Detection and digital quantitation of multiple targets
The disclosure provides compositions, methods, and systems for implementation of highly multiplexed molecular diagnostic assays involving color combinatorics, stimulus-responsive probes, tandem probes, conjugated polymer probes, and other mechanisms for increasing the number of targets that can be simultaneously detected in a digital assay. Multiplexed detection of targets is achieved in a rapid manner, with respect to sample partitioning and target detection using multiple color channels for detection. Implementation of methods described also achieve detection with significantly improved signal-to-noise ratio (SNR) values.
1. A method comprising:
performing a digital multiplexed analysis of a sample distributed across a set of partitions comprising more than 500,000 partitions stabilized in position, wherein performing the digital multiplexed analysis comprises:
reacting the sample with a set of processing materials within the set of partitions,
wherein each of the set of partitions has less than two targets, and
detecting signals from the set of partitions upon performing 3D scanning of the set of partitions with a set of channels, wherein each of the set of channels comprises a respective filter configuration,
wherein said signals correspond to a set of color combinatorics paired with targets of a set of targets potentially represented in the sample and contained within partitions of the set of partitions, and wherein the set of targets has a total number greater than a number of the set of channels used to detect signatures corresponding to the set of color combinatorics, and wherein the set of channels comprises at least four channels.
2. The method of claim 1 , wherein the set of partitions comprises droplets of an emulsion, stabilized in position along three axes within a closed container.
3. The method of claim 2 , wherein the closed container omits an outlet, and wherein there is no flow through the collecting container during the step of detecting signals.
4. The method of claim 1 , wherein the set of color combinatorics involves combinations of a set of fluorescent probes used to tag individual targets of the set of targets.
5. The method of claim 1 , wherein the set of fluorescent probes comprises a photobleachable probe.
6. The method of claim 1 , wherein the set of processing materials comprise, for a first target and a second target of the set of targets:
a primer set comprising: at least one primer structured to tag the first target with a first probe having a first fluorophore and the second target with a second probe having a second fluorophore.
7. The method of claim 6 , wherein the first fluorophore is a photo bleachable fluorophore, and wherein detecting signals from the set of partitions comprises:
detecting signals from the set of partitions in a first phase of analysis upon scanning the set of partitions with a first scanning event,
bleaching the first fluorophore, and
detecting signals from the set of partitions in a second phase of analysis upon scanning the set of partitions with a second scanning event, thereby enabling differential detection of the first target and the second target.
8. The method of claim 1 , wherein the set of processing materials further comprises a probe additive reagent structured to reduce background noise.
9. The method of claim 1 , wherein the set of partitions is characterized by less than 15% occupancy of partitions by the targets of the set of targets.
10. The method of claim 1 , wherein the digital multiplexed analysis has a dynamic range of at least 10 6 .
11. The method of claim 1 , wherein the set of partitions comprises more than 15 million partitions.
12. The method of claim 1 , wherein the set of partitions comprises a set of microwells.
13. The method of claim 1 , wherein the set of channels comprises 7 channels.
14. The method of claim 1 , wherein the set of processing materials comprises a set of non-hydrolysis probes, the method further comprising tagging the set of targets with a set of permutations of the set of non-hydrolysis probes, wherein detecting signals from the set of partitions comprises detecting signals corresponding to the set of permutations for differential detection of the set of targets.
15. The method of claim 1 , further comprising returning an output supporting at least one of: pathogen detection, non-invasive prenatal testing, and gene therapy, based upon the digital multiplexed analysis.
16. A method comprising:
performing a digital multiplexed analysis of a sample distributed across a set of droplets of an emulsion stabilized in position within a closed container,
wherein each droplet of the set of droplets comprises less than two targets of the sample, and
wherein the digital multiplexed analysis can simultaneously detect a set of targets comprising greater than 20 different targets from the sample using combinations of fluorescent labels used to differentially tag different targets of the set of targets.
17. The method of claim 16 , wherein the set of droplets comprises greater than 30 million droplets, and wherein performing the digital multiplexed analysis comprises scanning the set of droplets within the closed container with a lightsheet imaging operation.
18. The method of claim 16 , wherein the set of droplets is characterized by less than 15% occupancy of droplets by the targets of the set of targets, and wherein no droplet of the emulsion comprises multiple targets.
19. The method of claim 16 , further comprising returning an output supporting non-invasive prenatal testing based upon the digital multiplexed analysis.
20. The method of claim 16 , further comprising returning an output supporting a gene therapy application based upon the digital multiplexed analysis.