In most protocols, DNA is either restriction-digested or sheared and then ligated to adapters at both ends. Additional file 1: Table S1 lists a sampling of studies and research tools that employ these techniques in organisms as diverse as bacteria, yeast, plants, nematodes, insects and vertebrates. Furthermore, the ability of some mobile elements to retain activity between species, phyla, and even kingdoms, has led to a proliferation of their use for transgene delivery, gene trapping and mutagenesis screens. Applications include studying the integration site preferences of mobile genetic elements, the identification of transgene integration sites and the study of how remobilized endogenous genetic elements contribute to evolution and/or tumour development (reviewed in ). Ligation-mediated PCR methods have diverse applications in identifying the integration sites of a known DNA sequence at an unknown locus. The reproducible distinction of clonal and subclonal integration sites from each other allows for analysis of populations of cells undergoing selection, such as those found in insertional mutagenesis screens. Suggestions for optimizing the protocol to other target DNA sequences are included. We also include an informatics pipeline that maps reads, builds integration contigs and quantitates integration abundance using both fragment lengths and unique molecular identifiers. The protocol is robust enough for use in a 96 well format using an automated liquid handler and we include programs for use of a Beckman Biomek liquid handling workstation. Merging ligation and library generation steps can reduce total PCR amplification cycles without sacrificing coverage or fidelity. A dilution series of DNAs bearing integrations of MuLV or piggyBac transposon shows linearity of the quantitation over a range of concentrations. Replicate libraries of murine leukemia virus-infected spleen samples yielded highly reproducible quantitation of clonal integrations as well as a deep coverage of subclonal integrations. By inverting the forked adapter strands from a standard orientation, the integration-genome junction can be sequenced without affecting the sequence diversity required for cluster generation on the flow cell. This design reduces the number of PCR cycles required and improves relative quantitation of integration abundance for saturating sequencing coverage. Here we describe a modification of our previous splinkerette based ligation-mediated PCR using a novel Illumina-compatible adapter design that prevents amplification of non-target DNA and incorporates unique molecular identifiers. For approaches that employ NGS sequencing, the relative abundance of integrations within a complex mixture is typically determined through the use of read counts or unique fragment lengths from a ligation of sheared DNA however, these estimates may be skewed by PCR amplification biases and saturation of sequencing coverage. Ligation-mediated PCR protocols have diverse uses including the identification of integration sites of insertional mutagens, integrating vectors and naturally occurring mobile genetic elements.
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