Abstract: Next Generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of approximately 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, a method Duplex Consensus Sequencing (DCS) is provided. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand.

Abstract: Disclosed herein are devices and methods for amplifying double stranded DNA molecules. Methods for amplifying apoptotic cell-free DNA molecules can include performing end-repair and dA tailing of the cfDNA molecules, attachment of single-stranded hairpin adaptors to both ends of the end-repaired cfDNA molecules to produce adaptor-tagged, single-stranded, covalently closed DNA molecules, and amplification of the adaptor-tagged, single-stranded, covalently closed DNA molecules by a combination of rolling circle amplification and multiple displacement amplification using a PrimPol enzyme, a DNA polymerase with strand displacement activity and free nucleotides.

Abstract: Non-destructive methods and devices are disclosed herein for overcoming the limitations associated with analyzing shell casings and other cylindrical items for biomolecular and fingerprint extraction and analysis. In a preferred embodiment of these methodologies and devices, the open end of a spent shell casing (or like object) is plugged with a handle. This approach reduces the risk of sample contamination by gunpowder residue, while also providing a convenient means for handling the shell casing that reduces the risk of contamination or sample loss. Spin baskets (or extraction tubes) are provided which may be customized to different shell casing diameters, thereby substantially reducing the volume of extraction buffer required for sample collection and enable ease of centrifugation. A strong surfactant is preferably utilized during collection, which may reduce the amount of time needed for collection to just minutes.

Abstract: Assay cartridges are described that have purification, reaction, and detection zones and other fluidic components which can include sample chambers, waste chambers, conduits, vents, reagent chambers, reconstitution chambers and the like. The assay cartridges are used to conduct multiplexed nucleic acid measurements. Also described are kits including such cartridges, methods of using the same, and a reader configured to analyze an assay conducted using an assay cartridge.

Abstract: The present invention is directed to methods, reagents, kits, and compositions for identifying and quantifying target polynucleotide sequences. A linker probe comprising a 3? target specific portion, a loop, and a stem is hybridized to a target polynucleotide and extended to form a reaction product that includes a reverse primer portion and the stem nucleotides. A detector probe, a specific forward primer, and a reverse primer can be employed in an amplification reaction wherein the detector probe can detect the amplified target polynucleotide based on the stem nucleotides introduced by the linker probe. In some embodiments a plurality of short miRNAs are queried with a plurality of linker probes, wherein the linker probes all comprise a universal reverse primer portion a different 3? target specific portion and different stems. The plurality of queried miRNAs can then be decoded in a plurality of amplification reactions.

Abstract: Provided herein are methods for sequencing both strands of a double stranded nucleic acid fragment that improves fidelity and accuracy of a sequence determination compared to traditional next generation sequencing methods. Compositions and kits for use in the methods are also provided.

Abstract: Naturally occurring RNA pseudoknots fold into many topologies, yet their formation is poorly understood. Herein, by using high-resolution single-molecule force spectroscopy, the folding pathways of the H-type pseudoknot found in the preQ1-riboswitch in B. subtilis were investigated By holding a single riboswitch RNA molecule in the optical-trap, the structural rearrangements as the end-to-end distance change along the pulling direction, x at a force, F were followed. The data reveal a multistate folding, wherein the intermediate hairpin undergoes a unidirectional conformational switching in the presence of ligand to form the pseudoknot receptor. Specifically-designed mutant RNAs resisted the switching mechanism and resulted in a significantly reduced pseudoknot population (4.5%) compared to the wild-type (100%). The free-energy landscape highlighted two kinetic barriers (?G±) that interrupt the folding pathway.

Abstract: The present disclosure provides methods of generating supports (e.g., beads) comprising barcode molecules coupled thereto. A barcode molecule coupled to a support may comprise a barcode sequence and a functional sequence. A barcode molecule may be generated using two or more ligation reactions in a combinatorial fashion. A support comprising two or more different barcode molecules may be useful for analyzing or processing one or more analytes such as nucleic acid molecules, proteins, and/or perturbation agents.

Abstract: The present invention provides materials and methods useful for error correction of nucleic acid molecules. In one embodiment of the invention, a first plurality of double-stranded nucleic acid molecules having a nucleotide mismatch are fragmented by exposure to a molecule having unidirectional mismatch endonuclease activity. The nucleic acid molecules are cut at the mismatch site or near the mismatch site, leaving a double-stranded nucleic acid molecule having a mismatch at the end or near end of the molecule. The nucleic acid molecule is then exposed to a molecule having unidirectional exonuclease activity to remove the mismatched nucleotide. The missing nucleotides can then be filled in by the action of, e.g., a molecule having DNA polymerase activity. The result is double-stranded nucleic acid molecules with a decreased frequency of nucleotide mismatches.

Abstract: A system may include a horizontal actuator to move a tray, to which a microwell plate and a microfluidic chip may be coupled. The system may include a vertical actuator to move a support arm, to which a plurality of pipettes or pipette tips may be coupled. The system may include a rotational actuator to move an angle bracket, to which a magnet may be coupled. The system may include a heater, through which the pipettes may extend. The system may include a pump to control the flow of fluids through the pipettes. Disclosed methods include performing PCR within the described system.

Abstract: The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.

Abstract: System, including methods, apparatus, compositions, and kits, for making and using a stabilized emulsion. A method of generating a stabilized emulsion is provided. In the method, an aqueous phase may be provided. The aqueous phase may include an effective concentration of one or more skin-forming proteins. An emulsion may be formed. The emulsion may include droplets of a dispersed phase disposed in a continuous phase, with the aqueous phase being the continuous phase or the dispersed phase. The emulsion may be heated to create an interfacial skin between each droplet and the continuous phase, to transform the droplets into capsules.

Abstract: Methods and compositions for digital profiling of nucleic acid sequences present in a sample are provided.

Abstract: The present method of detection involves increasing an amount of analyte molecules by an isothermal molecular amplification approach. In the present approach a starting molecule of interest may be amplified through a reaction it induces with specifically engineered and functionalized particles, namely protected particles A and storage particles B. This reaction may result in a set of output DNA molecules that is larger in number than the input DNA molecules. Thus the reaction between nanoparticles for amplification of a certain DNA sequence (input DNA molecules) may occur when there is a match with a targeted molecule (stored molecules on storage particles B) and if the DNA sequence of the input DNA molecules does not match (partially or completely) the targeted molecule the reaction may not occur. Without a certain molecular input of the input DNA molecule the reaction may not occur.

Abstract: Methods are provided for performing antibiotic susceptibility testing based on the detection of RNA, such as tmRNA, from microbial cells after exposure to antibiotics. In some embodiments, aliquots are obtained from a sample, one of which contains a selected antibiotic. The aliquots, which include growth media, are incubated under conditions suitable for microbial growth, and the microbial cells in each aliquot are removed and lysed, and the lysate is subjected to reverse transcription and amplification in infer the effect of the selected antibiotic on the microbial cells. In one embodiment, a sample containing microbial cells is incubated in the presence of a selected antibiotic and a stimulus is provided to induce the production of m RNA within the microbial cells. The microbial cells are subsequently lysed without substantial degradation of the m RNA within the lysate, and the m RNA is detected to determine the effect of the antibiotic on the microbial cells.

Abstract: Provided herein are methods, compositions, and kits for removing a portion of a sequence in a member of a nucleic acid library.

Abstract: Methods for characterizing genome editing, clonal expansion and associated reagents for use in such methods are disclosed herein. Some embodiments of the technology are directed to characterizing a population of cells following an engineered genomic editing event, that includes in some embodiments characterizing genomic alterations occurring at both intended and unintended genomic loci within the genome of the populations of cells. Other embodiments are directed to utilizing Duplex Sequencing for assessing a clonal selection in mixed cell populations and/or cell populations following a genomic editing event. Further examples of the present technology are directed to methods for detecting and assessing clonal expansion of cells following a genomic editing event.

Abstract: A diagnostic system is configured to perform first and second, different nucleic acid amplification reactions. The system includes a bulk reagent container compartment configured to store first bulk reagent container containing a first bulk reagent for performing sample preparation processes with a first subset and a second subset of a plurality of samples and a second bulk reagent container containing a second bulk reagent for performing the first nucleic acid amplification reaction. The system includes a unit-dose reagent compartment storing a unit-dose reagent pack including unit-dose reagents for performing the second nucleic acid amplification reaction.

Abstract: A system may include a horizontal actuator to move a tray, to which a microwell plate and a microfluidic chip may be coupled. The system may include a vertical actuator to move a support arm, to which a plurality of pipettes or pipette tips may be coupled. The system may include a rotational actuator to move an angle bracket, to which a magnet may be coupled. The system may include a heater, through which the pipettes may extend. The system may include a pump to control the flow of fluids through the pipettes. Disclosed methods include performing PCR within the described system.

Abstract: Methods and compositions for digital profiling of nucleic acid sequences present in a sample are provided.