Abstract: Methods of partition-based analysis. In an exemplary method, a device having a port fluidically connected to a chamber may be selected. A sample-containing fluid may be placed into the port. The sample-containing fluid may be moved from the port to the chamber. Partitions of the sample-containing fluid may be formed. A monolayer of the partitions in the chamber may be created. At least a portion of the monolayer may be imaged.

Abstract: Method of haplotype analysis. In an exemplary method, an aqueous phase containing nucleic acid may be partitioned into a plurality of discrete volumes. At least one allele sequence may be amplified in the volumes from each of a first polymorphic locus and a second polymorphic locus that exhibit sequence variation in the nucleic acid. At least one measure of co-amplification of allele sequences from both loci in the same volumes may be determined. A haplotype of the first and second loci may be selected based on the at least one measure of co-amplification.

Abstract: System for performing a flow-based assay. The system may comprise a droplet generator to produce an emulsion including droplets in a carrier fluid. The system also may comprise a thermocycler including two or more temperature-controlled zones and also including a channel connected to the droplet generator for receiving the emulsion. The channel may form a single-pass continuous fluid route traversing the temperature-controlled zones multiple times, such that droplets passing through the channel are thermally cycled. The system further may comprise a detection station downstream from the thermocycler and configured to detect a signal from the droplets after such droplets have been thermally cycled by passing through the channel.

Abstract: Method of analysis. In the method, a first emulsion and a second emulsion substantially separated from one another by a spacer fluid may be formed. The first emulsion, the spacer fluid, and the second emulsion may be flowed in a channel from a fluid inlet to a fluid outlet of a heating and cooling station having two or more temperature-controlled zones, such that each emulsion is thermally cycled to promote amplification of a nucleic acid target in droplets of the emulsion. Amplification data may be collected from individual droplets of each emulsion downstream of the heating and cooling station. A level of the nucleic acid target present in each emulsion may be determined based on the amplification data collected from the individual droplets of the emulsion.

Abstract: The present disclosure provides methods and compositions for detecting polynucleotides in a sample and for quantifying polynucleotide load in a sample. The polynucleotides can be associated with a disease, disorder, or condition. In some applications, methylated DNA is quantified, e.g., in order to determine the load of polynucleotides in a sample. The present disclosure also provides methods and compositions for determining the load of fetal polynucleotides in a biological sample, e.g., the load of fetal polynucleotides (e.g., DNA, RNA) in maternal plasma. The present disclosure provides methods and compositions for detecting cellular processes such as cellular viability, growth rates, and infection rates. This disclosure also provides compositions and methods for detecting differences in copy number of a target polynucleotide. In some embodiments, the methods and compositions provided herein are useful for diagnosis of fetal genetic abnormalities, when the starting sample is maternal tissue (e.g.

Abstract: Method of haplotype analysis. In an exemplary method, an aqueous phase containing nucleic acid may be partitioned into a plurality of discrete volumes. At least one allele sequence may be amplified in the volumes from each of a first polymorphic locus and a second polymorphic locus that exhibit sequence variation in the nucleic acid. At least one measure of co-amplification of allele sequences from both loci in the same volumes may be determined. A haplotype of the first and second loci may be selected based on the at least one measure of co-amplification.

Abstract: This invention provides compositions and methods for detecting differences in copy number of a target polynucleotide. In some cases, the methods and compositions provided herein are useful for diagnosis of fetal genetic abnormalities, when the starting sample is maternal tissue (e.g., blood, plasma). The methods and materials described apply techniques for allowing detection of small, but statistically significant, differences in polynucleotide copy number.

Abstract: Methods of partition-based analysis. In an exemplary method, a device having a port fluidically connected to a chamber may be selected. A sample-containing fluid may be placed into the port. The sample-containing fluid may be moved from the port to the chamber. Partitions of the sample-containing fluid may be formed. A monolayer of the partitions in the chamber may be created. At least a portion of the monolayer may be imaged.

Abstract: Method of analysis. In the method, a microfluidic device defining a flow path extending from an inlet to an outlet may be selected. A sample-containing fluid may be introduced into the flow path via the inlet. Volumes of the sample-containing fluid may be isolated from one another on the flow path. A two-dimensional monolayer of the volumes may be imaged. The two-dimensional monolayer may be formed along the flow path between the inlet and the outlet.

Abstract: Method of detecting a target nucleic acid. In an exemplary method, at least two thermal zones of different temperature may be created using a heating assembly. A first emulsion and a second emulsion may be formed. The first and second emulsions may be thermally cycled by passing them through tubing in a spaced relation to one another, with the tubing being wound around a central axis of the heating assembly and extending through each thermal zone multiple times. Thermally cycling may promote amplification of the target nucleic acid in droplets of each emulsion. Droplets of each emulsion may be passed through a detection channel located downstream of the tubing. Fluorescence may be detected from the droplets being passed through the detection channel.

Abstract: Method of performing a droplet-based assay. The method may include obtaining droplets encapsulated by an immiscible liquid and packed closely together in a monolayer, performing a reaction in the droplets while packed closely together in the monolayer; and collecting data related to an analyte from a plurality of the droplets while the droplets remain closely packed together in the monolayer.

Abstract: System for performing a flow-based assay. The system may comprise a droplet generator to produce an emulsion including droplets in a carrier fluid. The system also may comprise a thermocycler including two or more temperature-controlled zones and also including a channel connected to the droplet generator for receiving the emulsion. The channel may form a single-pass continuous fluid route traversing the temperature-controlled zones multiple times, such that droplets passing through the channel are thermally cycled. The system further may comprise a detection station downstream from the thermocycler and configured to detect a signal from the droplets after such droplets have been thermally cycled by passing through the channel.

Abstract: Method of analysis. In the method, a first emulsion and a second emulsion substantially separated from one another by a spacer fluid may be formed. The first emulsion, the spacer fluid, and the second emulsion may be flowed in a channel from a fluid inlet to a fluid outlet of a heating and cooling station having two or more temperature-controlled zones, such that each emulsion is thermally cycled to promote amplification of a nucleic acid target in droplets of the emulsion. Amplification data may be collected from individual droplets of each emulsion downstream of the heating and cooling station. A level of the nucleic acid target present in each emulsion may be determined based on the amplification data collected from the individual droplets of the emulsion.

Abstract: System, including methods, apparatus, compositions, and kits, for the mixing of small volumes of fluid by coalescence of multiple emulsions.

Abstract: Method of detecting a target nucleic acid. In an exemplary method, at least two thermal zones of different temperature may be created using a heating assembly. A first emulsion and a second emulsion may be formed. The first and second emulsions may be thermally cycled by passing them through tubing in a spaced relation to one another, with the tubing being wound around a central axis of the heating assembly and extending through each thermal zone multiple times. Thermally cycling may promote amplification of the target nucleic acid in droplets of each emulsion. Droplets of each emulsion may be passed through a detection channel located downstream of the tubing. Fluorescence may be detected from the droplets being passed through the detection channel.

Abstract: Droplet-based methods of analysis. In an exemplary method, a device having a port connected to a chamber may be selected. A sample-containing fluid may be placed into the port. A pressure differential may be created that drives the sample-containing fluid from the port to the chamber and separates the sample-containing fluid into droplets. A two-dimensional monolayer of the droplets may be formed in the chamber. At least a portion of the monolayer may be imaged.

Abstract: Method of performing a droplet-based assay. The method may include obtaining droplets encapsulated by an immiscible liquid and packed closely together in a monolayer, performing a reaction in the droplets while packed closely together in the monolayer; and collecting data related to an analyte from a plurality of the droplets while the droplets remain closely packed together in the monolayer.

Abstract: Method of haplotype analysis. In an exemplary method, an aqueous phase containing nucleic acid may be partitioned into a plurality of discrete volumes. At least one allele sequence may be amplified in the volumes from each of a first polymorphic locus and a second polymorphic locus that exhibit sequence variation in the nucleic acid. At least one measure of co-amplification of allele sequences from both loci in the same volumes may be determined. A haplotype of the first and second loci may be selected based on the at least one measure of co-amplification.

Abstract: System, including methods, apparatus, compositions, and kits, for the mixing of small volumes of fluid by coalescence of multiple emulsions.

Abstract: Methods of generating droplets. In an exemplary method, a device including a sample well, a carrier well, a droplet well, and a plurality of microfluidic channels is selected. The microfluidic channels include a first channel, a second channel, and a third channel. A discrete volume of sample-containing fluid is placed into the sample well, and a discrete volume of carrier fluid is placed into the carrier well. A pressure differential is created after placing the discrete volumes, to cause fluid flow. Sample-containing fluid flows from the sample well to a droplet-generation region of the device via the first channel. Carrier fluid flows from the carrier well to the droplet-generation region via the second channel. Sample-containing droplets and carrier fluid flow from the droplet-generation region to the droplet well via the third channel.