Efficient and Sensitive Method for Detection of Transposon Insertions in Plants Based on Targeted Nanopore Amplicon Sequencing

Transposition of mobile elements is an important source of genetic diversity in plants. Rapid screening of plant populations for the presence of new insertions and identification of their localization sites in the genome are required when characterizing the collections of insertional mutants. We developed a new method onTEI-seq, which is based on specific amplification of mobile element insertion sites and subsequent nanopore sequencing of PCR products. The developed method was tested when sequencing endogenous EVD and ONSEN transposons of Arabidopsis thaliana. As a result, a high coverage of target transposons with nanopore reads (>1000×) was obtained in 2 h. The onTEI-seq technology was also used to search for ONSEN insertions in the population of A. thaliana insertional mutants containing the additional copies of LTR retrotransposons of this family. It was demonstrated that the sensitivity of the onTEI-seq method is sufficient to identify the insertions in plant population, where the portion of their carriers is only 2.5% (the ratio of insertion carriers to non-carriers was 1 : 39). Based on the results obtained, it can be concluded that the onTEI-seq is a sensitive and rapid method for the identification of new insertions and determination of their localization in the genome.