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 retained in position along three axes within a closed container, wherein upon performing a set of processes, the digital multiplexed analysis has a signal-to-noise ratio (SNR) greater than 10 4 , and wherein the set of processes comprises:
reacting the sample with the set of processing materials within the set of partitions, and
detecting signals from the set of partitions upon performing 3D scanning of the set of partitions with a number of color channels,
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 the number of color channels used to detect colors corresponding to the set of color combinatorics, and wherein the number of color channels comprises at least 4 color channels.
2. The method of claim 1 , wherein the digital multiplexed analysis has a dynamic range of at least 10 6 .
3. The method of claim 1 , wherein the set of partitions comprises droplets of an emulsion within the closed container, and wherein the set of color combinatorics comprises combinations of up to 6 colors detectable from each of the set of partitions.
4. The method of claim 1 , wherein the set of processing materials comprise, for a target of the set of targets:
a primer set comprising: a common primer and a set of target-specific primers structured to interact with a target region of the target, the set of target-specific primers comprising a first target-specific primer comprising a first flanking sequence, and
a first fluorophore-labeled oligonucleotide corresponding to the flanking sequence, the first fluorophore-labeled oligonucleotide comprising a first fluorophore configured to transmit a first target signal if the target region is amplified.
5. The method of claim 4 , wherein the set of target-specific primers further comprises a second target-specific primer comprising a second flanking sequence, and the set of processing materials further comprises a second fluorophore-labeled oligonucleotide corresponding to the second flanking sequence, the second fluorophore-labeled oligonucleotide comprising a second fluorophore configured to transmit a second target signal if the target region is amplified, such that the target can be positively detected based upon the first target signal and the second target signal.
6. The method of claim 1 , wherein the set of processing materials comprise, for a target of the set of targets:
a primer set comprising: at least one primer structured to tag the target with a first probe having a first fluorophore and a second probe having a second fluorophore, wherein the first fluorophore and the second fluorophore correspond to two color channels of the number of color channels.
7. The method of claim 6 , further comprising causing Foerster resonance energy transfer (FRET) from the first fluorophore to the second fluorophore upon exciting the first fluorophore with a first wavelength profile of light, wherein detecting signals from the set of partitions with the number of color channels comprises detecting the target from a partition upon scanning the set of partitions with a second wavelength profile of light corresponding to an emission spectrum of the second fluorophore.
8. 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.
9. The method of claim 8 , wherein the first fluorophore is a photo-bleachable fluorophore, and wherein detecting signals from the set of partitions comprises scanning the set of partitions with a first wavelength range of light and a second wavelength range of light configured to bleach the first fluorophore, the method further comprising:
detecting signals from the set of partitions in a first phase of analysis upon scanning the set of partitions with the first wavelength range of light, and
detecting signals from the set of partitions in a second phase of analysis upon scanning the set of partitions and bleaching the first fluorophore with the second wavelength range of light, thereby enabling differential detection of the first target and the second target.
10. 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: a first primer structured to tag the first target with a first probe having a first fluorophore and a second primer structured to tag the second target with a second probe having a second fluorophore, wherein the first fluorophore is a photo-bleachable fluorophore, and wherein detecting signals from the set of partitions comprises scanning the set of partitions with a first wavelength range of light and a second wavelength range of light configured to bleach the first fluorophore, thereby enabling differential detection of the first target and the second target.
11. The method of claim 1 , wherein the set of processing materials further comprises a probe additive reagent structured to reduce background noise.
12. The method of claim 11 , wherein the probe additive reagent includes probe additives having a melting temperature greater than 55° C.
13. The method of claim 11 , wherein the probe additive reagent comprises a quencher structured to interact with at least one of the 3′ region and the 5′ region of the first fluorophore-labeled oligonucleotide.
14. The method of claim 1 , wherein performing the set of processes comprises determining the SNR upon:
scanning a set of planes of the set of partitions,
determining a target signal value and a noise signal value for the set of planes, and
determining the SNR from the target signal value and the noise signal value.
15. The method of claim 14 ,
wherein determining the target signal value and the noise signal value comprises, for a plane of the set of planes:
determining a categorization of a set of categorizations based upon a profile of signal-positive partitions represented in the plane,
determining a target signal distribution for the plane based upon the categorization, and
determining a noise signal distribution based upon the categorization, wherein the SNR is determined from the target signal distribution and the noise signal distribution.
16. The method of claim 15 , wherein the set of categorizations correspond to different profiles of a set of profiles of signal-positive partitions observable in the set of planes.
17. The method of claim 14 , wherein the noise signal value is attributed to fluorescence from adjacent partitions and adjacent planes of the set of planes.
18. The method of claim 1 , wherein the set of partitions comprises greater than 500,000 partitions and wherein the set of partitions is characterized by less than 15% occupancy of partitions by said biological targets.
19. 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.
20. The method of claim 1 , wherein the set of processing materials comprises a set of hydrolysis probes, the method further comprising tagging the set of targets with a set of combinations of the set of hydrolysis probes, wherein detecting signals from the set of partitions comprises detecting signals corresponding to the set of combinations for differential detection of the set of targets.
21. The method of claim 1 , further comprising returning an output supporting at least one of: pathogen detection, non-invasive prenatal testing, organ transplantation analysis, forensics, and oncology, based upon the quantitative analysis.
22. The method of claim 1 , wherein the set of targets comprise hypervariable regions of at least one of 16S rRNA and ITS rRNA.