Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung
Citation: Gawronski, J.D., Wong, S.M.S., Giannoukos, G., Ward, D.V., and B. J. Akerley. Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung. Proc Natl Acad Sci U S A. 2009 September 22: 106: (38) 16422-16427. Epub 2009 Sep 4. Link to article on publisher's site: http://dx.doi.org/10.1073/pnas.0906627106.
See Related Files below to download the informational figures (.pdf), the raw data (.txt), and the processed database data (.xls).
Publisher PDF posted as allowed by the publisher's author rights policy at http://www.pnas.org/site/misc/authorfaq.shtml.
Rapid genome-wide identification of genes required for infection would expedite studies of bacterial pathogens. We developed genome-scale "negative selection" technology that combines high-density transposon mutagenesis and massively parallel sequencing of transposon/chromosome junctions in a mutant library to identify mutants lost from the library after exposure to a selective condition of interest. This approach was applied to comprehensively identify Haemophilus influenzae genes required to delay bacterial clearance in a murine pulmonary model. Mutations in 136 genes resulted in defects in vivo, and quantitative estimates of fitness generated by this technique were in agreement with independent validation experiments using individual mutant strains. Genes required in the lung included those with characterized functions in other models of H. influenzae pathogenesis and genes not previously implicated in infection. Genes implicated in vivo have reported or potential roles in survival during nutrient limitation, oxidative stress, and exposure to antimicrobial membrane perturbations, suggesting that these conditions are encountered by H. influenzae during pulmonary infection. The results demonstrate an efficient means to identify genes required for bacterial survival in experimental models of pathogenesis, and this approach should function similarly well in selections conducted in vitro and in vivo with any organism amenable to insertional mutagenesis.
Jeffrey D. Gawronski, Sandy M. S. Wong, Georgia Giannoukos, Doyle V. Ward, and Brian J. Akerley. "Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung" Proceedings of the National Academy of Sciences of the United States of America 106.38 (2009): 16422-16427.
Available at: http://works.bepress.com/brian_akerley/28
Supplemental information (PDF)
Table S1.xls (354 kB)
Table S1. Fitness data for all annotated genes (XLS)
Table S2.pdf (24 kB)
Table S2. Genes required for growth or survival in the lung model detected by HITS (PDF)
Table S3.pdf (11 kB)
Table S3. Distribution of genes required in vivo organized by functional categories (PDF)
Table S4.pdf (11 kB)
Table S4. Oligonucleotides used in this study (PDF)
SI computer script.doc (26 kB)
Custom PERL script for analysis and mapping of Illumina sequencing data (DOC)
SD1.txt (43536 kB)
Input Library Sample 1 (TXT)
SD2.txt (45265 kB)
Input Library Sample 2 (TXT)
SD3.txt (27485 kB)
Lung Output Library (TXT)