INTEGRATED SEQUENCING APPARATUSES AND METHODS OF USE

• US 20140073514A1
• Filed: 11/19/2013
• Published: 03/13/2014
• Est. Priority Date: 11/07/2011
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Shen, Min-Jui Richard, Kain, Robert C., Kuhn, Kenneth M., Talasaz, AmirAli Hajhossein, Jamshidi, Arash, Sakaldasis, George, Vermaas, Eric, Bohm, Sebastian, Khurana, Tarun, Eltoukhy, Helmy A., Gong, Jian
• Granted Patent: US 9,309,571 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/069 Absorbents; Gels to retain a fluid
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0851 Bottom walls
• CPC Class Codes: B01L 2300/165 Specific details about hydrophobic, oleophobic surfaces
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 3/502792 for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation

Abstract

Provided are methods and apparatuses for performing sequencing using droplet manipulation, for example, via electrowetting-based techniques. Also provided are integrated methods and apparatuses for performing sample preparation and sequencing on the same apparatus. In addition, provided are methods of reducing reagent waste and preloaded consumable cartridges comprising reagents for sample preparation and/or sequencing.

Claims

1. -10. (canceled)

11. A method of moving a droplet, comprising the steps of:

(a) providing a droplet manipulation apparatus for performing droplet operations, the droplet manipulation apparatus comprising a substrate surface comprising an array of dynamic pads, wherein a droplet dispensed onto the substrate surface moves along a desired path defined by the dynamic pads, and wherein at least one of the dynamic pads comprises a hydrophilic patch;

(b) moving a droplet on the substrate surface onto a first dynamic pad comprising a hydrophilic patch; and

(c) moving the droplet from the first dynamic pad to a second dynamic pad.

12. The method of claim 11, wherein the hydrophilic patch of step (b) comprises one or more primers.

13. The method of claim 12, wherein the droplet of step (b) comprises a plurality of nucleic acid molecules hybridized to the primers.

14. The method of claim 11, wherein the droplet of step (b) comprises one or more nucleic acid sequencing reagents.

15. The method of claim 11, wherein the droplet manipulation apparatus comprises a fill liquid that is immiscible to the droplets, and wherein the fill liquid flows past at least some of the droplets under a force that is independent of forces placed on the fluid by the droplets.

16. A method of sequencing a nucleic acid molecule, the method comprising the steps of:

(a) providing a droplet manipulation apparatus for performing droplet operations, the droplet apparatus comprising a substrate surface comprising an array of dynamic pads, wherein a droplet dispensed onto the substrate surface moves along a desired path defined by the dynamic pads, and wherein at least one of the pads comprises a hydrophilic patch;

(b) transporting a droplet comprising one or more nucleic acid molecules to be sequenced to the hydrophilic patch;

(c) immobilizing the one or more nucleic acid molecules;

(d) sequencing the one or more nucleic acid molecules.

17. A nucleic acid sequencing method comprising

(a) presenting a plurality of target nucleic acids on a detection surface;

(b) providing a collection of reagent droplets, individual droplets in the collection comprising different sequencing reagents, the collection of droplets comprising sufficient reagents to complete a cycle of the sequencing reaction;

(c) delivering droplets from the collection to the detection surface in a sequential order to complete the cycle of the sequencing reaction for the target nucleic acids, wherein at least a subset of individual droplets in the collection are discretely delivered to the detection surface and discretely removed from the detection surface; and

(d) re-using individual droplets in the subset in a subsequent cycle of the sequencing reaction.

18. The method of claim 17, wherein the droplet manipulation apparatus comprises a fill liquid that is immiscible to the droplets, and wherein the fill liquid comprises a scavenger that prevents active sequencing reagents or contaminants from passing through the fill liquid between droplets.

19. The method of claim 17, wherein a scavenger for a contaminant is immobilized at one or more of the dynamic pads.

20. The method of claim 17, wherein the droplets are delivered to the detection surface via movement of the droplets along dynamic pads in a fill liquid and wherein the droplets are further moved by being carried through a capillary channel from the dynamic pads to the detection surface.

21. The method of claim 20, wherein the capillary channel comprises a hydrophilic inlet surface that attracts the droplets and repels the fill fluid.

22. A sequencing system comprising a unit having reagent reservoirs, a detection surface, and one or more fluidic path connecting the reservoirs to the detection surface,

wherein the unit is composed of at least two cartridges that fit together to form the unit,

wherein the reagent reservoirs are loaded with a preselected amount of the reagents that are in a storage state, and

wherein the unit includes a mechanism for automatically introducing fluids to the reagent reservoirs to place the reagents in an active state.

23. The sequencing system of claim 22, wherein the one or more fluidic path comprises dynamic pads configured to move aqueous droplets through a chamber containing a non-aqueous fill fluid.

24. The sequencing system of claim 23, wherein the one or more fluidic path further comprises a channel configured to flow the aqueous droplets and the non-aqueous fill fluid to the detection surface.

25. The sequencing system of claim 24, wherein the aqueous droplets and the non-aqueous fill fluid flow from the chamber to the detection surface under the force of a pump, vacuum, gravity or capillary action.

26. The sequencing system of claim 22, wherein the one or more fluidic path further comprises a capillary channel configured to flow the aqueous droplets, wherein the capillary comprises a hydrophilic inlet surface that attracts the droplets and repels the non-aqueous fill fluid fill fluid.

27. The sequencing system of claim 22, wherein the one or more fluidic path further comprises a channel and a dynamic surface located at the entrance of the channel, whereby the dynamic surface is configured as a dynamic valve that controls entry of aqueous droplets into the channel.

28. The sequencing system of claim 27, wherein the channel is configured to have a path that is parallel to the direction of gravity, whereby an aqueous droplet that passes the dynamic valve will flow through the channel via gravitational force.