Provided herein are reagents, compositions, and methods for sequencing nucleic acids. Among the reagents disclosed are nucleotides labeled with cleavable linkers. Certain linkers are configured to undergo immolation reactions following cleavage, which can generate scars with enhanced properties for nucleic acid sequencing and synthesis. The present disclosure also provides reagents, compositions, and methods for capping scars generated through linker cleavage, which can alter scar properties, and in some cases can increase the rate and accuracy of nucleotide incorporations during polymerization.

The invention provides barcode libraries and methods of making and using them including obtaining a plurality of nucleic acid constructs in which each construct comprises a unique N-mer and a functional N-mer and segregating the constructs into a fluid compartments such that each compartment contains one or more copies of a unique construct. The invention further provides methods for digital PCR and for use of barcode libraries in digital PCR.

The invention provides barcode libraries and methods of making and using them including obtaining a plurality of nucleic acid constructs in which each construct comprises a unique N-mer and a functional N-mer and segregating the constructs into a fluid compartments such that each compartment contains one or more copies of a unique construct. The invention further provides methods for digital PCR and for use of barcode libraries in digital PCR.

The present disclosure provides methods and systems for sequencing nucleic acid molecules. Methods may include sequencing double-stranded nucleic acids or single-stranded nucleic acids. Sequencing may include the use of nucleotides coupled to electrostatic moieties. The electrostatic moieties may be detected by a sensor array. The electrostatic moieties may be reversible electrostatic moieties that are cleaved from the nucleic acid molecule after incorporation of the nucleotide. The electrostatic moieties may be irreversible electrostatic moieties. Nucleotides comprising irreversible electrostatic moieties may be incorporated into the nucleic acid molecule, detected by the sensor array, and exchanged for non-detectable nucleotides.

Provided herein are methods and materials for detecting and/or treating subject (e.g., a human) having cancer. In some embodiments, methods and materials for identifying a subject as having cancer (e.g., a localized cancer) are provided in which the presence of member(s) of two or more classes of biomarkers are detected. In some embodiments, methods and materials for identifying a subject as having cancer (e.g., a localized cancer) are provided in which the presence of member(s) of at least one class of biomarkers and the presence of aneuploidy are detected. In some embodiments, methods described herein provide increased sensitivity and/or specificity in the detection of cancer in a subject (e.g. a human).

The present disclosure provides a general strategy based on CRISPR and oligonucleotide-detectable marker conjugates that allows sensitive detection of nucleic acid and non-nucleic acid target analytes without stringent temperature requirements. In various aspects, this strategy can be used for rapid and routine detection of viral and bacterial infections, screening of diseases with known biomarkers, and tracking the progression of diseases or response to therapy over time.

The present disclosure provides a general strategy based on CRISPR and oligonucleotide-detectable marker conjugates that allows sensitive detection of nucleic acid and non-nucleic acid target analytes without stringent temperature requirements. In various aspects, this strategy can be used for rapid and routine detection of viral and bacterial infections, screening of diseases with known biomarkers, and tracking the progression of diseases or response to therapy over time.

Provided herein are mutant DNA-dependent polymerases which are derived from, or otherwise related to, wild type RB69 DNA polymerase. These mutant polymerases are capable of selectively binding labeled nucleotides. These mutant polymerases are also capable of incorporating a variety of naturally occurring and modified nucleotides, including, for example, terminator nucleotides.

Provided herein are mutant DNA-dependent polymerases which are derived from, or otherwise related to, wild type RB69 DNA polymerase. These mutant polymerases are capable of selectively binding labeled nucleotides. These mutant polymerases are also capable of incorporating a variety of naturally occurring and modified nucleotides, including, for example, terminator nucleotides.

The invention relates to methods and compositions, and systems for determining the identity of nucleic acids in nucleotide sequences, and in particular, sequences that contain consecutive repeats of a particular base. For example, a consecutive repeat of a particular base may be identified by a charged ion detection (e.g., hydrogen ion detection).