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:
(a) generating a plurality of droplets comprising at least 1 million droplets, wherein the plurality of droplets comprises:
(i) a plurality of target nucleic acid molecules; and
(ii) processing materials,
wherein a droplet of the plurality of droplets comprises two or fewer target nucleic acid molecules of the plurality of target nucleic acid molecules;
(b) in the plurality of droplets, reacting the processing materials with the plurality of target nucleic acid molecules; and
(c) using a detection subsystem to scan the plurality of droplets across a set of channels to detect signals from at least a subset of the plurality of droplets, wherein each channel of the set of channels comprises a different filter configuration, wherein the set of channels comprises at least 6 channels, and wherein the number of targets of the set of targets is greater than the number of channels of the set of channels.
2 . The method of claim 1 , wherein the set of channels comprise 7 channels.
3 . The method of claim 1 , wherein the number of targets comprises at least 15 targets.
4 . The method of claim 1 , wherein the plurality of droplets are contained within a set of wells.
5 . The method of claim 1 , wherein the plurality of droplets are provided within a vessel.
6 . The method of claim 1 , wherein the set of channels of the detection subsystem comprises a first blue channel, a second blue channel, a green channel, a yellow channel, and a red channel.
7 . The method of claim 1 , wherein the processing materials comprise: a FAM fluorophore, an Atto fluorophore, a ROX fluorophore, a Cy5™ fluorophore, and a Cy5.5™ fluorophore.
8 . The method of claim 1 , wherein a target nucleic acid molecule of the plurality of target nucleic acid molecules is tagged with a tandem probe comprising a first probe and a second probe.
9 . The method of claim 1 , wherein (b) comprises binding the plurality of target nucleic acid molecules with probes.
10 . The method of claim 9 , wherein the probes comprise hydrolysis probes.
11 . A system comprising:
a vessel comprising a plurality of partitions comprising at least 200,000 partitions and a plurality of target nucleic acid molecules distributed across the plurality of partitions, wherein a partition of the plurality of partitions comprises two or fewer target nucleic acid molecules of the plurality of target nucleic acid molecules;
a set of processing materials comprising a set of probes comprising FAM fluorophore probes, ROX fluorophore probes, and Cy5™ fluorophore probes, wherein the set of processing materials is distributed across the plurality of partitions; and
a detection subsystem comprising a set of channels comprising at least a blue channel, a red channel, a yellow channel, and a green channel, wherein each channel of the set of channels comprises a different filter configuration.
12 . The system of claim 11 , wherein the set of channels comprises seven channels.
13 . The system of claim 11 , wherein the partition of the plurality of partitions comprises a target nucleic acid molecule tagged with a first probe of the set of probes and a second probe of the set of probes.
14 . The system of claim 11 , wherein the set of probes comprises hydrolysis probes.
15 . The system of claim 11 , wherein the set of probes comprises TaqMan™ probes.
16 . The system of claim 11 , wherein the plurality of partitions comprises droplets.
17 . A method comprising:
(a) providing a plurality of partitions comprising at least 200,000 partitions, wherein the plurality of partitions comprises:
(i) a plurality of target nucleic acid molecules; and
(ii) a set of processing materials,
wherein a partition of the plurality of partitions comprises two or fewer target nucleic acid molecules of the plurality of target nucleic acid molecules;
(b) in the plurality of partitions, reacting the set of processing materials with the plurality of target nucleic acid molecules;
(c) using a detection subsystem to scan the plurality of partitions across a set of channels to detect signals from at least a subset of the plurality of partitions, wherein each channel of the set of channels comprises a different filter configuration, wherein each of the set of targets is detectable using at least one channel of the set of channels; and
(d) using the signals detected in (c) to identify at least a subset of the plurality of target nucleic acid molecules.
18 . The method of claim 17 , wherein the set of channels comprises at least a blue channel, a red channel, a yellow channel, and a green channel.
19 . The method of claim 17 , wherein the set of processing materials comprises a set of probes comprising FAM fluorophore probes, ROX fluorophore probes, and Cy5™ fluorophore probes, and wherein the partition of the plurality of partitions comprises a target nucleic acid molecule tagged with a first probe of the set of probes and a second probe of the set of probes.
20 . The method of claim 17 , wherein (d) comprises processing the signals detected in (c) against a set of pre-determined signal combinations to identify the at least the subset of the plurality of target nucleic acid molecules.
21 . A method comprising:
(a) providing a plurality of partitions comprising at least 300,000 partitions, wherein the plurality of partitions comprises:
(i) a plurality of target nucleic acid molecules, and
(ii) a set of processing materials comprising a forward primer corresponding to a target region and comprising an adapter sequence, a reverse primer, and a fluorophore-labeled oligonucleotide corresponding to the adapter sequence, wherein the fluorophore-labeled oligonucleotide comprises a fluorophore configured to transmit a target signal if the target region is amplified, and wherein a partition of the plurality of partitions comprises two or fewer target nucleic acid molecules of the plurality of target nucleic acid molecules;
(b) in the plurality of partitions, reacting the processing materials with the nucleic acid molecules;
(c) using a detection system to scan the plurality of partitions across a set of channels to detect signals from at least a subset of the plurality of partitions, wherein each channel of the set of channels comprises a different filter configuration, wherein the number of targets of the set of targets is greater than the number of channels of the set of channels; and
(d) using the signals detected in (c) to identify at least a subset of the plurality of target nucleic acid molecules.