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

Transposable elements (TEs), which occupy significant portions of most plant genomes, are a major source of genomic novelty, contributing to plant adaptation, speciation and new cultivar production. The often large, complex genomes of plants make identifying TE insertions from short reads challenging, while whole-genome sequencing remains expensive. To expand the toolbox for TE identification in plants, we used the recently developed Cas9-targeted Nanopore sequencing (CANS) approach.

Additionally, as no current bioinformatics tools automatically detect TE insertions after CANS, we developed NanoCasTE, a novel pipeline for target TE insertion discovery. We performed CANS of three copies of EVD retrotransposons in wild-type Arabidopsis thaliana and obtained up to 40× coverage of the targets after only a few hours of sequencing on a MinION sequencer. To estimate the ability to detect new TE insertions, we exploited the A. thaliana ddm1 mutant, which has elevated TE activity.

Using CANS, we detected 84% of these insertions in ddm1 after generating only 4420 Nanopore reads (0.2× genome coverage), and also unambiguously identified their locations, demonstrating the method’s sensitivity. CANS of pooled (∼50 plants) ddm1 plants captured >800 EVD insertions, especially in centromeric regions. CANS also identified insertions of a Ty3/Gypsy retrotransposon in the genomes of two Aegilops tauschii plants, a species with a large genome.