Circos could not find the configuration file. To run Circos, you need to specify this file using the -conf flag. The configuration file contains all the parameters that define the image, including input files, image size, formatting, etc. If you do not use the -conf flag, Circos will attempt to look for a file circos.conf in several reasonable places such as . etc/ ../etc To see how configuration files work, create the example image, whose configuration and data are found in example/. From the Circos distribution directory, cd example ../bin/circos -conf ./circos.conf or use the 'run' script (UNIX only). Configuration files are described here http://circos.ca/tutorials/lessons/configuration/configuration_files/ and the use of command-line flags, such as -conf, is described here http://circos.ca/tutorials/lessons/configuration/runtime_parameters/ Windows users unfamiliar with Perl should read http://circos.ca/tutorials/lessons/configuration/unix_vs_windows/