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International Journal of Genomics
Volume 2014, Article ID 434575, 8 pages
Research Article

Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA

1Los Alamos National Laboratory, P.O. Box 1663, MS M888, Los Alamos, NM 87545, USA
2Parkview Medical Center School for Medical Laboratory Science, 400 West 16th Street, Pueblo, CO 81003, USA

Received 29 May 2014; Accepted 2 October 2014; Published 13 November 2014

Academic Editor: Ian Dunham

Copyright © 2014 Momchilo Vuyisich et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Sequencing bacterial genomes has traditionally required large amounts of genomic DNA (~1 g). There have been few studies to determine the effects of the input DNA amount or library preparation method on the quality of sequencing data. Several new commercially available library preparation methods enable shotgun sequencing from as little as 1 ng of input DNA. In this study, we evaluated the NEBNext Ultra library preparation reagents for sequencing bacterial genomes. We have evaluated the utility of NEBNext Ultra for resequencing and de novo assembly of four bacterial genomes and compared its performance with the TruSeq library preparation kit. The NEBNext Ultra reagents enable high quality resequencing and de novo assembly of a variety of bacterial genomes when using 100 ng of input genomic DNA. For the two most challenging genomes (Burkholderia spp.), which have the highest GC content and are the longest, we also show that the quality of both resequencing and de novo assembly is not decreased when only 10 ng of input genomic DNA is used.