The present disclosure provides methods and systems for determining a target-activity of at least one resolved oligonucleotide encoded molecule. In an embodiment, a method includes providing a separation medium, wherein the separation medium contains at least one target molecule; and various methods of separating a mixture of at least two oligonucleotide encoded molecules by electrophoresis based on different target-activities of the oligonucleotide encoded molecules for a target molecule. Benefits of the methods disclosed herein can include, without limitation, collecting and calculating qualitative and quantitative data for the target-activity of an encoded portion of the oligonucleotide encoded molecule for a target molecule.

This invention relates to compositions of matter, methods, modules and instruments for automated mammalian cell growth and mammalian cell transduction followed by nucleic acid-guided nuclease editing in live mammalian cells.

Provided are methods of adding adapters to nucleic acids. The methods include combining in a reaction mixture a template ribonucleic acid (RNA), a template switch oligonucleotide including a 3′ hybridization domain and a sequencing platform adapter construct, a polymerase, and dNTPs. The reaction mixture components are combined under conditions sufficient to produce a product nucleic acid that includes the template RNA and the template switch oligonucleotide each hybridized to adjacent regions of a single product nucleic acid that includes a region polymerized from the dNTPs by the polymerase. Aspects of the invention further include compositions and kits.

A compound library may include a plurality of conjugate molecules, said conjugates comprising a small organic molecule covalently coupled to a nucleic acid moiety. The nucleic acid moiety may include or consist of 7-deazapurines and/or 7-deaza-8-azapurines, and, optionally, modified pyrimidine nucleotides and/or unmodified pyrimidine nucleotides. Further, a library for screening compounds binding to a target molecule and methods of synthesizing said library is also disclosed.

Rapid methods, capable of being performed in a single reaction tube, are described herein for constructing libraries for high-throughput polynucleotide sequencing applications, such as next generation sequencing (NGS) applications. Oligonucleotide probes include chemically-active groups at their 5′ or 3′ ends, or both, to facilitate the cleavage of their 5′ or 3′ ends, or both, following their hybridization to the single-stranded ends of frayed template fragments. Cleavage of probe ends reveal single-stranded regions at the ends of the hybridized fragments. Adaptors, specific to these ends, are ligated to the hybridized probe/template fragments, and blunt end fragments are ligated to blunt ends of hybridized probe/template fragments, if present, to generate the adaptor-ligated fragments of the library.

The present invention relates to a three-degree-of-freedom library preparation cassette and a method using the same. Two-degree-of-freedom movement capability is provided for mutual positioning between a pipette and reagent wells, within a limited small volume, by combining rotation of a mixing reagent tray and relative horizontal movement between the pipette and the mixing reagent tray, thus greatly increasing the number of reagent wells that can be provided, thereby increasing the functions and applications of the whole system; and the device is simple in structure, low in cost, and suitable for disposable use.

Compositions of matter, methods, modules, and automated instruments may relate to synthesizing a library including an editing cassette including a different gRNA and donor DNA pair, amplifying the editing cassette in a partition separate from other editing cassettes in the library, adding nuclease to the partition, and adding lipofectamine to the editing cassette and nuclease to form a lipofectamine/nucleic acid/nuclease complex. A microcarrier coated in extracellular matrix or a cell adhesion molecule coating may be added to the lipofectamine/nucleic acid/nuclease complex. Cell growth material, the microcarrier, and mammalian cells may be transferred to a growth module in an automated closed cell editing instrument via a liquid handling system. The mammalian cells may be allowed to seed on the microcarrier. Conditions may be provided for the mammalian cells to take-up and be edited by a payload associated with the lipofectamine/nucleic acid/nuclease complex. The mammalian cells may be detached from the microcarrier.

Methods and compositions are provided for amplifying a pool of oligonucleotides, such as dual guide oligonucleotide constructs comprising sequences encoding a first guide RNA segment and a sequence encoding a second guide RNA segment. An amplification mixture is formed comprising the pool of oligonucleotides, an amplification enzyme, deoxyribonucleotide triphosphates, and primers. The amplification mixture is thermocycled a sufficient number of times and under conditions to produce a library of oligonucleotide constructs. The present methods and compositions provide dual guide libraries, including libraries that are essentially free of scrambled library members.

Aspects of the invention relate to methods, compositions for synthesizing oligonucleotides having a predefined sequence.

The disclosure provides methods to calibrate polynucleotide seeding efficiency in flow cells.