Disclosed is a method for obtaining a bifunctional complex comprising a molecule linked to a single stranded identifier oligonucleotide, wherein a nascent bifunctional complex comprising a chemical reaction site and a priming site for enzymatic addition of a tag is a) reacted at the chemical reaction site with one or more reactants, and b) reacted enzymatically at the priming site with one or more tag(s) identifying the reactant(s).

Disclosed is a method for obtaining a bifunctional complex comprising a molecule linked to a single stranded identifier oligonucleotide, wherein a nascent bifunctional complex comprising a chemical reaction site and a priming site for enzymatic addition of a tag is a) reacted at the chemical reaction site with one or more reactants, and b) reacted enzymatically at the priming site with one or more tag(s) identifying the reactant(s).

The present invention discloses a sequencing library comprising a nucleotide sequence. The sequence comprises a linker sequence and two target sequences. Two ends of the linker sequence are respectively linked to the target sequences and the two target sequences are direct repeat sequences. The present invention further discloses preparation and use of the sequencing library. The present invention overcomes the high error rate problem of current DNA sequencing technologies, especially in a way of very low coverage bias, and can be used to detect low frequency mutations in different kinds of samples.

The present invention discloses a sequencing library comprising a nucleotide sequence. The sequence comprises a linker sequence and two target sequences. Two ends of the linker sequence are respectively linked to the target sequences and the two target sequences are direct repeat sequences. The present invention further discloses preparation and use of the sequencing library. The present invention overcomes the high error rate problem of current DNA sequencing technologies, especially in a way of very low coverage bias, and can be used to detect low frequency mutations in different kinds of samples.

The present invention relates to the analysis of complex single cell sequencing libraries. Disclosed are methods for enrichment of library members based on the presence of cell-of origin barcodes to identify and concentrate DNA that is relevant to interesting cells or components that would be expensive or difficult to study otherwise. Also, disclosed are methods of capturing cDNA library molecules by use of CRISPR systems, hybridization or PCR. The present invention allows for identifying the properties of rare cells in single cell RNA-seq data and accurately profile them through clustering approaches. Further information on transcript abundances from subpopulations of single cells can be analyzed at a lower sequencing effort. The methods also allow for linking TCR alpha and beta chains at the single cell level.

The present invention relates to the analysis of complex single cell sequencing libraries. Disclosed are methods for enrichment of library members based on the presence of cell-of origin barcodes to identify and concentrate DNA that is relevant to interesting cells or components that would be expensive or difficult to study otherwise. Also, disclosed are methods of capturing cDNA library molecules by use of CRISPR systems, hybridization or PCR. The present invention allows for identifying the properties of rare cells in single cell RNA-seq data and accurately profile them through clustering approaches. Further information on transcript abundances from subpopulations of single cells can be analyzed at a lower sequencing effort. The methods also allow for linking TCR alpha and beta chains at the single cell level.

A visual continuous spatial directed evolution method is disclosed. The host grows and moves in a solid culture space, the host carrying a foreign target gene to be evolved and containing a gene element that assists the evolution of the target gene, the target gene being correlated with the growth and movement of the host. Depending on different spatial distribution patterns formed in the solid culture space during the growth and movement of the host, screening is performed to obtain an evolved product. This method is carried out directly in the solid culture space. Depending on images of different spatial distribution morphologies visible to the naked eye that are locally formed, selection of evolved products is performed without the need for liquid fed-batch culture equipment. In addition, the evolution effect is visually observed through the infection spots formed during evolution, so that no real-time monitoring equipment is required.

A visual continuous spatial directed evolution method is disclosed. The host grows and moves in a solid culture space, the host carrying a foreign target gene to be evolved and containing a gene element that assists the evolution of the target gene, the target gene being correlated with the growth and movement of the host. Depending on different spatial distribution patterns formed in the solid culture space during the growth and movement of the host, screening is performed to obtain an evolved product. This method is carried out directly in the solid culture space. Depending on images of different spatial distribution morphologies visible to the naked eye that are locally formed, selection of evolved products is performed without the need for liquid fed-batch culture equipment. In addition, the evolution effect is visually observed through the infection spots formed during evolution, so that no real-time monitoring equipment is required.

A method for creating a plasmid for use in producing a chimeric antibody, comprising (a) receiving a FAB region of the antibody; (b) receiving a fluorescent protein; (c) receiving a linker having length of at least 5 amino acids; (d) using the Gibson assembly process to join the FAB region, the fluorescent protein, and the linker into an expression plasmid.

A method for creating a plasmid for use in producing a chimeric antibody, comprising (a) receiving a FAB region of the antibody; (b) receiving a fluorescent protein; (c) receiving a linker having length of at least 5 amino acids; (d) using the Gibson assembly process to join the FAB region, the fluorescent protein, and the linker into an expression plasmid.