305 Thomas Peterson (ed.), Plant Transposable Elements: Methods and Protocols, Methods in Molecular Biology, vol. 1057, DOI 10.1007/978-1-62703-568-2_22, © Springer Science+Business Media New York 2013 Chapter 22 TEnest 2.0: Computational Annotation and Visualization of Nested Transposable Elements Brent A. Kronmiller and Roger P. Wise Abstract Grass genomes harbor a diverse and complex content of repeated sequences. Most of these repeats occur as abundant transposable elements (TEs), which present unique challenges to sequence, assemble, and annotate genomes. Multiple copies of Long Terminal Repeat (LTR) retrotransposons can hinder sequence assembly and also cause problems with gene annotation. TEs can also contain protein-encoding genes, the ancient remnants of which can mislead gene identification software if not correctly masked. Hence, accu- rate assembly is crucial for gene annotation. We present TEnest v2.0. TEnest computationally annotates and chronologically displays nested transposable elements. Utilizing organism-specific TE databases as a reference for reconstructing degraded TEs to their ancestral state, annotation of repeats is accomplished by iterative sequence alignment. Subsequently, an output consisting of a graphical display of the chrono- logical nesting structure and coordinate positions of all TE insertions is the result. Both linux command line and Web versions of the TEnest software are available at www.wiselab.org and www.plantgdb.org/ tool/, respectively. Key words Transposable elements, Retroelements, Bioinformatics, Sequence analysis, Annotation, Molecular evolution 1 Introduction Transposable elements (TEs) contribute to a significant fraction of plant genomes, with considerable diversity in content, even between closely related species. Grass genomes are highly repetitive (e.g., Oryza sativa (rice) is 35 % repetitive [1], Zea mays (maize) is 75 % [2, 3], and Triticum aestivum (wheat) is approximately 80 % [4]. Classes of TEs also vary between grass species; rice contains 13 % DNA transposons and 19.3 % retrotransposons [1], while maize is made up of only 1.3 % DNA transposons and 63.3 % retrotransposons [3]. The high degree of TEs makes grass genomes difficult to sequence, assemble, and annotate [5]. Multiple copies of TEs can cause regions of the genome to collapse during sequence assembly.