Abstract: The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called “Safe-SeqS” for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant (“super-mutants”) if ?95% of them contain the identical mutation.

Abstract: Provided herein is method for, among other things, estimating the number of sequence variations in a sample of DNA. In some embodiments, the method can be used to estimate the mutational load of a sample. In some embodiments, the method makes use of a set of primers that have 3? ends that specifically hybridizes to a sequence that is repeated multiple times in the genome. Thermocycling a reaction mix containing the primers may produce a reaction product comprising at least 50 amplicons having a total length of at least 100 kb. This product can be sequenced to provide an estimate of the number of sequence variations in the sample, and thus the mutational load of the sample.

Abstract: A method of identifying a subject having Kawasaki disease (KD), which includes discriminating the subject from a subject having another condition, for example other infectious and inflammatory conditions, such as those that present similar symptoms to KD. Also provided is a minimal gene signature employed in the method, as well as primers, probes and gene chips for use in the method.

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: The present disclosure provides methods, compositions and kits as well as systems for manipulating nucleic acids, including implementing isothermal amplification, such as recombinase-polymerase amplification (RPA), of a nucleic acid template using a pre-seeded solid support. Provided are rapid and efficient methods for generating template nucleic acid molecules comprising specific nucleotide sequence bound to solid support. Such methods can be used, for example, in manipulating nucleic acids in preparation for analysis methods that utilize monoclonal populations of nucleic acids.

Abstract: Provided is a method capable of simply and exponentially amplifying circular DNA, and particularly, long-chain circular DNA, in a cell-free system. Specifically, provided herein is a method for amplifying circular DNA which comprises mixing circular DNA having a replication origin sequence (origin of chromosome (oriC)) with a reaction solution comprising: a first enzyme group that catalyzes replication of circular DNA; a second enzyme group that catalyzes an Okazaki fragment maturation and synthesizes two sister circular DNAs constituting a catenane; a third enzyme group that catalyzes a separation of two sister circular DNAs; and also, a buffer, NTP, dNTP, a magnesium ion source, and an alkali metal ion source, to form a reaction mixture, which is then reacted.

Abstract: Disclosed herein are methods for detecting presence of a target nucleic acid (such as an influenza virus nucleic acid) in a sample. In some embodiments, the methods include contacting the sample with a first probe capable of hybridizing to the target nucleic acid and a second probe capable of hybridizing to the target nucleic acid, contacting the resulting complex with one or more gap filling reagents, thereby producing a gap-filled target nucleic acid, isolating and amplifying the gap-filled target nucleic acid. The amplified gap-filled target nucleic acid covalently linked to the substrate is then detected, for example with a detectably labeled probe. Also disclosed herein are probes capable of hybridizing to influenza virus nucleic acids. The disclosure also includes kits for detecting and/or discriminating influenza virus nucleic acids in a sample. In some examples, the kits include two or more of the disclosed probes.

Abstract: In some aspects, provided herein are methods for analyzing a nucleic acid comprising first and second regions of interest flanking an adaptor region, comprising hybridizing an anchor to the adaptor region, analyzing the first region of interest from one end of the anchor using probe ligation (e.g., sequencing-by-ligation), and binding a polymerase to the other end of the anchor and optionally incorporating a nucleotide and/or analog thereof into the anchor by the polymerase using the second region of interest or a probe bound thereto as a template. In some embodiments, the second region of interest is used as a template for sequencing-by-synthesis. In some embodiments, spatially resolved detections of analytes are performed at a cellular or sub-cellular resolution which involve correlating signals associated with analytes with specific spatial locations in a biological sample.

Abstract: Disclosed herein are “dual-priming” isothermal amplification method (including “self-priming” and “pairing-priming” strand extension, termed “DAMP”) for rapid nucleic acid detection.

Abstract: Provided herein are compositions, kits, and methods for detecting at least one of a C. diffcile tcdA, tcdB, tcdC, cdtA, or cdtB nucleic acid in a sample. In son embodiments, one or more alleles of tcdC such as 117del tcdC or 184T tcdC are detected.

Abstract: A method for constructing a library of cell-free DNAs in body fluids, comprising directly acting a transposase or an endonuclease on a body fluid sample, fragmenting the cell-free DNAs within, and performing amplification to obtain a library. Also provided is a test kit using the present method for prenatal diagnosis or early detection of cancer.

Abstract: The present invention is directed to methods and compositions for adding tails of specific lengths to a substrate polynucleotide. The invention also contemplates methods and compositions for immobilization of tailed substrates to a solid support. The disclosure contemplates that the attenuator molecule is any biomolecule that associates with a tail sequence added to a substrate polynucleotide and controls the addition of a tail sequence to the 3? end of the substrate polynucleotide. The sequence that is added to the substrate polynucleotide is referred to herein as a tail sequence, or simply a tail, and the process of adding a nucleotide to a substrate polynucleotide is referred to herein as tailing.

Abstract: Methods and compositions for making DNA libraries for massive parallel next generation sequencing (NGS), comprises two parts. These methods may be referred to as Triseq sequencing. The first part includes ligating a UMI adapter, amplifying the DNA fragments in the presence of dUTP, enriching the target molecules through primer extension by using a panel of both forward and reverse primers, and removing the dU-containing template DNA. The DNA molecules are organized to primary clones and subclones, labeled by the UMI on 5? and 3? end of the DNA fragments, respectively. The second part includes sequencing the DNA library by NGS, deducing consensus sequence from each subclone, and from within each primary clone, and between the consensus sequences obtained from both forward and reverse primers.

Abstract: Embodiments of a method for accurate determination of biological target abundance can include generating a first set of molecules associated with a target sequence, where the first set of molecules includes a first set of dilution tags associated with a relative concentration profile; generating a second set of molecules including a second set of dilution tags associated with the first set of dilution tags; generating a dilution tagged mixture; amplifying the subsets of dilution tagged genetic targets using the second set of molecules; generating a modified dilution tagged mixture from the amplified subsets; determining, for the biological sample, a count of the distinct molecules including the target sequence; and/or determining, for the biological sample, an assessment of relative concentrations distinct species, such as over a vast dynamic range.

Abstract: A method for amplifying a target nucleic acid including providing a system having a crRNA or a derivative thereof, and a Cas protein or a variant thereof. The crRNA or the derivative thereof contains a target-specific nucleotide region substantially complementary to a region of the target nucleic acid, and contacting the target nucleic acid with the system to form a complex.

Abstract: Provided is a method of gene sequencing on a microwell array chip, including: Step 1: adding a PCR amplification system containing DNA fragments to be sequenced into the microwell array chip, allowing microwells to each individually form reaction spaces and allowing one DNA fragment to be contained in one microwell; Steps 2 to 3: subjecting the microwell array chip to PCR amplification, and denaturing amplified double-stranded DNAs in individual microwells; and Steps 4 and 5: sequencing the DNA fragments with sequencing primer S2 molecules and dNTPs in each microwell with a sensor at the bottom of the microwell.

Abstract: A buffered suspension includes a surfactant and a solid buffer particulate having a point of zero charge at least 1.2 pH units different that the pH of the buffered suspension. The buffered suspension can be prepared by mixing a stock solution with the solid buffer particulate and titrating. A method of preforming a pH sensitive process includes drawing the buffered suspension from a reservoir, filtering the solid buffer particulate from the buffered suspension, and applying the filtered solution to a sensor.

Abstract: Provided herein is technology for neoplasia screening, and particularly, but not exclusively, to methods, compositions, and related uses for detecting the presence of cancer, in particular, colorectal cancer.

Abstract: The present disclosure provides improved methods for isolating asymmetrically-primed and/or asymmetrically-tagged nucleic acid complexes that find use in downstream analytical analyses, including sequence analysis. Compositions comprising such complexes and kits and systems for generating such complexes are also provided.

Abstract: The present disclosure relates to oligonucleotide sequences for amplification primers and their use in performing nucleic acid amplifications of HCV, in particular regions that encode the NS3 polypeptide. In some embodiments the primers are used in nested PCR methods for the detection or sequencing of HCV NS3. The oligonucleotide sequences are also provided assembled as kits that can be used to amplify and detect or sequence HCV NS3.