Email updates

Keep up to date with the latest news and content from BMC Genomics and BioMed Central.

Open Access Research article

Systematic overrepresentation of DNA termini and underrepresentation of subterminal regions among sequencing templates prepared from hydrodynamically sheared linear DNA molecules

Sherri L Schwartz* and Mark L Farman

Author Affiliations

Department of Plant Pathology, University of Kentucky, 1405 Veterans Dr, Lexington, KY 40546, USA

For all author emails, please log on.

BMC Genomics 2010, 11:87  doi:10.1186/1471-2164-11-87

Published: 2 February 2010

Abstract

Background

Analysis of fungal genome sequence assemblies reveals that telomeres are poorly represented even though telomeric reads tend to be superabundant. We surmised that the problem might lie in the DNA shearing conditions used to create clone libraries for genome sequencing.

Results

A shotgun strategy was used to sequence and assemble circular and linear cosmid DNAs sheared using conditions typical for a genome project. The DNA sheared in circular form assembled into a single sequence contig. However, the linearized cosmid produced an incomplete assembly because the two DNA termini, though greatly overrepresented in the clone library used for sequencing, were separated from neighboring sequences by gaps of ~1.4 and 1.8 kb. These gap sizes were reduced, but not eliminated, by shearing the linear cosmid into smaller fragments. Mapping of shearing breakpoints revealed a paucity of breaks in the subterminal regions of the linearized cosmid and also near chromosome ends of the fungus Neurospora crassa.

Conclusion

Together, our data indicate that the ends of linear DNA molecules are recalcitrant to hydrodynamic shearing. We propose that this causes DNA termini to be overrepresented in the resulting fragment population but ultimately prevents their incorporation into sequence assemblies.