This invention pertains to improved methods for the synthesis of long, double stranded nucleic acid sequences containing difficult to clone or variable regions.

Disclosed herein are compositions, methods and kits useful for epigenetic analysis based on the use of transposons that are targeted to specific regions of chromatin based on DNA-DNA interactions, protein-protein interactions, RNA-RNA interactions, and nucleic acid-protein interactions.

Provided herein are methods and compositions relating to variant nucleic acid libraries encoding for antibodies including single domain antibodies. Libraries generated using methods described herein have improved characteristics including improved binding affinity. Libraries described herein include variegated libraries comprising nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. Further described herein are protein libraries generated when the nucleic acid libraries are translated. Further described herein are cell libraries expressing variegated nucleic acid libraries described herein.

Disclosed herein are methods for producing DNA libraries by incorporating dUTPs into DNA fragments and treating with uracil-DNA glycosylase and kits for preparing the DNA libraries. The DNA libraries are advantageous for next generation sequencing.

Systems and methods for identifying and using hybrid/capture probes are provided. cDNA sequences from poly-adenylated mRNA are obtained. Each cDNA sequence in a first subset maps to a gene in a plurality of genes. Each cDNA sequence in a second subset maps to a reference genome portion not represented by the plurality of genes. Each gene has a corresponding plurality of transcripts. The cDNA sequences are exposed to at least 210.sup.3 nucleic acid baits between K.sub.1 and K.sub.2 residues long, forming nucleic acid baitsequence read complexes. Each nucleic acid bait that hybridizes to a cDNA sequence mapping to a gene selectively hybridizes to a first subset or another subset of transcripts corresponding to the gene. Each transcript of each gene is hybridizable to a nucleic acid bait in the at least 210.sup.3 nucleic acid baits. The nucleic acid baitsequence read complexes are captured and analyzed.

Ordered assembly of large numbers of fragments into a single large DNA have been improved in both frequency and fidelity of the assembled product. This has been achieved by novel compositions and methods that are utilized in a computer system that integrates comprehensive ligation data from multiple sources to provide optimized synthetic overhangs or overhangs from restriction endonuclease cleavage on DNA fragments for assembly by ligation. Intragenic cut sites are avoided by the use of a novel restriction endonuclease which recognizes 7 nucleotides (bases) and cuts DNA to create 4-base overhangs with the help of a synthetic activator oligonucleotide. Variations in ligation preferences by different ligases provide extra precision in assembly reactions. The use of the improved methods are exemplified by the successful assembly from 52 fragments of a viral genome and also a 52 fragment ordered assembly of a bacteria operon.

The invention is a novel method of making and using a template for nucleic acid sequencing. The templates include circular and linear templates with symmetric and asymmetric adaptors. The methods include utilizing the templates in an asymmetric fashion.

Provided are methods for preparing a library of target nucleic acid sequences, as well as compositions and uses therefor. Methods comprise contacting a nucleic acid sample with a plurality of adaptors capable of amplification of one or more target nucleic acid sequences under conditions wherein the target nucleic acid(s) undergo a first amplification; digesting the resulting first amplification products; repairing the digested target amplicons; and amplifying the repaired products in a second amplification, thereby producing a library of target nucleic acid sequence. Each of the plurality of adaptor compositions comprise a handle and a targeted nucleic acid sequence and optionally one or more tag sequences. Provided methods may be carried out in a single, addition only workflow reaction, allowing for rapid production of highly multiplexed targeted libraries, optionally including unique tag sequences. Resulting library compositions are useful for a variety of applications, including sequencing applications.

Provided is a method for efficiently preparing a library. The method for preparing a library includes the steps of (a) synthesizing single-stranded cDNA from 10 pg or more of template RNA; (b) synthesizing double-stranded cDNA from the single-stranded cDNA; and (c) preparing a library using the double-stranded cDNA that is unpurified.

The disclosure provides methods and reagents for preparing DNA libraries from biological materials for targeted sequencing. The approach can enhance the efficiency and sensitivity of targeted sequencing applications, such as liquid biopsy analyses to assess genetically driven conditions. In an embodiment, the disclosed method comprises attaching the 5 end of an oligonucleotide adapter to the 3 end of double-stranded DNA fragment to produce an adapter/fragment chimeric molecule; producing at least one complementary strand of the adapter/fragment chimeric molecule by linear amplification; hybridizing the at least one complementary strand with an oligonucleotide probe, wherein the oligonucleotide probe comprises a hybridization domain with a sequence that hybridizes to a target sequence in the complement strand to produce a targeted complement strand/probe duplex; purifying the targeted complement strand/probe duplex; and extending the probe in the purified targeted complement strand/probe duplex and amplifying with PCR to produce a plurality of sequencing template molecules.