [0343] the bottom is rotated so that w9 is placed under the transfer pipette; [0344] the liquid is transferred to w9; [0345] the transfer pipette is moved to a fixed height; [0346] the bottom disc is rotated so that w8 is under the transfer pipette; [0347] the transfer pipette sucks a certain volume of washing solution; [0348] the transfer pipette is moved to a fixed height; [0349] the disc is rotated so that w2 is under the transfer pipette; [0350] the transfer pipette transfers the liquid into w2; [0351] the transfer pipette is moved to a fixed height; [0352] standing is performed for certain time; [0353] the transfer pipette is moved into w2 and draws a certain amount of liquid; [0354] the transfer pipette is moved to a fixed height; [0355] the bottom disc is rotated so that w10 is moved to under the transfer pipette; [0356] the liquid is released to w10; [0357] the transfer pipette returns to a specified position; [0358] hot lid is pressed down to over w10; and [0359] the process is completed.

[0360] Described above are only preferred specific implementations of the present invention, but the protection scope of the present invention is not limited thereto, and all changes or substitutions that are readily conceivable to those skilled in the art within the technical scope disclosed by the present invention should be encompassed within the protection scope of the present invention. Thus, the protection scope of the present invention should be defined by the claims.