Integrated sequencing apparatuses and methods of use
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.
1. 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.
2. The method of claim 1 , 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.
3. The method of claim 2 , wherein the capillary channel comprises a hydrophilic inlet surface that attracts the droplets and repels the fill fluid.
4. The method of claim 1 , wherein the plurality of target nucleic acids comprises an array of at least 10 features/cm 2 .
5. The method of claim 4 , wherein each of the features comprises copies of a template nucleic acid attached to a hydrogel layer.
6. The method of claim 1 , wherein the detection surface comprises a CMOS detector.
7. The method of claim 1 , wherein the collection of droplets comprises reagents to complete a cycle of a sequencing by synthesis reaction.
8. The method of claim 7 , wherein the individual droplets comprise at least one reagent selected from the group consisting of a polymerase, a reversibly blocked nucleotide, and a deblocking agent.
9. The method of claim 8 , wherein the individual droplets comprise a polymerase.
10. The method of claim 8 , wherein the individual droplets comprise a reversibly blocked nucleotide.
11. The method of claim 8 , wherein the individual droplets comprise a deblocking agent for a reversibly blocked nucleotide.
12. The method of claim 1 , wherein the droplets are discretely delivered to the detection surface and discretely removed from the detection surface by electrowetting.
13. The method of claim 1 , further comprising providing a collection of amplification reagent droplets,
wherein step (a) comprises delivering droplets from the collection of amplification reagent droplets to the detection surface in a sequential order to amplify nucleic acids on the detection surface, thereby presenting the plurality of target nucleic acids on the detection surface.
14. The method of claim 13 , wherein individual droplets in the collection of amplification reagent droplets comprise different amplification reagents, the collection of amplification reagent droplets comprising sufficient reagents to complete a cycle of amplification.
15. The method of claim 14 , wherein at least a subset of individual droplets in the collection of amplification reagent droplets are discretely delivered to the detection surface and discretely removed from the detection surface during the cycle of amplification.
16. The method of claim 1 , wherein the delivering of the droplets to the detection surface occurs in a droplet manipulation apparatus that comprises a fill liquid that is immiscible to the droplets.
17. The method of claim 16 , wherein the fill liquid comprises a scavenger that prevents active sequencing reagents or contaminants from passing through the fill liquid between droplets.
18. The method of claim 16 , wherein the droplet manipulation apparatus comprises a substrate surface comprising an array of dynamic pads, wherein the droplets move along a desired path defined by the dynamic pads.
19. The method of claim 18 , wherein a scavenger for a contaminant is immobilized at one or more of the dynamic pads.
20. The method of claim 1 , wherein the detection surface comprises a hydrophilic patch.
21. The method of claim 20 , wherein the target nucleic acids are attached to the hydrophilic patch.