Bio-Rad Laboratories, Inc.

Lawrence Livermore National Security, LLC

University of California

Billy Wayne Colston, Jr

Benjamin J Hindson

Kevin D Ness

Donald Masquelier

Fred Paul Milanovich

Phillip Belgrader

Anthony Joseph Makarewicz, Jr

Samuel Burd

Serge Saxonov

Douglas N Modlin

Michael Ybarra Lucero

Neil Reginald Beer

Simant Dube

Jeffrey Mellen

John Frederick Regan

Vincent Riot

Nicholas Heredia

Austin So

Steve B Brown

Don Masquelier

Raymond P Mariella, Jr

Duane K Wolcott

Brian L Anderson

Joseph P Fitch

Graham D Marshall

John Chang

Richard G Langlois

J Chance Carter

Kodomudi Venkateswaran

Pedro Estacio

Chris Elkin

Benjamin Joseph Hindson

Donald Arthur Masquelier

Kevin Dean Ness

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.

Partition-based method of analysis

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.

Droplet-based assay system

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.

Method of performing droplet-based assays

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.

Analysis of nucleic acids

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.

Detection method for a target nucleic acid

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.

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

System for mixing fluids by coalescence of multiple emulsions

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.

Droplet-based analysis method

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.

Method of generating droplets

Devices and methods for generating droplets. An exemplary device comprises a substantially planar base portion including a bottom surface having a plurality of microfluidic channels formed therein as recessed regions of the bottom surface. The device also comprises a plurality of protrusions projecting from a top surface of the base portion and each formed integrally with the base portion. The device further comprises a sample well, a carrier well, and a droplet well. Each well has an upper portion created by one of the protrusions. A cover layer is attached to the bottom surface of the base portion and seals a bottom side of each microfluidic channel.

Device for generating droplets

Methods of making a droplet-generating device. In an exemplary method, an upper member is injection molded. The upper member includes a bottom surface and also includes a first microfluidic channel, a second microfluidic channel, and a third microfluidic channel each formed in the bottom surface. The upper member has a plurality of openings each extending completely through the upper member from the bottom surface and creating a side wall region of a sample well, a carrier well, and a droplet well. A cover layer is attached to the bottom surface of the upper member, such that the cover layer seals a bottom side of each microfluidic channel. The microfluidic channels meet one another to create a droplet-generation region. The sample well, the carrier well, and the droplet well are connected to the droplet-generation region via the first, second, and third microfluidic channels, respectively.

Method of making a device for generating droplets

Provided herein are improved methods, compositions, and kits for analysis of nucleic acids. The improved methods, compositions, and kits can enable copy number estimation of a nucleic acid in a sample. Also provided herein are methods, compositions, and kits for determining the linkage of two or more copies of a target nucleic acid in a sample (e.g. whether the two or more copies are on the same chromosome or different chromosomes) or for phasing alleles.

System, including apparatus and methods, for performing droplet-based assays. The system may comprise a droplet transporter configured to pick up droplets from each emulsion of an array of reacted emulsions and to drive flow of the droplets through a detection region. The system also may comprise a detector configured to collect data related to one or more analytes from individual droplets of the reacted emulsions as such individual droplets travel through the detection region. The system further may comprise a controller programmed to determine, based on the data collected, an aspect of the one or more analytes in one or more samples included in droplets of the emulsions.

System for transporting emulsions from an array to a detector

A method of rapid, genome and proteome based identification of unknown pathogenic or non-pathogenic organisms in a complex sample. The entire sample is analyzed by creating millions of emulsion encapsulated microdroplets, each containing a single pathogenic or non-pathogenic organism sized particle and appropriate reagents for amplification. Following amplification, the amplified product is analyzed.

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San Ramon, CA, United States of America

Billy Wayne Colston, Jr