The present disclosure provides methods and compositions for molecular tagging of complex populations of nucleic acid molecules. The disclosure provides methods and compositions to obtain phase information of tagged nucleic acid molecules from high-throughput nucleic acid sequencing data.

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.

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.

The present disclosure describes technologies that permit sensitive detection of nucleic acids of interest (i.e., nucleic acids whose nucleotide sequence is or includes a target sequence). Among other things the disclosure provides a system comprising: a plurality of nucleic acid molecules having different nucleotide sequences; a set of ligation oligonucleotides, comprising: a first ligation oligonucleotide whose nucleotide sequence includes a templating element and a first target hybridization element; and a second ligation oligonucleotide whose nucleotide sequence includes a second target hybridization element and optionally a second templating element; wherein the target hybridization elements bind to different portions of a common target site, to form a gapped nucleic acid strand susceptible to ligation with a ligase to generate a ligated strand that is amenable to lateral flow assessment.

The present disclosure describes technologies that permit sensitive detection of nucleic acids of interest (i.e., nucleic acids whose nucleotide sequence is or includes a target sequence). Among other things the disclosure provides a system comprising: a plurality of nucleic acid molecules having different nucleotide sequences; a set of ligation oligonucleotides, comprising: a first ligation oligonucleotide whose nucleotide sequence includes a templating element and a first target hybridization element; and a second ligation oligonucleotide whose nucleotide sequence includes a second target hybridization element and optionally a second templating element; wherein the target hybridization elements bind to different portions of a common target site, to form a gapped nucleic acid strand susceptible to ligation with a ligase to generate a ligated strand that is amenable to lateral flow assessment.

Methods and systems are provided for massively parallel genetic analysis of single cells in emulsion droplets or reaction containers. Genetic loci of interest are targeted in a single cell using a set of probes, and a fusion complex is formed by molecular linkage and amplification techniques. Methods are provided for high-throughput, massively parallel analysis of the fusion complex in a single cell in a population of at least 10,000 cells. Also provided are methods for tracing genetic information back to a cell using barcode sequences.

Methods and systems are provided for massively parallel genetic analysis of single cells in emulsion droplets or reaction containers. Genetic loci of interest are targeted in a single cell using a set of probes, and a fusion complex is formed by molecular linkage and amplification techniques. Methods are provided for high-throughput, massively parallel analysis of the fusion complex in a single cell in a population of at least 10,000 cells. Also provided are methods for tracing genetic information back to a cell using barcode sequences.

This present disclosure describes hybridization probes modularly constructed from several oligonucleotides with a pattern of designed complementary interactions, allowing the probes to sequence-specifically capture or analyze nucleic acid target sequences that are long and/or complex.