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 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 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 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.
2. The method of claim 1 , wherein the hydrophilic patch comprises one or more primers.
3. The method of claim 2 , wherein step (c) includes hybridizing the nucleic acid molecules to the primers.
4. The method of claim 1 , wherein prior to step (d) the nucleic acid molecules are amplified.
5. The method of claim 1 , wherein the size of the patch is smaller than the pad.
6. The method of claim 1 , wherein the dynamic pads comprise electrowetting control pads.
7. The method of claim 6 , wherein the hydrophilic patch remains hydrophilic regardless of whether the electrowetting control pad is active.
8. The method of claim 6 , wherein the electrowetting control pad comprises an electrode covered by a hydrophobic layer.
9. The method of claim 8 , wherein the hydrophilic patch is on the same plane as the hydrophobic layer.
10. The method of claim 8 , wherein the electrowetting control pad is the same size as the electrode.
11. The method of claim 1 , wherein the hydrophilic patch is comprised of one or more hydrophilic areas being separated from one another.
12. The method of claim 1 , wherein the sequencing step comprises:
(i) 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, and
(ii) delivering droplets from the collection to a detection surface in a sequential order to complete the cycle of the sequencing reaction for the one or more nucleic acid molecules, wherein at least a subset of individual droplets in the collection are discretely delivered to the one or more immobilized nucleic acid molecules.
13. The method of claim 12 , wherein the one or more nucleic acid molecules are immobilized on the detection surface.
14. The method of claim 13 , wherein the detection surface comprises a CMOS detector.
15. The method of claim 12 , wherein the one or more immobilized nucleic acid molecules comprises an array of at least 10 features/cm 2 .
16. The method of claim 15 , wherein each of the features comprises copies of a template nucleic acid attached to a hydrogel layer.
17. The method of claim 12 , 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.