Disclosed herein include systems, methods, compositions, and kits for multiplexing single-cell RNA-sequencing (scRNA-seq) samples. In some embodiments, the methods comprise chemically tagging cells with identifying sample tags (e.g., barcoded DNA oligonucleotides).

Localized detection of RNA in a tissue sample that includes cells is accomplished on an array. The array include a number of features on a substrate. Each feature includes a different capture probe immobilized such that the capture probe has a free 3′ end. Each feature occupies a distinct position on the array and has an area of less than about 1 mm.sup.2. Each capture probe is a nucleic acid molecule, which includes a positional domain including a nucleotide sequence unique to a particular feature, and a capture domain including a nucleotide sequence complementary to the RNA to be detected. The capture domain can be at a position 3′ of the positional domain.

Provided herein are compositions and methods using mutant Phi29 polymerases for nucleic acid amplification. Further provided herein are methods for accurate and scalable Primary Template-Directed Amplification (PTA) nucleic acid amplification and sequencing methods, and their applications for mutational analysis in research, diagnostics, and treatment using mutant Phi29 polymerases.

The invention is directed to a method for detecting RNA, DNA or protein target sequences by a) Hybridizing a library of probes having the general formula (I)

P—(CL-D).sub.x  (I)  With P: probes having at least 10 nucleotides or amino acids CL: cleavable linker D: fluorescent dye X: integer between 1 and 5  to RNA, DNA or protein target sequences wherein the library comprises probes P having different sequences of nucleotides or amino acids and cleavable linkers CL of different groups which are cleavable with different means b) Removing unhybridized probes and detecting the hybridized probes via the fluorophores D by a first image c) Cleaving sequentially by different means each group of chemical linkers CL from the hybridized probes; removing the thus cleaved fluorophores D and detecting the remaining hybridized probes via their fluorophores D by a second image d) Detecting the removed fluorophores D by comparing the first and second image. e) Obtaining a part of the sequence information of the target sequences via the sequence information of the probes P associated with the removed fluorophores D f) Repeating step c) until all groups of chemical linkers CL are cleaved.

Multiple polynucleotides with random sequences are collectively used as a molecular anti-counterfeiting tag. The polynucleotides are placed on an item as a molecular identifier of authenticity. Each position within the random sequences is synthesized using a predetermined ratio of nucleoside bases. With this technique the sequence of each polynucleotide is random but the ratio of nucleoside bases over the collection of synthetic polynucleotides is not. Verification of authenticity is achieved by sequencing a portion of the polynucleotides collected from the item and calculating the ratio of nucleoside bases at each position. If these ratios are the same or similar to the ratios used for synthesizing the polynucleotides, then the item is identified as authentic. The ratios of nucleoside bases and a description of the item may be stored in an electronic record that is used for validating authenticity of the item.

The present invention relates to methods for diagnosing a disease by determining via multiplex fluorescence in situ hybridization (FISH) whether or not mRNA species and/or at least one miRNA species of disease-associated biomarkers ar present in a sample obtained from a subject, and by determining by multiplex sequencing whether or not said mRNA species of disease-associated biomarkers and/or said miRNA species of disease-associated biomarkers of step (a) are present in said sample. The present invention also relates to kits for performing the methods for diagnosis as described and provided herein as well as use of such kits for performing the methods for diagnosis as described and provided herein.

The present invention relates to a method of spatially barcoding a given location on a substrate, and further to spatially barcoding detection probes present in a sample such as a biological tissue specimen for the purposes of analysing molecular features present in the tissue. Such analysis may include: i) the spatial expression of one or more biological molecules, specifically; ii) the spatial analysis of the transcriptome and/or iii) the spatial analysis of the proteome, including post-translational protein modifications. The invention further relates to various component products for performing such methods that include reagents kits, instrumentation and software.

The present invention relates to a method of spatially barcoding a given location on a substrate, and further to spatially barcoding detection probes present in a sample such as a biological tissue specimen for the purposes of analysing molecular features present in the tissue. Such analysis may include: i) the spatial expression of one or more biological molecules, specifically; ii) the spatial analysis of the transcriptome and/or iii) the spatial analysis of the proteome, including post-translational protein modifications. The invention further relates to various component products for performing such methods that include reagents kits, instrumentation and software.

The present invention provides a method of identifying the strength of one or more unique regulatory elements (URE) having conformational effect on a transcribable reporter sequence.

A method for spatially tagging nucleic acids of a biological specimen, including steps of (a) providing a solid support comprising different nucleic acid probes that are randomly located on the solid support, wherein the different nucleic acid probes each includes a barcode sequence that differs from the barcode sequence of other randomly located probes on the solid support; (b) performing a nucleic acid detection reaction on the solid support to locate the barcode sequences on the solid support; (c) contacting a biological specimen with the solid support that has the randomly located probes; (d) hybridizing the randomly located probes to target nucleic acids from portions of the biological specimen; and (e) modifying the randomly located probes that are hybridized to the target nucleic acids, thereby producing modified probes that include the barcode sequences and a target specific modification, thereby spatially tagging the nucleic acids of the biological specimen.