A method and system for constructing sequencing library, includes: obtaining a transformed DNA sample with a universal sequence; performing amplification using a first specific primer located upstream of the target region and a first universal primer at least partially matching or overlapping the universal sequence; and performing amplification using a second specific primer, a second universal primer and a tagged primer. The second specific primer is located downstream of the first specific primer and upstream of the target region, the second universal primer overlaps at least a partial sequence of the second specific primer, and the tagged primer overlaps a partial sequence of the first universal primer. Alternatively, the second specific primer is located downstream of the target region, the second universal primer overlaps at least a partial sequence of the first specific primer, and the tagged primer overlaps a partial sequence of the second specific primer.

A method and system for constructing sequencing library, includes: obtaining a transformed DNA sample with a universal sequence; performing amplification using a first specific primer located upstream of the target region and a first universal primer at least partially matching or overlapping the universal sequence; and performing amplification using a second specific primer, a second universal primer and a tagged primer. The second specific primer is located downstream of the first specific primer and upstream of the target region, the second universal primer overlaps at least a partial sequence of the second specific primer, and the tagged primer overlaps a partial sequence of the first universal primer. Alternatively, the second specific primer is located downstream of the target region, the second universal primer overlaps at least a partial sequence of the first specific primer, and the tagged primer overlaps a partial sequence of the second specific primer.

The present invention provides a method of detecting and a method of quantifying oligonucleotides with more excellent specificity and quantitativity as compared to conventional signal amplification (PALSAR) measurement methods.

In the present invention, the problem is solved by hybridizing a target oligonucleotide to be measured with a complementary nucleic acid probe (3′-complementary sequence of target sequence-5′), or hybridizing the target oligonucleotide to be measured having given bases such as poly(A) added thereto with a complementary nucleic acid probe (3′-complementary sequence of target oligonucleotide+complementary sequence of given bases-5′), decomposing and removing an incomplete hybridization product by using a single-strand-specific nuclease such as Si nuclease, and measuring the nucleic acid probe contained in a remaining complete hybridization product by a PALSAR method.

The present invention provides a method of detecting and a method of quantifying oligonucleotides with more excellent specificity and quantitativity as compared to conventional signal amplification (PALSAR) measurement methods.

In the present invention, the problem is solved by hybridizing a target oligonucleotide to be measured with a complementary nucleic acid probe (3′-complementary sequence of target sequence-5′), or hybridizing the target oligonucleotide to be measured having given bases such as poly(A) added thereto with a complementary nucleic acid probe (3′-complementary sequence of target oligonucleotide+complementary sequence of given bases-5′), decomposing and removing an incomplete hybridization product by using a single-strand-specific nuclease such as Si nuclease, and measuring the nucleic acid probe contained in a remaining complete hybridization product by a PALSAR method.

Provided herein are methods of determining a location of a biological analyte in a biological sample.

Provided herein are methods of determining a location of a biological analyte in a biological sample.

A system, methods, and apparatus are described to collect and prepare single cells and groups of cells from microsamples of specimens and encode spatial information of the physical position of the cells in the specimen. In some embodiment, beads or surfaces with oligonucleotides containing spatial barcodes are used to analyze DNA or RNA. The spatial barcodes allow the position of the cell to be defined and the nucleic acid sequencing information, such as target sequencing, whole genome, gene expression, used to analyze the cells in a microsample for cell type, expression pattern, DNA sequence, and other information, in the context of the cell's physical position in the specimen. In other embodiment, markers such as isotopes are added to a microsample to encode spatial position with mass spectoscopy or other analysis. The spatial encoded information is then readout by analysis such as DNA sequencing, mass spectrometry, fluorescence, or other methods.

A system, methods, and apparatus are described to collect and prepare single cells and groups of cells from microsamples of specimens and encode spatial information of the physical position of the cells in the specimen. In some embodiment, beads or surfaces with oligonucleotides containing spatial barcodes are used to analyze DNA or RNA. The spatial barcodes allow the position of the cell to be defined and the nucleic acid sequencing information, such as target sequencing, whole genome, gene expression, used to analyze the cells in a microsample for cell type, expression pattern, DNA sequence, and other information, in the context of the cell's physical position in the specimen. In other embodiment, markers such as isotopes are added to a microsample to encode spatial position with mass spectoscopy or other analysis. The spatial encoded information is then readout by analysis such as DNA sequencing, mass spectrometry, fluorescence, or other methods.

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization. Such polynucleotide processing may be useful for a variety of applications, including analyte characterization by polynucleotide sequencing. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes including, for example, protein (e.g., cell surface or intracellular proteins), genomic DNA, and RNA (e.g., mRNA or CRISPR guide RNAs). Also described herein, are barcoded labelling agents and oligonucleotide molecules useful for “tagging” analytes for characterization

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization. Such polynucleotide processing may be useful for a variety of applications, including analyte characterization by polynucleotide sequencing. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes including, for example, protein (e.g., cell surface or intracellular proteins), genomic DNA, and RNA (e.g., mRNA or CRISPR guide RNAs). Also described herein, are barcoded labelling agents and oligonucleotide molecules useful for “tagging” analytes for characterization