SYSTEMS AND METHODS FOR GENERATING DROPLETS AND PERFORMING DIGITAL ANALYSES
This disclosure provides for devices, methods, and systems for generating a plurality of droplets within a collecting container at an extremely high rate (e.g., of at least 1 million droplets per minute, etc.), each of the plurality of droplets comprising an aqueous mixture for a digital analysis, wherein upon generation, the plurality of droplets is stabilized in position within a region of the collecting container. The inventions enable partitioning of samples for digital analyses at unprecedented rates, where readout of signals from targets within such partitions can still be achieved in accordance with various assays.
1 . A method comprising:
(a) providing a sample comprising a plurality of nucleic acid molecules, wherein the sample has a volume of less than 50 microliters;
(b) generating a plurality of partitions comprising processing materials and the plurality of nucleic acid molecules of the sample, upon flowing the sample through a hole and into a container, wherein the container is coupled with a supporting body within a centrifuge during generation of the plurality of partitions;
(c) amplifying the plurality of nucleic acid molecules or derivatives thereof within the container; and
(d) scanning the plurality of partitions in the container to detect signals corresponding to the plurality of nucleic acid molecules or derivatives thereof.
2 . The method of claim 1 , wherein (b) comprises flowing the sample through a plurality of holes, wherein the plurality of holes comprises the hole.
3 . The method of claim 1 , wherein the plurality of nucleic acid molecules comprises a target nucleic acid molecule.
4 . The method of claim 3 , wherein the target nucleic acid molecule comprises a viral nucleic acid molecule.
5 . The method of claim 1 , wherein the supporting body comprises a swing bucket positioned within the centrifuge.
6 . The method of claim 1 , wherein the container is a well a 96-well plate.
7 . The method of claim 1 , wherein the plurality of partitions comprises at least 200,000 partitions.
8 . The method of claim 1 , wherein the plurality of partitions comprises a plurality of droplets, wherein a droplet of the plurality of droplets is surrounded by an immiscible fluid.
9 . The method of claim 1 , wherein (b) comprises generating the plurality of partitions within a duration of 50 minutes.
10 . The method of claim 1 , wherein (d) comprises scanning partitions of the plurality of partitions across a plurality of channels comprising at least 4 color channels.
11 . The method of claim 1 , further comprising, after (d), analyzing signals generated from (d) indicative of rejection events of transplanted organs.
12 . A method comprising:
(a) providing a sample comprising a plurality of nucleic acid molecules, wherein the sample has a volume of less than 50 microliters; and
(b) generating a plurality of partitions comprising processing materials and the plurality of nucleic acid molecules of the sample upon flowing the sample through a hole and into a container, wherein the container is a well of a 96-well plate and coupled with a supporting body within a centrifuge during generation of the plurality of partitions, wherein the plurality of partitions comprises at least 500,000 partitions.
13 . The method of claim 12 , wherein (b) comprises flowing the sample through a plurality of holes, wherein the plurality of holes comprises the hole.
14 . The method of claim 12 , further comprising amplifying the plurality of nucleic acid molecules or derivatives thereof within the collecting container.
15 . The method of claim 14 , wherein amplifying the plurality of nucleic acids or derivatives thereof comprises performing isothermal amplification.
16 . The method of claim 12 , further comprising scanning the plurality of partitions to detect signals corresponding to the plurality of nucleic acid molecules or derivatives thereof.
17 . The method of claim 16 , wherein scanning the plurality of partitions to detect signals corresponding to the plurality of nucleic acid molecules comprises scanning partitions across a plurality of channels comprising at least 6 color channels.
18 . The method of claim 16 , wherein scanning the plurality of partitions comprises detecting signals indicative of rejection events of transplanted organs.
19 . The method of claim 16 , wherein scanning the plurality of partitions comprises detecting signals indicative of cancerous material of the sample.
20 . The method of claim 13 , wherein the plurality of nucleic acid molecules comprises a viral nucleic acid molecule.
21 . The method of claim 13 , wherein the supporting body comprises a swing bucket positioned within the centrifuge.