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Extrachromosomal linear DNA (eclDNA) is the reverse-transcribed cDNA intermediate derived from long terminal repeat (LTR) transposable elements (TEs) (Cho et al., Nat Plants 5:26-33, 2018). Given that the eclDNAs are the final intermediate of LTR-TE life cycle prior to integration to the host chromosomes, their presence is considered a strong indication of active LTR retrotransposons (Cho et al., Nat Plants 5:26-33, 2018; Lanciano et al., PLoS Genet 13:e1006630, 2017). Here, we describe a method of amplification of LTR extrachromosomal DNA followed by sequencing (ALE-seq) which determines the 5' LTR sequences of eclDNAs. Briefly, ALE-seq consists of two steps of amplification, in vitro transcription of adaptor-ligated eclDNAs and subsequent reverse transcription to cDNAs primed at the conserved primer binding site (PBS) (Cho et al., Nat Plants 5:26-33, 2018). ALE-seq allows the high-throughput identification of novel LTR-TEs which are active in plants that could be potentially useful for crop biotechnology.

ALE-seq is a method devised to identify pre-integration intermediates of LTR retrotransposons called extrachromosomal linear DNA, which can be used to predict retrotransposition activity. We describe here a bioinformatic methodology to process reads obtained from the ALE-seq protocol for the effective annotation of novel and active retroelements.

Spontaneous proliferation of transposable elements contributes to genetic diversity at varying levels such as somatic mosaicism, genetic divergence in population, and genome evolution. Such genetic diversity is essential for plants' adaptation to changing environment and serves as a valuable resource for crop improvement. Therefore, measuring the copy number variation of transposable elements with precision and efficiency is important to understand the extent of their proliferation. Droplet Digital PCR (ddPCR) is an accurate and sensitive technique that allows measurement of copy number variation of a transposon. Briefly, genomic DNA is extracted, digested, and partitioned into thousands of nanoliter-scale droplets. The TaqMan real-time PCR followed by the end-point fluorescence detection enables the quantitative measurement of copy number of template DNAs. Here in this chapter, we describe the step-by-step procedure of ddPCR using EVADE retrotransposon of Arabidopsis as an example.

Mapping the genomic location to which transposons jumped is of greatest interest to transposon biologists. Transposon display (TD) is the technique of choice that is easy and fast in determining the neo-insertion positions of a target transposon. Essentially, tagging of transposon is performed by digesting genomic DNA, ligating adaptors to digested DNA ends and PCR amplifying genomic regions flanking the transposon of interest. In this chapter, the experimental procedure of TD is described using Onsen retrotransposon of Arabidopsis as an example.