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New sequencing technologies now allow de novo genome assembly of complex genomes and, in particular, regions absent in published assemblies. Methods for combining different sequencing technologies are particularly attractive, allowing a significant overall reduction of cost. This Special Issue features significant advances in de novo assembly methods, application of new sequencing and scaffolding technologies, and the analyses of genomic variation from de novo assemblies. The issue is Guest Edited by Dr. Adam Phillippy, head of the Genome Informatics Section and investigator in the Computational and Statistical Genomics Branch at the National Human Genome Research Institute. (Cover illustration by Elena Strelnikova, Center for Algorithmic Biotechnology, St. Petersburg State University.)