© The Authors, 2010. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Genome Biology 11 (2010): R119, doi:10.1186/gb-2010-11-12-r119. ; We characterize and extend a highly efficient method for constructing shotgun fragment libraries in which transposase catalyzes in vitro DNA fragmentation and adaptor incorporation simultaneously. We apply this method to sequencing a human genome and find that coverage biases are comparable to those of conventional protocols. We also extend its capabilities by developing protocols for sub-nanogram library construction, exome capture from 50 ng of input DNA, PCR-free and colony PCR library construction, and 96-plex sample indexing. ; This work was supported in part by grants from the National Institutes of Health/National Heart Lung and Blood Institute (R01 HL094976 to JS), the National Institutes of Health/National Human Genome Research Institute (R21 HG004749 to JS), the National Institutes of Health/National Institute of Allergy and Infectious Disease Northwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases at the University of Washington (3U54AI05714), the Ministry of Science and Technology of China, 863 program (2006AA02A301), and an NSF Graduate Research Fellowship (to JOK).