This disclosure describes compositions, methods, and systems for constructing defined variation in a contiguous functional genetic unit in association with a unique sequence identifier (“a barcode”) in a combinatorial manner.

Methods and compositions for preparing a nucleic acid sequencing library are described including (a)transposing an insertion sequence into first fragments of the target nucleic acid, wherein the insertion sequence comprises a hybridization sequence, and wherein the transposing produces nicks in the first fragments; (b) combining in a single mixture (i) the first fragments of the target nucleic acid from (a), (ii) a splint oligonucleotide, and (iii) a population of beads, wherein each bead comprises capture oligonucleotides immobilized thereon, and (c) ligating capture oligonucleotides of individual beads to inserted hybridization sequences of individual first fragments.

The present disclosure provides particles (e.g., beads) and methods, kits, and systems involving the same for sample processing or analysis. Such particles may include one or more analytes, one or more reagents, and two or more gel components and/or walled components. The particles described herein may be formed, for example, by polymerization of a polymerizable material in proximity to a gel or walled component.

The invention is directed to methods for synthesizing oligonucleotides direction on biomolecules or cells living or fixed. In some embodiments, template-free enzymatic synthesis is implemented under biological conditions with successive cycles of (i) enzymatic addition of a 3-O-blocked nucleoside triphosphate and (ii) enzymatic deblocking of the incorporated nucleotide to regenerate a free 3 hydroxyl. The invention has applications in single-cell cDNA library construction and analysis.

A method of characterizing candidate agents including steps of (a) providing a library of candidate agents attached to nucleic acid tags; (b) contacting the library with a solid support to attach the candidate agents to the solid support, whereby an array of candidate agents is formed; (c) contacting the array with a screening agent, wherein one or more candidate agents in the array react with the screening agent; (d) detecting the array to determine that at least one candidate agent in the array reacts with the screening agent; (e) sequencing the nucleic acid tag to determine the tag sequences attached to candidate agents in the array; and (f) identifying the at least one candidate agent in the array that reacts with the screening agent based on the tag sequence that is attached to the at least one candidate agent.

A method for sequencing a polynucleotide sample having a barcode sequence, includes: introducing a series of nucleotides to the polynucleotide sample according to a predetermined flow ordering; obtaining a series of signals resulting from the introducing of nucleotides to the polynucleotide sample; and resolving the series of signals over the barcode sequence to render a flowspace string, wherein the flowspace string is a codeword of an error-tolerant code capable of distinguishing the barcode sequence from other barcode sequences in the presence of one or more errors.

A method of synthesizing an oligonucleotide using a nanofluidic device including a plurality of nanopore channels, a plurality of electrodes, and an electrolyte solution, includes coupling a primer to an inner wall of a nanopore channel of the plurality of nanopore channels, the primer having a protecting group. The method also includes applying a voltage to an electrode of the plurality of electrodes that corresponds to the nanopore channel to produce an acid from the electrolyte solution at the electrode. The electrode includes an anode and a cathode disposed at opposite sides of the nanopore channel. The method further includes the acid removing the protecting group from the primer. Moreover, the method includes coupling a nucleotide to the primer with the protecting group removed to form an intermediate product. In addition, the method includes repeating the steps on the intermediate product until the oligonucleotide is synthesized.

A method of retrieving a subset of polynucleotide molecules from a mixture of polynucleotide molecules includes receiving a mixture of nucleotide sequences comprising one or more polynucleotide molecules, synthesizing one or more identifier (ID) regions onto the one or more polynucleotide molecules, and sequencing members of the population of polynucleotide molecules to associate the sequence of one or more of the molecules (the Polynucleotide Sequence) with the sequence of the attached ID region (the ID Sequence). The method also includes generating a bead-bound library of one or more beads comprising subsets of identical polynucleotide molecules. Each bead is identified by the ID Sequence of the associated Polynucleotide Sequence. The method further includes sequencing the one or more ID regions of each bead to generate ID Sequence information for each bead, combining the Polynucleotide Sequence information, the one or more ID Sequences, and coordinates of each bead to identify the Polynucleotide Sequence on the bead, and retrieving the bead with its associated Polynucleotide Sequence from the flow cell based on the absolute coordinate position of the bead.

Provided herein are methods directed to multiplexed detection of the modulation of transcriptional activity using perturbation elements.

A kit for use with a nucleic acid sequencing instrument can include a plurality of combinatorial barcodes sequences meeting the following criteria: each of the combinatorial barcode sequences comprise a plurality of iterations of a sequence motif, where the sequence motif comprises a first nucleotide base from a first group of nucleotide bases followed by a second nucleotide base from a second group of nucleotide bases, the first group and the second group differing from each other; and the plurality of combinatorial barcode sequences is at least 1,000,000 different barcode sequences.