Illuminating the plant transposon insertion landscape in real time using Cas9-targeted Nanopore sequencing and a novel pipeline

Abstract Transposable element insertions (TEIs) are an important source of genomic innovation by contributing to plant adaptation, speciation, and the production of new varieties. The often large, complex plant genomes make identifying TEIs from short reads difficult and expensive. Moreover, rare somatic insertions that reflect mobilome dynamics are difficult to track using short reads. To address these challenges, we combined Cas9-targeted Nanopore sequencing (CANS) with the novel pipeline NanoCasTE to trace both genetically inherited and somatic TEIs in plants. We performed CANS of the EVADÉ ( EVD ) retrotransposon in wild-type Arabidopsis thaliana and rapidly obtained up to 40x sequence coverage. Analysis of hemizygous T-DNA insertion sites and genetically inherited insertions of the EVD transposon in the ddm1 genome uncovered the crucial role of DNA methylation in shaping EVD insertion preference. We also investigated somatic transposition events of the ONSEN transposon family, finding that genes that are downregulated during heat stress are preferentially targeted by ONSEN s. Finally, we detected hypomethylation of novel somatic insertions for two ONSEN s. CANS and NanoCasTE are effective tools for detecting TEIs and exploring mobilome organization in plants in response to stress and in different genetic backgrounds, as well as screening T-DNA insertion mutants and transgenic plants.