Labeled probes, and methods of use thereof, comprise a Cas polypeptide conjugated to gRNA that is specific for target nucleic acid sequences, including genomic DNA sequences. The probes and methods can be used to label nucleic acid sequences without global DNA denaturation. The presently-disclosed subject matter meets some or all of the above identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

Labeled probes, and methods of use thereof, comprise a Cas polypeptide conjugated to gRNA that is specific for target nucleic acid sequences, including genomic DNA sequences. The probes and methods can be used to label nucleic acid sequences without global DNA denaturation. The presently-disclosed subject matter meets some or all of the above identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

A nucleic acid probe, a method of immobilizing the nucleic acid probe to a solid support and the solid support including the immobilized probes using UV light. The nucleic acid probe includes a terminus anchor chain portion, and a capture portion wherein the terminus anchor chain portion includes a sequence of at least 18 nucleotides composed of stretches of up to 5 nucleotides of base type X with intermediate nucleotide(s) of base type Cytosine (C) and optionally one nucleotide of base type Guanine (G) or a sequence with at least 90% similarity thereto, wherein each base type X independently of each other designate base type Thymine (T) or base type Uracil (U).

A nucleic acid probe, a method of immobilizing the nucleic acid probe to a solid support and the solid support including the immobilized probes using UV light. The nucleic acid probe includes a terminus anchor chain portion, and a capture portion wherein the terminus anchor chain portion includes a sequence of at least 18 nucleotides composed of stretches of up to 5 nucleotides of base type X with intermediate nucleotide(s) of base type Cytosine (C) and optionally one nucleotide of base type Guanine (G) or a sequence with at least 90% similarity thereto, wherein each base type X independently of each other designate base type Thymine (T) or base type Uracil (U).

The invention relates to methods of detecting a genetic variation in a genetic sample from a subject using labeled probes and counting the number of labels in the probes.

The invention relates to methods of detecting a genetic variation in a genetic sample from a subject using labeled probes and counting the number of labels in the probes.

The invention provides methods for assessment of tumor biomarkers using target-enriched multiplexed parallel analysis. The methods of the invention utilize Target Capture Sequences (TACS) to thereby enrich for target sequences of interest, followed by massive parallel sequencing and statistical analysis of the enriched population. The methods can be used with DNA samples from a patient, such as a tissue biopsy or plasma sample (liquid biopsy), for detection of the presence of tumor biomarkers, e.g., for purposes of diagnosis, screening, therapy selection and/or treatment monitoring. Kits for carrying out the methods of the invention are also provided.

The invention provides methods for assessment of tumor biomarkers using target-enriched multiplexed parallel analysis. The methods of the invention utilize Target Capture Sequences (TACS) to thereby enrich for target sequences of interest, followed by massive parallel sequencing and statistical analysis of the enriched population. The methods can be used with DNA samples from a patient, such as a tissue biopsy or plasma sample (liquid biopsy), for detection of the presence of tumor biomarkers, e.g., for purposes of diagnosis, screening, therapy selection and/or treatment monitoring. Kits for carrying out the methods of the invention are also provided.

The invention relates to a method for enriching a target polynucleotide sequence containing a genetic variation said method comprising: (a) providing two primers targeted to said target polynucleotide sequence; (b) providing a target specific xenonucleic acid clamp oligomer specific for a wildtype polynucleotide sequence; (c) generating multiple amplicons using PCR under specific temperature cycling conditions; and (d) detecting said amplicons. We introduce a novel molecule, Xenonucleic Acid (XNA) for the NGS library preparation. XNA is able to selectively suppress amplification of DNA with wild type alleles and amplify DNA containing mutant alleles. Mutants with low allelic frequency will be easily detectable without deep sequencing after enrichment by adding XNA in multiplex PCR. The 17 actionable mutants related to lung or colorectal cancer diseases at different variant allelic frequency (VAF) % were investigated. Clinical sensitivity is significantly improved with XNA in various types of samples.

Disclosed herein are methods for single-cell sequencing. In some examples, the methods include enriching a sample comprising a plurality of cells for cells of interest to produce an enriched cell sample; isolating one or more cells of interest in the enriched cell sample; and obtaining sequence information of one or more polynucleotides from each of the one or more isolated cells. Obtaining sequence information may include generating a molecularly indexed polynucleotide library from the one or more isolated cells. Enriching the sample may include focusing cells of interest in the sample using acoustic focusing.