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.

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.

The invention relates to methods of in situ detection of a nucleic acid variation of a target nucleic acid in a sample, including single nucleotide variations, multi-nucleotide variations or splice sites. The method can comprise the steps of contacting the sample with a probe that detects the nucleic acid variation or splice site and a neighbor probe; contacting the sample with pre-amplifiers that bind to the nucleic acid variation probe or splice site probe and neighbor probe, respectively; contacting the sample with a collaboration amplifier that binds to the pre-amplifiers; and contacting the sample with a label probe system, wherein hybridization of the components forms a signal generating complex (SGC) comprising a target nucleic acid with the nucleic acid variation or splice site, the probes and amplifiers; and detecting in situ signal from the SGC on the sample. The invention also provides samples, tissue slides, and kits relating to detection of nucleic acid variations, including single nucleotide variations, multi-nucleotide variations or splice sites, of a target nucleic acid.

The invention relates to methods of in situ detection of a nucleic acid variation of a target nucleic acid in a sample, including single nucleotide variations, multi-nucleotide variations or splice sites. The method can comprise the steps of contacting the sample with a probe that detects the nucleic acid variation or splice site and a neighbor probe; contacting the sample with pre-amplifiers that bind to the nucleic acid variation probe or splice site probe and neighbor probe, respectively; contacting the sample with a collaboration amplifier that binds to the pre-amplifiers; and contacting the sample with a label probe system, wherein hybridization of the components forms a signal generating complex (SGC) comprising a target nucleic acid with the nucleic acid variation or splice site, the probes and amplifiers; and detecting in situ signal from the SGC on the sample. The invention also provides samples, tissue slides, and kits relating to detection of nucleic acid variations, including single nucleotide variations, multi-nucleotide variations or splice sites, of a target nucleic acid.

Methods and compositions for detecting genetic instability using digital amplification assays. The methods may be performed in a set of isolated volumes and generally may involve competitive hybridization of a competitor and a probe/primer with a normal allele and one or more mutant alleles of a microsatellite locus. The competitor may be configured to compete similarly with, or to outcompete, the primer/probe for hybridization with the normal allele. The primer/probe may be configured to outcompete the competitor for hybridization with various mutant alleles of the locus that alter the length of the repetitive sequence by different amounts. Isolated volumes in which the primer/probe outcompetes the competitor may be enumerated, and represent one or more of the mutant alleles. The methods may enable diagnosing microsatellite instability and treating a subject based on the diagnosis.

Methods and compositions for detecting genetic instability using digital amplification assays. The methods may be performed in a set of isolated volumes and generally may involve competitive hybridization of a competitor and a probe/primer with a normal allele and one or more mutant alleles of a microsatellite locus. The competitor may be configured to compete similarly with, or to outcompete, the primer/probe for hybridization with the normal allele. The primer/probe may be configured to outcompete the competitor for hybridization with various mutant alleles of the locus that alter the length of the repetitive sequence by different amounts. Isolated volumes in which the primer/probe outcompetes the competitor may be enumerated, and represent one or more of the mutant alleles. The methods may enable diagnosing microsatellite instability and treating a subject based on the diagnosis.

Methods for multiplexed detection of a nucleic acid sequence in a sample including the use of a plurality of oligonucleotide target-specific probes (TSPs) configured to bind to a distinct target nucleic acid sequence, where each of the TSPs includes one or more copies of a first fluorescent probe (FP) binding region and one or more copies of a second FP binding region, and where a predetermined ratio of the one or more copies of the first FP binding region to the one or more copies of the second FP binding region is indicative of the distinct target nucleic acid sequence the TSP is configured to bind to.

Methods for multiplexed detection of a nucleic acid sequence in a sample including the use of a plurality of oligonucleotide target-specific probes (TSPs) configured to bind to a distinct target nucleic acid sequence, where each of the TSPs includes one or more copies of a first fluorescent probe (FP) binding region and one or more copies of a second FP binding region, and where a predetermined ratio of the one or more copies of the first FP binding region to the one or more copies of the second FP binding region is indicative of the distinct target nucleic acid sequence the TSP is configured to bind to.

Disclosed is an enhanced method for rapid and cost-effective analysis of sequences of a microorganism by qPCR. These methods identify allelic variation, SNPs, and genetic mutations of a particular gene such as those responsible for conferring resistance or sensitivity to an antibiotic, chemotherapy, or another chemical compound. By selection of appropriate gene regions, mutation loci that confer resistance to key antibiotics can be identified by qPCR. Additionally, the approach can identify heteroresistant strains, e.g., populations of strains from a sample that contain both mutation and wild-type nucleotides. By selecting appropriate that bind efficiently to the area of mutation can identify resistance conferring mutations. Methods are useful to sequences derived from viral agents, such as influenza virus, bacterial agents, such as tuberculosis bacteria, and cancer cells.

Disclosed is an enhanced method for rapid and cost-effective analysis of sequences of a microorganism by qPCR. These methods identify allelic variation, SNPs, and genetic mutations of a particular gene such as those responsible for conferring resistance or sensitivity to an antibiotic, chemotherapy, or another chemical compound. By selection of appropriate gene regions, mutation loci that confer resistance to key antibiotics can be identified by qPCR. Additionally, the approach can identify heteroresistant strains, e.g., populations of strains from a sample that contain both mutation and wild-type nucleotides. By selecting appropriate that bind efficiently to the area of mutation can identify resistance conferring mutations. Methods are useful to sequences derived from viral agents, such as influenza virus, bacterial agents, such as tuberculosis bacteria, and cancer cells.