Harvard College

University of California

Bio-rad Laboratories, Inc.

Universitat Augsburg

Basf Se Corporation

Abate, Adam

Kiani, Sepehr

Esmail, Adnan, Moez

Mary, Pascaline

Hung, Tony

Adam R Abate

David A Weitz

Tony Hung

Adam R Sciambi

Pascaline Mary

Sepehr Kiani

Adnan Moez Esmail

Dennis Jay Eastburn

Jeremy Agresti

Mark Benjamin Romanowsky

Christian Holtze

Freeman Lan

Russell S Cole

Michael Weiner

Adnan Esmail

Achim Wixforth

Thomas Franke

John R Haliburton

Lothar Schmid

Zev Jordan Gartner

Assaf Rotem

Scott Powers

John Lawrence Emhoff

Shaun Lim

Joshua Blouwolff

Jason Hung

David Sukovich

Maithreyan Srinivasan

Julia Z Sharpe

Daeyeon Lee

Shea Thompson Lance

Ralph Alexander Sperling

Leqian Liu

Benjamin Demaree

Dennis Jay Eastbum

Iain Clark

Thao Do

Amber T Krummel

Justin Madrigal

Christian Siltanen

Julian W P Thiele

Cyrus Modavi

Samuel Kim

Michael P Weiner

Russell Cole

Makiko N Hatori

Julian WP Thiele

Peter Stokes

Microfluidic methods for barcoding nucleic acid target molecules to be analyzed, e.g., via nucleic acid sequencing techniques, are provided. Also provided are microfluidic, droplet-based methods of preparing nucleic acid barcodes for use in various barcoding applications. The methods described herein facilitate high-throughput sequencing of nucleic acid target molecules as well as single cell and single virus genomic, transcriptomic, and/or proteomic analysis/profiling. Systems and devices for practicing the subject methods are also provided.

Sequencing of nucleic acids via barcoding in discrete entities

Methods for selectively combining discrete entities including, e.g. cells, reagents, drugs, hydrogels, extracellular matrices beads, particles, biological materials, media, or a combination thereof, are provided. In certain aspects, the methods include sorting a plurality of discrete entities and trapping two or more discrete entities for a time sufficient for the two or more discrete entities to combine to form a combined discrete entity. In certain aspects, the methods include making the plurality of discrete entities. In certain aspects, the methods include detecting or analyzing the discrete entities, e.g. via optical detection. In certain aspects, the methods include manipulating or analyzing the combined discrete entity or a component therein, e.g. imaging, sequencing, culturing, e.g., three-dimensional culturing, and measuring cell-cell interactions. Systems and devices for practicing the subject methods are also provided.

Platform for the deterministic assembly of microfluidic droplets

The present invention is generally related to systems and methods for producing droplets. The droplets may contain varying species, e.g., for use as a library. In some cases, at least one droplet is used to create a plurality of droplets, using techniques such as flow-focusing techniques. In one set of embodiments, a plurality of droplets, containing varying species, can be divided to form a collection of droplets containing the various species therein. A collection of droplets, according to certain embodiments, may contain various subpopulations of droplets that all contain the same species therein. Such a collection of droplets may be used as a library in some cases, or may be used for other purposes.

Droplet creation techniques

Methods for the detection of components from biological samples are provided. In certain aspects, the methods may be used to detect and/or quantify specific components in a biological sample, such as tumor cells (e.g., circulating tumor cells). Systems and devices for practicing the subject methods are also provided.

Methods and systems for detecting biological components

The present invention generally relates to systems and methods for the control of fluids and, in some cases, to systems and methods for flowing a fluid into and/or out of other fluids. As examples, fluid may be injected into a droplet contained within a fluidic channel, or a fluid may be injected into a fluidic channel to create a droplet. In some embodiments, electrodes may be used to apply an electric field to one or more fluidic channels, e.g., proximate an intersection of at least two fluidic channels. For instance, a first fluid may be urged into and/or out of a second fluid, facilitated by the electric field. The electric field, in some cases, may disrupt an interface between a first fluid and at least one other fluid. Properties such as the volume, flow rate, etc. of a first fluid being urged into and/or out of a second fluid can be controlled by controlling various properties of the fluid and/or a fluidic droplet, for example curvature of the fluidic droplet, and/or controlling the applied electric field.

Fluid injection

Parallel uses of microfluidic methods and devices for focusing and/or forming discontinuous sections of similar or dissimilar size in a fluid are described. In some aspects, the present invention relates generally to flow-focusing-type technology, and also to microfluidics, and more particularly parallel use of microfluidic systems arranged to control a dispersed phase within a dispersant, and the size, and size distribution, of a dispersed phase in a multi-phase fluid system, and systems for delivery of fluid components to multiple such devices.

Scale-up of microfluidic devices

The present invention generally relates to systems and techniques for manipulating fluids and/or making droplets. In certain aspects, the present invention generally relates to droplet production. The droplets may be formed from fluids from different sources. In one set of embodiments, the present invention is directed to a microfluidic device comprising a plurality of droplet-making units, and/or other fluidic units, which may be substantially identical in some cases. Substantially each of the fluidic units may be in fluidic communication with a different source of a first fluid and a common source of a second fluid, in certain embodiments. In one aspect, substantially the same pressure may be applied to substantially all of the different sources of fluid, which may be used to cause fluid to move from the different sources into the microfluidic device. In some cases, the fluids may interact within the fluidic units, e.g., by reacting, or for the production of droplets within the microfluidic device. In some cases, the droplets may be used, for example, to form a library of droplets.

Systems and methods for droplet production and/or fluidic manipulation

The methods described herein, referred to as PCR-Activated Sorting (PAS), allow nucleic acids contained in biological systems to be sorted based on their sequence as detected with nucleic acid amplification techniques, e.g., PCR. The nucleic acids can be free floating or contained within living or nonliving structures, including particles, viruses, and cells. The nucleic acids can include, e.g., DNA or RNA. Systems and devices for use in practicing methods of the invention are also provided.

PCR-activated sorting (PAS)

Various aspects of the present invention relate to the control and manipulation of fluidic species, for example, in microfluidic systems. In one set of embodiments, droplets may be sorted using surface acoustic waves. The droplets may contain cells or other species. In some cases, the surface acoustic waves may be created using a surface acoustic wave generator such as an interdigitated transducer, and/or a material such as a piezoelectric substrate. The piezoelectric substrate may be isolated from the microfluidic substrate except at or proximate the location where the droplets are sorted, e.g., into first or second microfluidic channels. At such locations, the microfluidic substrate may be coupled to the piezoelectric substrate (or other material) by one or more coupling regions. In some cases, relatively high sorting rates may be achieved, e.g., at rates of at least about 1,000 Hz, at least about 10,000 Hz, or at least about 100,000 Hz, and in some embodiments, with high cell viability after sorting.

Acoustic waves in microfluidics

The methods and systems described herein provide an improved emulsion droplet based nucleic acid amplification method, which allows nucleic acids contained in biological systems to be detected, quantitated and/or sorted based on their sequence as detected with nucleic acid amplification techniques, e.g., polymerase chain reaction (PCR). The nucleic acids can be free floating or contained within living or nonliving structures, including particles, viruses, and cells. The nucleic acids can include, e.g., DNA or RNA.

Combined multiple-displacement amplification and PCR in an emulsion microdroplet

The present disclosure provides ultrahigh-throughput single cell genomic sequencing methods, referred to herein as 'SiC-seq', which methods include encapsulating single cells in molten gel droplets to facilitate bulk cell lysis and purification of genomic DNA in microgels. Systems and devices for practicing the subject methods are also provided.

Single cell genomic sequencing using hydrogel based droplets

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San Francisco, CA, United States of America

Adam R Abate