This disclosure relates to analyzing the end-to-end sequence and the relative distributions in heterogeneous mixtures of polynucleotides and methods and enabling reagents related thereto. In certain embodiments this method relates to the complete full length sequencing and quantitative profiling of mRNAs present in the transcriptomes of cells or tissues of, but not limited to, higher multicellular organisms that possess interrupted genes subject to complex post-transcriptional RNA processing

This disclosure relates to analyzing the end-to-end sequence and the relative distributions in heterogeneous mixtures of polynucleotides and methods and enabling reagents related thereto. In certain embodiments this method relates to the complete full length sequencing and quantitative profiling of mRNAs present in the transcriptomes of cells or tissues of, but not limited to, higher multicellular organisms that possess interrupted genes subject to complex post-transcriptional RNA processing

The present invention relates to a method for detecting a target RNA polynucleotide by using a catcher polynucleotide which anneals to at least a portion of the target RNA polynucleotide, and a catalytically-inactive crRNA-guided (CRISPR RNA) RNA-binding protein which binds to the target RNA polynucleotide. Further envisaged is a kit for detecting a specific target RNA polynucleotide comprising inter alia one or more catcher polynucleotides complementary to at least a portion of the target RNA polynucleotide, a mature crRNA molecule which is specific for a target sequence on the target RNA polynucleotide and a catalytically-inactive crRNA-guided RNA-binding protein; as well as the use of these ingredients for the detection of the target RNA polynucleotide.

The present invention relates to a method for detecting a target RNA polynucleotide by using a catcher polynucleotide which anneals to at least a portion of the target RNA polynucleotide, and a catalytically-inactive crRNA-guided (CRISPR RNA) RNA-binding protein which binds to the target RNA polynucleotide. Further envisaged is a kit for detecting a specific target RNA polynucleotide comprising inter alia one or more catcher polynucleotides complementary to at least a portion of the target RNA polynucleotide, a mature crRNA molecule which is specific for a target sequence on the target RNA polynucleotide and a catalytically-inactive crRNA-guided RNA-binding protein; as well as the use of these ingredients for the detection of the target RNA polynucleotide.

The present disclosure provides a method for reversibly protecting and separation DNA, comprising phosphorylating the 5′-terminal of a target DNA molecule, modifying the 5′-terminal by adenylation; adding adenosine DNA-sensitive exonuclease to samples obtained after termination of the reaction to digest the template; finally, the obtained adenylated modified DNA, that is, the obtained target DNA is separated, and subjected to technical analysis such as sequencing and identification, and the 5′end of the obtained sequence is the site of adenylation modification. The method provided by the present disclosure fills the gap that the prior art cannot accurately locate the break site on genomic DNA, can realize the quantitative and positioning analysis of the break site on DNA samples of different lengths and different sources, and is simple to use and easy to operate. And there are no special requirements for samples, high accuracy, low detection background influence, and high resolution.

The present disclosure provides a method for reversibly protecting and separation DNA, comprising phosphorylating the 5′-terminal of a target DNA molecule, modifying the 5′-terminal by adenylation; adding adenosine DNA-sensitive exonuclease to samples obtained after termination of the reaction to digest the template; finally, the obtained adenylated modified DNA, that is, the obtained target DNA is separated, and subjected to technical analysis such as sequencing and identification, and the 5′end of the obtained sequence is the site of adenylation modification. The method provided by the present disclosure fills the gap that the prior art cannot accurately locate the break site on genomic DNA, can realize the quantitative and positioning analysis of the break site on DNA samples of different lengths and different sources, and is simple to use and easy to operate. And there are no special requirements for samples, high accuracy, low detection background influence, and high resolution.

Provided herein are methods of identifying the origin of a nucleic acid sample. The methods include forming a reaction mixture comprising a nucleic acid sample comprising nucleic acid molecules from a single cell and a set of barcodes, incorporating the set of barcodes into the nucleic acid molecules of the sample, and identifying the set of barcodes incorporated into the nucleic acid molecules of the single cell thereby identifying the origin of the nucleic acid sample.

Provided herein are methods of identifying the origin of a nucleic acid sample. The methods include forming a reaction mixture comprising a nucleic acid sample comprising nucleic acid molecules from a single cell and a set of barcodes, incorporating the set of barcodes into the nucleic acid molecules of the sample, and identifying the set of barcodes incorporated into the nucleic acid molecules of the single cell thereby identifying the origin of the nucleic acid sample.

Disclosed herein include systems, methods, compositions, and kits for 5′-based gene expression profiling. Some embodiments provide synthetic particles (e.g., beads) associated with a first plurality of oligonucleotide barcodes and a second plurality of oligonucleotide barcodes. In some embodiments, nucleic acid targets (e.g., mRNAs) are initially barcoded on the 3′ end with the first plurality of oligonucleotide barcodes and subsequently barcoded on the 5′ end following a template switching reaction and intermolecular hybridization with the first plurality of oligonucleotide barcodes and extension. Immune repertoire profiling methods are also provided in some embodiments.

Disclosed herein include systems, methods, compositions, and kits for 5′-based gene expression profiling. Some embodiments provide synthetic particles (e.g., beads) associated with a first plurality of oligonucleotide barcodes and a second plurality of oligonucleotide barcodes. In some embodiments, nucleic acid targets (e.g., mRNAs) are initially barcoded on the 3′ end with the first plurality of oligonucleotide barcodes and subsequently barcoded on the 5′ end following a template switching reaction and intermolecular hybridization with the first plurality of oligonucleotide barcodes and extension. Immune repertoire profiling methods are also provided in some embodiments.