Systems, methods, and compositions provided herein relate to preparation of beads encapsulating genetic material. Some embodiments include preparation of nucleic acid libraries within the bead, wherein the bead includes pores that allow diffusion of reagents while retaining genetic material.

A method of preparing a library of library constructs by multiplex amplification for use in targeted next generation sequencing is described. The method comprises the steps of: (a) providing a reaction vessel comprising (i) a plurality of different target sequences, (ii) a plurality of target capture primer pairs, and (iii) one or more tagging primer pairs, (b) performing sequential rounds of amplification at sequential annealing temperatures configured to amplify the target sequences, generate target sequences comprising first or second read sequences, and provide a reaction product comprising library constructs in a sequential manner; and (c) capture of the library of constructs from the reaction product. One of the forward and reverse tagging primers comprises a purification label at the 5′ end, and is provided at a limiting concentration whereby the library constructs comprises an abundance of partial constructs containing only one indexing sequences and only one adapter sequences, and a limited number of full (complete) constructs containing the first and second indexing sequences, the first and second adapter sequences and the purification label. The capture step comprises capturing the full (complete) constructs from the reaction product using the purification label.

Constructs and methods are described for producing sparse and stochastic labeling of cells expressing one or more site-specific recombinases in a host mammal. Described is a nucleic acid construct comprising, in operable linkage, a translation start site, an optional spacer, a polycytosine mononucleotide repeat, and an open reading frame (ORF), wherein the polycytosine repeat and the ORF are out of frame with respect to the translation start site. The simple, general, and scalable solution for genetically-directed sparse cell labeling allows the visualization of the complete cellular morphology of cells. Representative examples of cells to be visualized using sparse labeling include, but are not limited to, neurons or non-neuronal cells in the central nervous system (including brain), peripheral nervous systems, and other peripheral tissues.

Compositions and methods for detecting nucleic acid-protein interactions, or more generally interactions between a nucleic acid and another molecule. A Cas protein (e.g., a catalytically dead Cas13) is fused to a proximity tagging enzyme (e.g., a Pup ligase) and thus brings the proximity tagging enzyme to the proximity of a protein that binds to a nucleic acid, when the Cas protein recognizes the nucleic acid, e.g., through a guide RNA. The proximity tagging enzyme then tags the protein enabling it to be identified as a protein that interacts with the nucleic acid.

Methods for performing procedures on single analytes at single-analyte resolution are disclosed. The methods utilize an iterative approach to performing a sequence of steps during a single-analyte process. Control of the single-analyte process is achieved by implementing actions during each iteration based upon one or more determined process metrics. Systems are also detailed for implementing the disclosed methods at single-analyte resolution.

Provided herein are methods for preparing a sequencing library that includes nucleic acids from a plurality of single cells. In one embodiment, the sequencing library includes whole genome nucleic acids from the plurality of single cells. In one embodiment, the method includes generating nucleosome-depleted nuclei by chemical treatment while maintaining integrity of the nuclei. Also provided herein are compositions, such as compositions that include chemically treated nucleosome-depleted isolated nuclei.

Methods and systems for security, authentication, tagging, and tracking using nucleic acid (e.g., deoxyribonucleic acid) molecules encoding information. Unique nucleic acid molecules are efficiently produced from pre-fabricated fragments to quickly produce libraries of nucleic acid molecules encoding encrypted or randomized information. Physical objects or artifacts can be tagged with libraries to authenticate the objects, grant access to secured assets or locations, or track the objects or entities. Chemical methods can be applied to verify authenticity, decrypt, or decode information stored in the libraries.

A system and method are provided for simplifying and accelerating the screening and characterization of molecular interactions by high-throughput functional screening and sequencing of single cells. More specifically, a platform is provided which combines a solid support and an innovative method for capturing and barcoding of nucleic acids that allows simultaneous phenotyping and genotyping of >100, 000s of cells.

Single-cell multi-omics by co-encapsulating a single cell with two beads, the first an RNA barcoding bead having barcoded mRNA capture primer oligonucleotides attached on the bead surface; and the second a DNA barcoding bead having two types of oligonucleotides releasably attached to the surface: (1) barcoded adapter oligonucleotides that are complementary to oligonucleotides bound to the transposase that are eventually incorporated into gDNA fragments and (2) polyadenylated barcoded oligonucleotides containing the same barcode sequence as the adapters. In addition, integrated analysis of RNA and protein, including intracellular protein, from individual cells using similar co-encapsulation of a single cell, an RNA barcoding bead, and with/without a specific or non-specific protein binding bead in a microwell, to avoid protein fixation by first lysing the cell to liberate intracellular contents, and then capturing protein either on a solid surface or in solution with barcoded affinity reagents.

This invention provides methods for near-instantaneously separating cells that have undergone RNA guided genome modifications into pre-templated instant partitions (PIPs) and using the PIPs to associate the guide RNAs with the gene expression level changes that resulted from the genome modification.