The invention provides systems and methods for rapid automated identification of microbes and antimicrobial susceptibility testing (AST) directly from a patient specimen, without specimen preparation. Specimens are loaded into an analytical cartridge for processing. Analytical cartridges are preloaded with species-specific labels that are used to identify and enumerate microbes in the specimen. Instruments, such as analyzers can be used to interact with analytical cartridges to carry out methods of the invention all within the cartridge.

The invention provides systems and methods for rapid automated identification of microbes and antimicrobial susceptibility testing (AST) directly from a patient specimen, without specimen preparation. Specimens are loaded into an analytical cartridge for processing. Analytical cartridges are preloaded with species-specific labels that are used to identify and enumerate microbes in the specimen. Instruments, such as analyzers can be used to interact with analytical cartridges to carry out methods of the invention all within the cartridge.

Disclosed herein are methods, compositions and systems for the interrogation of macromolecules, more particularly for preparation of isolated single macromolecules for subsequent processing of specific regions of interest within said macromolecule based on an analysis of the molecule's physical map. The disclosure is further related to the controlled segmentation of long nucleic acid parent molecules into smaller child molecules in a targeted manner such that further processing on said children may be performed with the knowledge of their origin within the parent, in a controlled environment enabled by purposefully designed microfluidic devices. Also disclosed is binding of regional specific barcodes along the length of a long nucleic acid molecule such that upon cleavage of said molecule into child molecules, the regional origin of the children can be tracked, in a controlled environment enabled by purposefully designed microfluidic devices. Finally, the disclosure is further related to droplet devices and method to control the encapsulation of long nucleic acid molecules or specific subregions thereof into a droplet, and further tracking said droplets with their contents.

Disclosed herein are methods, compositions and systems for the interrogation of macromolecules, more particularly for preparation of isolated single macromolecules for subsequent processing of specific regions of interest within said macromolecule based on an analysis of the molecule's physical map. The disclosure is further related to the controlled segmentation of long nucleic acid parent molecules into smaller child molecules in a targeted manner such that further processing on said children may be performed with the knowledge of their origin within the parent, in a controlled environment enabled by purposefully designed microfluidic devices. Also disclosed is binding of regional specific barcodes along the length of a long nucleic acid molecule such that upon cleavage of said molecule into child molecules, the regional origin of the children can be tracked, in a controlled environment enabled by purposefully designed microfluidic devices. Finally, the disclosure is further related to droplet devices and method to control the encapsulation of long nucleic acid molecules or specific subregions thereof into a droplet, and further tracking said droplets with their contents.

The present disclosure provides methods, systems, and kits for processing nucleic acid molecules. A method may comprise providing a template nucleic acid fragment (e.g., within a cell, cell bead, or cell nucleus) within a partition (e.g., a droplet or well) and subjecting the template nucleic acid fragment to one or more processes including a barcoding process and a single primer extension or amplification process. The processed template nucleic acid fragment may then be recovered from the partition and subjected to further amplification to provide material for subsequent sequencing analysis. The methods provided herein may permit simultaneous processing and analysis of both DNA and RNA molecules originating from the same cell, cell bead, or cell nucleus.

The present disclosure provides methods, systems, and kits for processing nucleic acid molecules. A method may comprise providing a template nucleic acid fragment (e.g., within a cell, cell bead, or cell nucleus) within a partition (e.g., a droplet or well) and subjecting the template nucleic acid fragment to one or more processes including a barcoding process and a single primer extension or amplification process. The processed template nucleic acid fragment may then be recovered from the partition and subjected to further amplification to provide material for subsequent sequencing analysis. The methods provided herein may permit simultaneous processing and analysis of both DNA and RNA molecules originating from the same cell, cell bead, or cell nucleus.

In an amplification reaction in a microfluidic apparatus, the reaction is carried out using starting substances tagged with fluorophore and quencher. The detection of reaction products occurs according to the disclosure by a separation of fluorophore and quencher occurring in the context of the amplification reaction. For the detection reaction, at least one energy-transferring substance is added and the evaluation occurs on the basis of the fluorescence emission of the fluorophores which occurs.

In an amplification reaction in a microfluidic apparatus, the reaction is carried out using starting substances tagged with fluorophore and quencher. The detection of reaction products occurs according to the disclosure by a separation of fluorophore and quencher occurring in the context of the amplification reaction. For the detection reaction, at least one energy-transferring substance is added and the evaluation occurs on the basis of the fluorescence emission of the fluorophores which occurs.

A microfluidic reaction chamber with a reaction chamber circuit includes a microfluidic reaction chamber to contain a reaction fluid for amplification of nucleic acids, and a reaction chamber circuit disposed within the microfluidic reaction chamber. The microfluidic reaction chamber includes a base wall, a top wall parallel to the base wall and defined in part by a transparent lid, a first side wall, and a second side wall. The reaction chamber circuit is disposed within the microfluidic reaction chamber, and includes a top surface, a bottom surface, a first side wall, and a second side wall. The reaction chamber circuit is in fluidic contact with the reaction fluid and includes a photodetector to detect a fluorescence signal from a labeled fluorescent tag in the reaction fluid.

A microfluidic reaction chamber with a reaction chamber circuit includes a microfluidic reaction chamber to contain a reaction fluid for amplification of nucleic acids, and a reaction chamber circuit disposed within the microfluidic reaction chamber. The microfluidic reaction chamber includes a base wall, a top wall parallel to the base wall and defined in part by a transparent lid, a first side wall, and a second side wall. The reaction chamber circuit is disposed within the microfluidic reaction chamber, and includes a top surface, a bottom surface, a first side wall, and a second side wall. The reaction chamber circuit is in fluidic contact with the reaction fluid and includes a photodetector to detect a fluorescence signal from a labeled fluorescent tag in the reaction fluid.