This invention provides methods for near-instantaneously separating cells that have undergone RNA guided genome modifications into pre-templated instant partitions (PIPs) and using the PIPs to associate the guide RNAs with the gene expression level changes that resulted from the genome modification.

Provided herein is a method for detecting the presence of a COVID-19 virus RNA or other pathogenic respiratory viruses, such as an influenza virus, or other RNA of interest in a sample. Nucleic acids are obtained from the sample and are used as a template in a combined isothermal reverse transcription, RNAse H and isothermal amplification reaction to generate single stranded RNA amplicons containing sequences complementary to fluorescent labeled detector probes. The single-stranded RNA amplicons hybridize to the detector probe and to hybridization probes with sequences complementary to a sequence determinant in the COVID-19 or other virus RNAs. The microarray is imaged to detect fluorescent signals thereby identifying the virus.

Provided herein is a method for detecting the presence of a COVID-19 virus RNA or other pathogenic respiratory viruses, such as an influenza virus, or other RNA of interest in a sample. Nucleic acids are obtained from the sample and are used as a template in a combined isothermal reverse transcription, RNAse H and isothermal amplification reaction to generate single stranded RNA amplicons containing sequences complementary to fluorescent labeled detector probes. The single-stranded RNA amplicons hybridize to the detector probe and to hybridization probes with sequences complementary to a sequence determinant in the COVID-19 or other virus RNAs. The microarray is imaged to detect fluorescent signals thereby identifying the virus.

The methods, compositions, and kits of the disclosure provide a novel approach for a whole genome, unbiased DNA analysis method that can be performed on limited amounts of DNA. can be used to analyze DNA to determine its modification status. Aspects of the disclosure relate to a method for amplifying bisulfite-treated deoxyribonucleic acid (DNA) molecules comprising: (a) ligating an adaptor to the DNA molecules, wherein the adaptor comprises a RNA polymerase promoter comprising bisulfite-protected cytosines; (b) treating the ligated DNA molecules with bisulfite; (c) hybridizing the bisulfite-treated DNA molecules with a primer; (d) extending the hybridized primer to make double stranded DNA; and (e) in vitro transcribing the double-stranded DNA to make RNA.

The methods, compositions, and kits of the disclosure provide a novel approach for a whole genome, unbiased DNA analysis method that can be performed on limited amounts of DNA. can be used to analyze DNA to determine its modification status. Aspects of the disclosure relate to a method for amplifying bisulfite-treated deoxyribonucleic acid (DNA) molecules comprising: (a) ligating an adaptor to the DNA molecules, wherein the adaptor comprises a RNA polymerase promoter comprising bisulfite-protected cytosines; (b) treating the ligated DNA molecules with bisulfite; (c) hybridizing the bisulfite-treated DNA molecules with a primer; (d) extending the hybridized primer to make double stranded DNA; and (e) in vitro transcribing the double-stranded DNA to make RNA.

Disclosed herein are methods and systems for correcting errors in sample amplification, including the errors occurred in determining the number of targets in samples. In some embodiments, the method comprises: stochastically barcoding a plurality of targets in the samples using oligonucleotides comprising stochastic barcodes to generate stochastically barcoded targets; contacting one or more defined barcoded primers with each of the one or more samples; and determining an amplification noise.

Disclosed herein are methods and systems for correcting errors in sample amplification, including the errors occurred in determining the number of targets in samples. In some embodiments, the method comprises: stochastically barcoding a plurality of targets in the samples using oligonucleotides comprising stochastic barcodes to generate stochastically barcoded targets; contacting one or more defined barcoded primers with each of the one or more samples; and determining an amplification noise.

The present invention refers to a method directed to RT-qPCR reactions, preferably performed in a one or two-step approach combining the reverse transcription and subsequent PCR in a single tube and buffer, using a reverse transcriptase along with a DNA polymerase with mandatory 3′.fwdarw.5′ exonuclease activity, which corrects miss-incorporated nucleotides. In particular, in this invention we present a One-step RT-PCR, preferably qPCR, method with a novel priming strategy that utilizes a novel bivalent reverse primer, wherein this primer is used for both, i) the generation of cDNA and ii) the completion of the subsequent amplification using that same cDNA as template. This bivalent reverse primer also allows end-tagging the amplicon(s) obtained so that they can be used in a variety of applications including, standard sequencing, next generation sequencing (NGS), gene expression analysis, RNAi validation, microarray validation, pathogen detection, genetic testing, and disease research.

The present invention refers to a method directed to RT-qPCR reactions, preferably performed in a one or two-step approach combining the reverse transcription and subsequent PCR in a single tube and buffer, using a reverse transcriptase along with a DNA polymerase with mandatory 3′.fwdarw.5′ exonuclease activity, which corrects miss-incorporated nucleotides. In particular, in this invention we present a One-step RT-PCR, preferably qPCR, method with a novel priming strategy that utilizes a novel bivalent reverse primer, wherein this primer is used for both, i) the generation of cDNA and ii) the completion of the subsequent amplification using that same cDNA as template. This bivalent reverse primer also allows end-tagging the amplicon(s) obtained so that they can be used in a variety of applications including, standard sequencing, next generation sequencing (NGS), gene expression analysis, RNAi validation, microarray validation, pathogen detection, genetic testing, and disease research.

The disclosure provides methods for processing nucleic acid populations containing different forms (e.g., RNA and DNA, single-stranded or double-stranded) and/or extents of modification (e.g., cytosine methylation, association with proteins). These methods accommodate multiple forms and/or modifications of nucleic acid in a sample, such that sequence information can be obtained for multiple forms. The methods also preserve the identity of multiple forms or modified states through processing and analysis, such that analysis of sequence can be combined with epigenetic analysis.