Provided herein are methods of developing biomolecule variants (such as proteins, RNA, or DNA) with improved characteristics, for example by developing libraries of variants with alterations to one or more specific monomer locations and screening said libraries for characteristics of interest. These alterations can include deletion, substitution, and insertion, and variants may comprise one alteration or a combination of alterations. Said methods may include further iterative cycles of library construction and evaluation to develop, for example, a biomolecule variant with improved characteristics compared to a reference biomolecule. The methods can also provide information that may be used in the rational design of variants.

Provided are methods and systems useful for screening large libraries of effector molecules. Such methods and systems are particularly useful in microfluidic systems and devices. The methods and systems provided herein utilize encoded effectors to screen large libraries of effectors.

Provided are methods and systems useful for screening large libraries of effector molecules. Such methods and systems are particularly useful in microfluidic systems and devices. The methods and systems provided herein utilize encoded effectors to screen large libraries of effectors.

The present invention provides a method for constructing a next-generation sequencing library for detecting low-frequency mutations, and a kit thereof. The constructing method comprises steps of obtaining blunt-end DNA fragments, obtaining DNA fragments with A-tail at the 3′ end, obtaining adapter-added DNA fragments using a specific nucleotide sequence and obtaining amplification products using a specific nucleotide sequence.

The present invention provides a method for constructing a next-generation sequencing library for detecting low-frequency mutations, and a kit thereof. The constructing method comprises steps of obtaining blunt-end DNA fragments, obtaining DNA fragments with A-tail at the 3′ end, obtaining adapter-added DNA fragments using a specific nucleotide sequence and obtaining amplification products using a specific nucleotide sequence.

Methods and compositions of single tube preparation of sequencing libraries from a target DNA are provided. The methods include contacting the DNA with a composition comprising Cas9 endonuclease, a first and a second guide RNAs, a ligase, and sequencing adapters, subjecting the composition to thermal cycling to cleave the DNA at the sites flanking the regions of interest by the RNA guided endonuclease, and subjecting the composition to a temperature to allow ligation of the cleaved DNA fragments including the regions of interest with the sequencing adapters to generate the sequencing libraries.

Methods and compositions of single tube preparation of sequencing libraries from a target DNA are provided. The methods include contacting the DNA with a composition comprising Cas9 endonuclease, a first and a second guide RNAs, a ligase, and sequencing adapters, subjecting the composition to thermal cycling to cleave the DNA at the sites flanking the regions of interest by the RNA guided endonuclease, and subjecting the composition to a temperature to allow ligation of the cleaved DNA fragments including the regions of interest with the sequencing adapters to generate the sequencing libraries.

The present invention addresses the problem of providing a novel method which is for preparing a DNA fragment for microbial cell transformation, and by which the combinatorial library of a long-chain DNA can be efficiently constructed and confirmation of the genotype of the obtained clone is facilitated. The present invention is a method for preparing a DNA fragment, which is for microbial cell transformation and has at least one insert DNA unit that includes a DNA containing an effective replication origin in a host microorganism and an insert DNA in which unit DNAs are linked, the method being characterized by including: (A) a step for preparing, through an OGAB method, a plurality of types of plasmids having an insert DNA unit in which a plurality of types of unit DNAs capable of being linked in a specific linking order are linked; (B) a step for decomposing a plasmid into unit DNAs by treating the plurality of types of plasmids prepared in the step (A) with a restriction enzyme suitable for each plasmid and preparing a mixed liquid of a plurality of types of unit DNAs; and (C) a step for preparing a long-chain DNA fragment by re-assembling the unit DNAs through the OGAB method by using the mixed liquid of a plurality of types of unit DNAs obtained in the step (B).

The present invention provides a nucleic acid which encodes an adeno-associated virus (AAV) capsid protein mutant that contains a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID Nos. 15 to 62 or a peptide comprising an amino acid sequence produced by substituting, deleting, inserting and/or adding one or several amino acid residues in an amino acid sequence selected from the group consisting of SEQ ID Nos. 15 to 62; DNA comprising the nucleic acid; a cell harboring the DNA; and a method for producing the cell.

The present invention discloses a method for detecting the mutation and methylation of tumor-specific genes in ctDNA, and this method can simultaneously detect the mutation (including point mutation, insertion-deletion mutation, HBV integration and other mutation forms) and/or methylation of tumor-specific genes in ctDNA in one sample. Not only the sample size requirement is low, but the MC library prepared by this method can support 10-20 subsequent detections. The results of each test can represent the mutation status of all the original ctDNA specimens and the methylation modification status of the region covered by the restriction sites, without reducing the sensitivity and specificity. The present invention has important clinical significance for early tumor screening, disease tracking, efficacy evaluation, prognosis prediction and the like, and has great application value.