The invention relates to a high-throughput method for characterizing the genome-wide activity of editing nucleases in vitro.

The invention provides systems, methods, reagents, apparatuses, vectors, and host cells for the continuous evolution of nucleic acids. For example, a lagoon is provided in which a population of viral vectors comprising a gene of interest replicates in a stream of host cells, wherein the viral vectors lack a gene encoding a protein required for the generation of infectious viral particles, and wherein that gene is expressed in the host cells under the control of a conditional promoter, the activity of which depends on a function of the gene of interest to be evolved. Some aspects of this invention provide evolved products obtained from continuous evolution procedures described herein. Kits containing materials for continuous evolution are also provided.

Described herein are high coverage single tube Long Fragment Read (stLFR) technology which uses performs stLFR on target DNA fragments that have already been amplified before they are co-barcoded, which provides higher amount of DNA for sequencing and increases sequencing coverage. In some embodiments, the high coverage stLFR described in this application uses two rounds of stLFR. In some embodiments, the target DNA fragments are transposed with transposons having particular positional barcodes that can be used to order sequence reads.

Described herein are high coverage single tube Long Fragment Read (stLFR) technology which uses performs stLFR on target DNA fragments that have already been amplified before they are co-barcoded, which provides higher amount of DNA for sequencing and increases sequencing coverage. In some embodiments, the high coverage stLFR described in this application uses two rounds of stLFR. In some embodiments, the target DNA fragments are transposed with transposons having particular positional barcodes that can be used to order sequence reads.

The disclosure provides DNA library preparation methods that do not require a purification between adapter ligation and PCR amplification. Adaptors are added to DNA fragments to form oligonucleotide extension products and the oligonucleotide extension products are amplified without stopping or interruption for a cleanup step. Excess materials, such as enzymes, adaptors, or co-factors, from the adaptor addition step do not interfere with the amplification step and the amplification step proceeds without regards to the presence of reagents from the ligation step. In preferred embodiments, the ligation and amplification step make use of a common priming sequence e.g., in the form of one of the adaptor oligos.

The disclosure provides DNA library preparation methods that do not require a purification between adapter ligation and PCR amplification. Adaptors are added to DNA fragments to form oligonucleotide extension products and the oligonucleotide extension products are amplified without stopping or interruption for a cleanup step. Excess materials, such as enzymes, adaptors, or co-factors, from the adaptor addition step do not interfere with the amplification step and the amplification step proceeds without regards to the presence of reagents from the ligation step. In preferred embodiments, the ligation and amplification step make use of a common priming sequence e.g., in the form of one of the adaptor oligos.

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.

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.

The present invention relates to a method of preparing a DNA library derived from FFPE tissue using endonucleases, and the majority of general clinical samples are FFPE tissues and in case of old FFPE tissues, DNA library preparation often fails and NGS analysis becomes difficult and therefore there is a desperate need to overcome these problems. S1 nucleases are enzymes which specifically cleave only single-stranded DNA and in almost all FFPEs, there is a nick in the dsDNA everywhere, which can be the target of S1 nucleases. In the process of extracting gDNA from FFPE tissues, since when S1 nucleases are treated and eluted under appropriate conditions during elution, a DNA fragment can be obtained with DNA having a size suitable for DNA library preparation together with the DNA extraction, the Covaris fragmentation process can be omitted and cost and time can be reduced.

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).