阅读说明：本技术 用于制备测序设备的系统和方法 (System and method for preparing sequencing device ) 是由 H.于 X.杨 R.郑 C.T.A.王 J.格雷 于 2019-08-16 设计创作，主要内容包括：本公开一般涉及用于磁珠加载的系统,方法和装置。本公开的一个示例性实施例涉及将磁珠与测序珠子混合以形成溶液。将含有两种珠子的溶液注射到具有多个微井的微芯片上。磁珠可以具有比微井更大的直径,而测序珠子可以具有更小的直径,允许它们进入并存在于微井中。位于微芯片下方的一个或多个磁体在微芯片表面上来回移动。磁珠形成一条线并跟随磁体的运动。在扫描轮次期间,测序珠子加载到相应的井中。在加载测序珠子之后可以使磁体脱离并且可以洗掉磁珠。(The present disclosure relates generally to systems, methods, and devices for magnetic bead loading. One exemplary embodiment of the present disclosure relates to mixing magnetic beads with sequencing beads to form a solution. The solution containing both beads was injected onto a microchip having a plurality of microwells. The magnetic beads may have a larger diameter than the microwells, while the sequencing beads may have a smaller diameter, allowing them to enter and reside in the microwells. One or more magnets located below the microchip are moved back and forth across the surface of the microchip. The magnetic beads form a line and follow the movement of the magnet. During the scan round, sequencing beads were loaded into the respective wells. The magnet can be detached after loading the sequencing beads and the magnetic beads can be washed away.)

1. A method of loading a bead support into a reaction well of a plurality of reaction wells of a substrate, each reaction well having an inlet opening at a first surface of the substrate, the method comprising:

introducing a suspension having a plurality of bead complexes onto a substrate, a bead complex of the plurality of bead complexes comprising a magnetic bead coupled to a bead support;

moving the magnetic device parallel to a second surface of the substrate, the second surface being opposite the first surface, the magnetic beads being attracted to the first surface, the bead support entering a reaction well of the plurality of reaction wells;

separating the magnetic beads from the bead support; and

the magnetic beads were washed off the substrate.

2. The method of claim 1, wherein the bead diameter of the magnetic bead is larger than the opening of the plurality of reaction wells, and wherein the bead diameter of the bead support is smaller than the opening of the plurality of reaction wells.

3. The method of claim 1, wherein the magnetic device comprises a pair of magnets separated by an inert material.

4. The method of claim 3, wherein a first magnet of the pair of magnets has a north pole disposed adjacent the second surface of the substrate and a second magnet of the pair of magnets has a south pole disposed adjacent the second surface of the substrate.

5. The method of claim 1, wherein the bead support is a sequencing bead having a polynucleotide thereon.

6. The method of claim 5, further comprising amplifying the polynucleotide to provide multiple copies of the polynucleotide on the sequencing bead.

7. The method of claim 5, further comprising sequencing polynucleotides attached to the bead supports in the reaction wells of the substrate.

8. The method of claim 1, wherein moving the magnetic device parallel to the second surface of the substrate comprises moving the magnetic device in different directions parallel to the second surface of the substrate.

9. The method of claim 1, wherein the bead support is coupled to a polynucleotide having a linker moiety disposed distally of the bead support, and the magnetic bead has a complementary linker moiety, and wherein a bead complex is formed when the linker moiety of the bead support is linked to the complementary linker moiety of the magnetic bead.

10. The method of claim 9, wherein the polynucleotide having a linker moiety is hybridized to a second polynucleotide covalently bound to a bead support, wherein separating the magnetic bead from the bead support comprises separating the polynucleotide from the second polynucleotide.

11. The method of claim 10, wherein separating the polynucleotide from the second polynucleotide comprises washing with an aqueous solution of low ionic strength.

12. The method of claim 10, wherein separating the polynucleotide from the second polynucleotide comprises heating the substrate.

13. The method of claim 1, further comprising:

generating a template nucleic acid comprising a capture sequence portion, a template portion, and a primer portion modified with a linker portion;

capturing the template nucleic acid on a bead support, the bead support having a plurality of capture primers complementary to a capture sequence portion of the template nucleic acid, the capture primers hybridizing to the capture sequence portion of the template nucleic acid; and

the captured template nucleic acid is ligated to a magnetic bead having a second linker moiety attached to the first linker moiety to form a bead complex.

14. The method of claim 13, further comprising extending a capture primer complementary to the template nucleic acid to form a sequence target nucleic acid attached to the bead support.

15. The method of claim 14, further comprising denaturing the template nucleic acid and the sequence target nucleic acid to release the magnetic beads from the bead support.

16. The method of claim 15, wherein denaturing comprises enzymatic denaturation.

17. The method of claim 15, wherein denaturing comprises denaturing in the presence of an ionic solution.

18. The method of claim 15, further comprising amplifying the sequence target nucleic acid to form a population of sequence target nucleic acids on the bead support in the reaction well.

19. The method of claim 18, wherein amplifying comprises performing Recombinase Polymerase Amplification (RPA).

20. The method of claim 19, wherein performing RPA comprises performing RPA for a first period of time, washing, performing RPA for a second period of time, the first period of time being shorter than the second period of time.

21. The method of claim 13, wherein generating comprises extending a primer comprising an adaptor modification complementary to the target nucleic acid.

22. The method of claim 13, wherein the generating comprises amplifying the target nucleic acid having the first primer portion, the target portion, and the second primer portion in the presence of a bead support having a capture primer, an adaptor-modified first primer complementary to the first primer portion, and a second primer having a portion complementary to at least a portion of the second primer portion, the second primer having a capture primer portion attached to the portion and complementary to the capture primer, wherein the bead support capture primer is extended to include a sequence of the target nucleic acid.

23. The method of claim 22, wherein amplifying comprises performing three Polymerase Chain Reaction (PCR) cycles.

Background

Biological and medical research is increasingly turning to sequencing for enhanced biological research and medicine. For example, biologists and zoologists are turning to sequencing to study the migration of animals, the evolution of species, and the origin of traits. Sequencing is being turned to by the medical community to study the origin of the disease, sensitivity to drugs and origin of infection. However, sequencing has historically been an expensive process, thus limiting its practice.

Among other problems, there are challenges in loading beads modified with nucleic acid molecules into a confinement region or receptacle (e.g., microwell or well) to form an array for sequencing. Placing sequencing beads in an organized, close-packed manner, for example, in small microwells, can increase throughput per cycle and reduce customer costs. As microwell density increases or microwell size decreases, bead loading becomes difficult, resulting in more open microwells and low bead counts in the wells. Too many open microwells provide reduced base reads and therefore poor sequencing performance.

Disclosure of Invention

In one example, a method of preparing a sequencing apparatus includes linking a bead support having captured template nucleic acids modified with a linker moiety to a magnetic bead having a complementary linker moiety to form a bead assembly, and loading the bead assembly into a well of the sequencing apparatus using a magnetic field. The bead assembly can be denatured to release the magnetic beads, leaving the bead supports attached to the target nucleic acids in the wells. The target nucleic acid can be amplified to provide a population of cloned target nucleic acids that can be used to sequence the target nucleic acid.

In another example, an apparatus includes a plate including a surface for receiving a substrate having a plurality of wells; a bar magnet adjacent to a surface of the plate opposite to the surface of the receiving substrate; and a driving mechanism for moving the bar magnet parallel to the surface of the plate. The substrate is used to receive a solution comprising magnetic beads, such as sequencing beads, coupled to a bead support. The diameter of the magnetic bead is greater than the diameter of a well of the plurality of wells. The movement of the magnet facilitates the deposition of the bead support into the well.

Drawings

The present disclosure may be better understood, and its numerous features and advantages made apparent to those skilled in the art by referencing the accompanying drawings.

FIG. 1 includes a schematic representation of an exemplary sequencing system.

Fig. 2 includes an illustration of an exemplary system that includes a sensor array.

FIG. 3 includes an illustration of an example sensor and associated well.

Fig. 4 includes an illustration of an exemplary method for preparing a sequencing device.

Fig. 5,6 and 7 show exemplary protocols for preparing a bead assembly.

Fig. 8 and 9 include illustrations of exemplary bead configurations.

FIG. 10 includes a schematic diagram of an exemplary magnetic loading system.

Figure 11 schematically shows the movement of a solution containing magnetic beads relative to a magnetic enclosure at a first speed.

Figure 12 schematically illustrates the movement of a solution containing magnetic beads relative to a magnetic package at a second speed.

Figure 13 schematically shows the movement of a solution containing magnetic beads in the opposite direction with respect to the magnetic enclosure.

Figure 14 shows the beads mounted on the microchip.

Fig. 15 schematically shows a magnetic loading model.

Fig. 16, 17,18, and 19 include illustrations of exemplary loading devices.

FIG. 20 shows an exemplary flow cell (flowcell).

FIG. 21 illustrates another exemplary flow cell having a coverslip and a slide and moving in a first direction relative to a magnet.

FIG. 22 shows another exemplary flow cell having a coverslip and a slide and moving in a second direction relative to a magnet.

Figure 23 includes a photographic illustration of the edge of the stack within the reagent solution as it moves across the array surface.

Fig. 24 schematically shows the alignment of beads with magnetic field lines.

Figure 25 shows an example embodiment in which a magnet is placed above a microchip.

Figure 26 shows the movement of the bead stack relative to the magnets of the magnetic device of figure 25.

The use of the same reference symbols in different drawings indicates similar or identical items.

Embodiments generally relate to loading one or more sequencing beads into one or more respective microwells of an array, e.g., one or more respective microwells formed on a microchip. In certain embodiments, after clonal amplification, each sequencing bead can contain multiple copies of the same polynucleotide fragment.