The template brain (FCWB) was constructed by screening for whole brains within the FlyCircuit dataset, and manually selecting a pool of brains that appeared of good quality when the stacks were inspected. Separate average female and average male template brains were constructed from 17 and 9 brains, respectively using the CMTK (http://www.nitrc.org/projects/cmtk) avg_adm function which takes a single brain as a seed. After five iterations the resultant average male and average female brains were placed in an affine symmetric position within their image stacks so that a simple horizontal (x -axis) flip of either template brain resulted in an almost perfect overlap of left and right hemispheres. Finally the two sex-specific template brains were then averaged (with equal weight) to make an intersex template brain using the same procedure. Since the purpose of this template was to provide an optimal registration target for the flycircuit.tw dataset, no attempt was made to correct for the obvious disparity between the XY and Z voxel dimensions common to all the images in the dataset. The scripts used for the construction of the template are available at https://github.com/jefferislab/MakeAverageBrain. Images from FlyCircuit were obtained from the NCHC (National Center for High-performance Computing) and NTHU (National Tsing Hua University), Hsinchu, Taiwan. If you use this template brain, to acknowledge the original raw data, please ensure that you additionally cite: Three-dimensional reconstruction of brain-wide wiring networks in Drosophila at single-cell resolution Ann-Shyn Chiangemail, Chih-Yung Lin11, Chao-Chun Chuang11, Hsiu-Ming Chang11, Chang-Huain Hsieh11, Chang-Wei Yeh, Chi-Tin Shih, Jian-Jheng Wu, Guo-Tzau Wang, Yung-Chang Chen, Cheng-Chi Wu, Guan-Yu Chen, Yu-Tai Ching, Ping-Chang Lee, Chih-Yang Lin, Hui-Hao Lin, Chia-Chou Wu, Hao-Wei Hsu, Yun-Ann Huang, Jing-Yi Chen, Hsin-Jung Chiang, Chun-Fang Lu, Ru-Fen Ni, Chao-Yuan Yeh, Jenn-Kang Hwang Current Biology 2011, 21:1-11. http://dx.doi.org/10.1016/j.cub.2010.11.056