The invention provides novel reverse transcriptases (RTs) with desirable properties such as increased thermostability, increased thermoreactivity and/or increased resistance to inhibitors. In certain embodiments, the invention provides methods of producing, amplifying and/or sequencing nucleic acid molecules (particularly cDNA molecules) using kits, compositions and/or reactions mixtures containing such novel reverse transcriptase enzymes.

The present invention features, inter alia, compositions and methods for preparing, from a plurality of original, nucleic acid-containing samples, a unified library of linear, non-selectively amplified DNA fragments in which the proportional representation of the fragments from each of the plurality of original samples is normalized and the library is created in a highly parallelized, pool-based fashion. The invention is particularly useful for preparing libraries in which specific information is encoded that allows shorter sequencing reads derived from high-throughput sequencing of the library to be analyzed or assembled into longer scale sequences that are fully traceable to an original, nucleic acid-containing sample within a potentially very large collection of samples. The compositions of the invention encompass the various constructs described herein, which may be variously packaged with one or more additional reagents useful in the present methods and instructions for use.

Methods, devices and systems for analyzing precious samples of cells, including single cells are provided. The methods, devices, and systems in various embodiments of the invention are used to assess genomic heterogeneity, which has been recognized as a central feature of many cancers and plays a critical role in disease initiation, progression, and response to treatment. The methods devices and systems are also used to analyze embryonic biopsies for reimplantation genetic diagnosis (PGD). In one embodiment, the devices, systems and methods provided herein allow for the construction of genomic and RNA-seq libraries without a pre-amplification step.

The invention features methods of identifying and validating affinity reagents, such as antibodies. The methods of the invention generally involve screening an antibody library by, for example, phage display on bacteria (e.g., E. coli) to identify particular antibody clones capable of binding a desired target polypeptide. Clones identified in this way can then be validated using yeast 2-hybrid. In some instances, antibodies identified by their capacity to binding a partial antigen can be validated by their capacity to bind to the full-length antigen. Validated clones can be further screened by additional rounds of phage display and/or yeast 2-hybrid. Between each round, additional variants of particular antibody clones can be generated and screened to identify variants that demonstrate higher binding affinity to the target of interest.

Disclosed herein is a phage-displayed single-chain variable fragment (scFv) library, that comprised a plurality of phage-displayed scFvs characterized with (1) a specific CS combination; (2) a specific distribution of aromatic residues in each CDR; and (3) a specific sequence in each CDR. The present scFv library could be used to efficiently produce different antibodies with binding affinity to different antigens. Accordingly, the present disclosure provides a potential means to generate different antigen-specific antibodies promptly in accordance with the need in experimental researches and/or clinical applications.

The present invention provides nucleic acid constructs, expression vectors, transgenic cell and methods of making and using the same, wherein the nucleic acid construct includes a synthetic promoter designed from the endogenous promoter of BIRC5 and LAMC2. In illustrative working embodiments of the invention, an exogenous nucleic acid fragment encoding thymidine kinase is operably linked to the synthetic promoter which is then shown to regulate the expression of this polypeptide.

Disclosed herein are methods and compositions for inactivating mutant genes associated with LCA, using engineered nucleases comprising a DNA binding domain and a cleavage domain or cleavage half-domain in conditions promoting the cleavage of the mutant genes. Polynucleotides encoding nucleases, vectors comprising polynucleotides encoding nucleases, and cells comprising polynucleotides encoding nucleases and/or cells comprising nucleases are also provided.

Methods of coupling adaptors to a target nucleic acid include coupling a first adaptor to a first end of the target nucleic acid to form a coupled first adaptor. A portion of a second adaptor is hybridized to a portion of the coupled first adaptor to form a hybridized second adaptor having a single-stranded 3′-end. The hybridized second adaptor is coupled to a second end of the target nucleic acid to form an adaptor-flanked product having at least a part of the first adaptor coupled to the first end of the target nucleic acid and at least a part of the second adaptor coupled to the second end of the target nucleic acid. These methods can minimize the formation of adaptor-dimers that may be problematic in subsequent complementary nucleic acid strand synthesis, amplification, and sequencing.

The present disclosure provides methods and systems for identification of genomic regions for therapeutic targeting. A method for identifying one or more genomic regions for therapeutic targeting, which may facilitate re-programming of a cell from one phenotypic state to another, may comprise: providing single-cell RNA-seq data for a plurality of diseased cells and a plurality of normal cells of a cell type; mapping the single-cell RNA-seq data for the plurality of diseased cells and the plurality of normal cells into a latent space corresponding to a plurality of phenotypic states of the cell type; identifying, based at least in part on a topology of the latent space, the one or more genomic regions for therapeutic targeting; and electronically outputting the one or more genomic regions for therapeutic targeting.

The present disclosure relates to methods, compositions, and kits for treating target nucleic acids, including methods and compositions for fragmenting and tagging nucleic acid (e.g., DNA) using transposome complexes bound to a solid support.