Nanopore Cas9-targeted sequencing enables accurate and simultaneous identification of transgene integration sites, their structure and epigenetic status in recombinant Chinese hamster ovary cells

In this study, we present a fast and robust nanopore Cas9-targeted sequencing (nCats) pipeline to identify integration sites, the composition of the integrated sequence and its DNA methylation status in CHO cells that can be obtained from a single sequencing run. A Cas9-enrichment step during library preparation enables targeted and directional nanopore sequencing with up to 724x median on-target coverage and up to 153 Kb long reads. The data generated by nCats provides sensitive, detailed and correct information on the transgene integration sites and the expression vector structure, which could only partly be produced by traditional Targeted Locus Amplification-Seq data. Moreover, with nCats the DNA methylation status can be analyzed from the same raw data without prior DNA amplification.