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Georgios A. Pavlopoulos, Maria Secrier, Reinhard Schneider, Jan Aerts (Katholieke Universiteit Leuven, Faculty of Engineering - ESAT/SCD, Kasteelpark Arenberg, 3001 Leuven-Heverlee, Belgium)Arena3D presents a new multi-layer concept in 3D. Users can divide datasets into multiple 2D layers. 3D allows more ‘space’ for the vertices between layers, hence reducing the overlap and intersection that occurs in 2D. Arena3D handles few thousands of nodes and connections. It tries to integrate information obtained from various sources. Experiments with time lapse data capturing crucial time points of an underlining biological processes is also supported. Besides the basic 3D operations (zoom, rotations, and translations), the tool allows to change the order, location, size, and orientation of individual layers. We implemented several layout algorithms to distribute the nodes on each layer. Individual layers: 2D grid, spherically, or in a circular way. Force-directed methods: Fruchterman-Reingold algorithm and a distance geometry based algorithm (Crippen and Havel, 1988). Arena3D supports k-means, affinity propagation and MCL clustering methods. Clustering can be applied on individual layers or among layers. Single linkage hierarchical clustering, neighbor joining and UPGMA are also supported.