Provided herein are methods, compositions, and kits for preparing biological samples for multiplex spatial gene expression and proteomic analysis, such as determining a location of a nucleic acid analyte and a protein analyte in a biological sample.

Provided is a composition and method for suppressing the reverse transcription of ribosomal RNA. The composition for suppressing the reverse transcription of ribosomal RNA contains at least one nucleic acid polymer selected from the group consisting of inosinic acid polymers, cytidylic acid polymers, guanylic acid polymers, adenylic acid polymers, thymidylic acid polymers, uridylic acid polymers, deoxyinosinic acid polymers, deoxycytidylic acid polymers, deoxyguanylic acid polymers, deoxyadenylic acid polymers, deoxythymidylic acid polymers, and deoxyuridylic acid polymers.

Provided is a composition and method for suppressing the reverse transcription of ribosomal RNA. The composition for suppressing the reverse transcription of ribosomal RNA contains at least one nucleic acid polymer selected from the group consisting of inosinic acid polymers, cytidylic acid polymers, guanylic acid polymers, adenylic acid polymers, thymidylic acid polymers, uridylic acid polymers, deoxyinosinic acid polymers, deoxycytidylic acid polymers, deoxyguanylic acid polymers, deoxyadenylic acid polymers, deoxythymidylic acid polymers, and deoxyuridylic acid polymers.

Provided is a PCR-free library construction and sequencing method. A PCR-free high-throughput sequencing method is provided, including the following steps: obtaining a DNA fragment of target size by performing or not performing, based on a size of a nucleic acid sample, fragmentation on the nucleic acid sample; performing end repair and an A-tailing reaction; ligating an adapter containing a barcode; obtaining DNA nanoballs by performing single-strand cyclization and rolling circle replication; and loading and sequencing.

Provided is a PCR-free library construction and sequencing method. A PCR-free high-throughput sequencing method is provided, including the following steps: obtaining a DNA fragment of target size by performing or not performing, based on a size of a nucleic acid sample, fragmentation on the nucleic acid sample; performing end repair and an A-tailing reaction; ligating an adapter containing a barcode; obtaining DNA nanoballs by performing single-strand cyclization and rolling circle replication; and loading and sequencing.

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.

Compartment-free single cell genetic analysis methods are provided. Aspects of the methods include: (a) combining a cellular sample with a plurality of distinct barcoded beads comprising barcoded reverse primers under conditions sufficient to produce a liquid composition comprising a plurality of separated cell/barcoded bead complexes; (b) hybridizing template binding domains of barcoded reverse primers to template nucleic acids of the cells to produce primed template nucleic acids; and (c) subjecting the primed template nucleic acids to primer extension reaction conditions sufficient to produce barcoded nucleic acids, e.g., for subsequent amplification and analysis, such as by Next Generation Sequencing (NGS) protocols. Also provided are compositions that find use in practicing embodiments of the methods.

Compartment-free single cell genetic analysis methods are provided. Aspects of the methods include: (a) combining a cellular sample with a plurality of distinct barcoded beads comprising barcoded reverse primers under conditions sufficient to produce a liquid composition comprising a plurality of separated cell/barcoded bead complexes; (b) hybridizing template binding domains of barcoded reverse primers to template nucleic acids of the cells to produce primed template nucleic acids; and (c) subjecting the primed template nucleic acids to primer extension reaction conditions sufficient to produce barcoded nucleic acids, e.g., for subsequent amplification and analysis, such as by Next Generation Sequencing (NGS) protocols. Also provided are compositions that find use in practicing embodiments of the methods.

The present invention provides methods, compositions and kits for enriching and determining nucleotide sequences of a plurality of target loci from a sample comprising nucleic acids. The methods comprise one or more cycles of primer extension followed by PCR amplification of target sequences using nested target-specific primers.