The present disclosure in some aspects relate to a direct RNA (dRNA) detection approach incorporating the use of circularizable probes (e.g., padlock probes) having asymmetric arms, rolling circle amplification of the ligated circularizable probes, and in situ detection (e.g., using hybridization-based in situ sequencing) for multiplexed spatial analysis of nucleic acid sequences (e.g., short sequences such as SNPs and point mutations) in a biological sample. In some aspects, compositions and methods disclosed herein improve detection specificity, reduce false positive signal detection, and/or maintain or improve detection efficiency.

Single-cell RNA sequencing (scRNAseq) allows the identification, characterization, and quantification of cell types in a tissue. When focused on the adaptive immune system's T and B cells, scRNAseq carries the potential to track the clonal lineage of each analyzed cell through the unique rearranged sequence of its antigen receptor (TCR or BCR, respectively), and link it to the functional state inferred from transcriptome analysis. Computational approaches to infer clonality and maturation status (for BCR only) from scRNAseq datasets of T and B cells have been developed but there are cumbersome and not costly effective. The inventors have now developed a FACS-based 5′-end RNAseq method, in particular a FACS-based 5′-end scRNAseq method, for cost effective integrative analysis of B and T cell transcriptome and paired BCR and TCR repertoire in phenotypically defined B and T cell subsets. In particular, the method of the present invention includes a reverse transcription step that uses a number of different well specific template switching oligonucleotides (TSO) to introduce a well-specific DNA barcode in the 5′-end of cDNAs.

Single-cell RNA sequencing (scRNAseq) allows the identification, characterization, and quantification of cell types in a tissue. When focused on the adaptive immune system's T and B cells, scRNAseq carries the potential to track the clonal lineage of each analyzed cell through the unique rearranged sequence of its antigen receptor (TCR or BCR, respectively), and link it to the functional state inferred from transcriptome analysis. Computational approaches to infer clonality and maturation status (for BCR only) from scRNAseq datasets of T and B cells have been developed but there are cumbersome and not costly effective. The inventors have now developed a FACS-based 5′-end RNAseq method, in particular a FACS-based 5′-end scRNAseq method, for cost effective integrative analysis of B and T cell transcriptome and paired BCR and TCR repertoire in phenotypically defined B and T cell subsets. In particular, the method of the present invention includes a reverse transcription step that uses a number of different well specific template switching oligonucleotides (TSO) to introduce a well-specific DNA barcode in the 5′-end of cDNAs.

The present application provides methods for detecting a target nucleic acid molecule in a sample comprising contacting said sample with a ligatable probe comprising one or more parts and allowing said probe to hybridise to the target nucleic acid molecule, ligating any probe which has hybridised to the target nucleic acid molecule, amplifying the ligated probe, and detecting the amplification product, thereby to detect the target nucleic acid molecule, wherein said probes comprise at least one ribonucleotide at or near to a ligation site and/or wherein the probe or a probe part comprises an additional sequence 5′ to a target-specific binding site which is not hybridised to the target nucleic acid molecule upon hybridisation of the probe to the target nucleic acid molecule and forms a 5′ flap containing one or more nucleotides at its 3′ end that is cleaved prior to ligation, and methods of synthesising a DNA molecule with Phi29 DNA polymerase using a template nucleic acid molecule comprising at least one ribonucleotide. Probes for use in the detection methods are provided.

The present application provides methods for detecting a target nucleic acid molecule in a sample comprising contacting said sample with a ligatable probe comprising one or more parts and allowing said probe to hybridise to the target nucleic acid molecule, ligating any probe which has hybridised to the target nucleic acid molecule, amplifying the ligated probe, and detecting the amplification product, thereby to detect the target nucleic acid molecule, wherein said probes comprise at least one ribonucleotide at or near to a ligation site and/or wherein the probe or a probe part comprises an additional sequence 5′ to a target-specific binding site which is not hybridised to the target nucleic acid molecule upon hybridisation of the probe to the target nucleic acid molecule and forms a 5′ flap containing one or more nucleotides at its 3′ end that is cleaved prior to ligation, and methods of synthesising a DNA molecule with Phi29 DNA polymerase using a template nucleic acid molecule comprising at least one ribonucleotide. Probes for use in the detection methods are provided.

Disclosed is a hybridisation capture method based on the pyrophosphorolysis reaction. According to the present invention, there is provided a method for increasing the ratio of a first nucleic add sequence to second nucleic add sequence in a sample.

Disclosed is a hybridisation capture method based on the pyrophosphorolysis reaction. According to the present invention, there is provided a method for increasing the ratio of a first nucleic add sequence to second nucleic add sequence in a sample.

Isolation or in vitro assembly of the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system and use for cleavage of DNA bearing a nucleotide sequence complementary to the crRNA and a proto-spacer adjacent motif. Methods for site-specific modification of a target DNA molecule using an RNA-guided DNA endonuclease comprising at least one RNA sequence and at least one of an RuvC active site motif and an HNH active site motif; for conversion of Cas9 polypeptide into a nickase cleaving one strand of double-stranded DNA by inactivating one of the active sites (RuvC or HNH) in the polypeptide by at least one point mutation; for assembly of active polypeptide-polyribonucleotides complex in vivo or in vitro; and for re-programming a Cas9-crRNA complex specificity in vitro or using a cassette containing a single repeat-spacer-repeat unit.

Isolation or in vitro assembly of the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system and use for cleavage of DNA bearing a nucleotide sequence complementary to the crRNA and a proto-spacer adjacent motif. Methods for site-specific modification of a target DNA molecule using an RNA-guided DNA endonuclease comprising at least one RNA sequence and at least one of an RuvC active site motif and an HNH active site motif; for conversion of Cas9 polypeptide into a nickase cleaving one strand of double-stranded DNA by inactivating one of the active sites (RuvC or HNH) in the polypeptide by at least one point mutation; for assembly of active polypeptide-polyribonucleotides complex in vivo or in vitro; and for re-programming a Cas9-crRNA complex specificity in vitro or using a cassette containing a single repeat-spacer-repeat unit.

The present invention relates to the field of genotyping samples containing short tandem repeat (STR) loci. More specifically, the present invention discloses a composition of matter containing an array of probes and a method to genotype these loci relying on the recognition of RNA:DNA base pairing followed by cleavage of the RNA containing strand. By measuring the temperature at which the chimeric DNA-RNA-DNA probe is cleaved, resulting in an increase of fluorescence of the probe, it can be assessed whether or not the probe and the sample share the same amount of repeats. An array of probes is utilised, covering all possible alleles of the investigated STR-locus. The probes and method of the present invention are well-suited to be used in a portable, less-expensive DNA analysis device and can be applied in other fields than forensics, like food fraud, diagnostics and many others.