High-resolution definition of the Vibrio cholerae essential gene set with hidden Markov model-based analyses of transposon-insertion sequencing data.

Nucleic Acids Res
Authors
Keywords
Abstract

The coupling of high-density transposon mutagenesis to high-throughput DNA sequencing (transposon-insertion sequencing) enables simultaneous and genome-wide assessment of the contributions of individual loci to bacterial growth and survival. We have refined analysis of transposon-insertion sequencing data by normalizing for the effect of DNA replication on sequencing output and using a hidden Markov model (HMM)-based filter to exploit heretofore unappreciated information inherent in all transposon-insertion sequencing data sets. The HMM can smooth variations in read abundance and thereby reduce the effects of read noise, as well as permit fine scale mapping that is independent of genomic annotation and enable classification of loci into several functional categories (e.g. essential, domain essential or 'sick'). We generated a high-resolution map of genomic loci (encompassing both intra- and intergenic sequences) that are required or beneficial for in vitro growth of the cholera pathogen, Vibrio cholerae. This work uncovered new metabolic and physiologic requirements for V. cholerae survival, and by combining transposon-insertion sequencing and transcriptomic data sets, we also identified several novel noncoding RNA species that contribute to V. cholerae growth. Our findings suggest that HMM-based approaches will enhance extraction of biological meaning from transposon-insertion sequencing genomic data.

Year of Publication
2013
Journal
Nucleic Acids Res
Volume
41
Issue
19
Pages
9033-48
Date Published
2013 Oct
ISSN
1362-4962
URL
DOI
10.1093/nar/gkt654
PubMed ID
23901011
PubMed Central ID
PMC3799429
Links
Grant list
AI R37-42347 / AI / NIAID NIH HHS / United States
T32 GM007753 / GM / NIGMS NIH HHS / United States
F32 GM108355 / GM / NIGMS NIH HHS / United States
T32 AI007638 / AI / NIAID NIH HHS / United States
R37 AI042347 / AI / NIAID NIH HHS / United States
HHSN272200900018C / PHS HHS / United States
Howard Hughes Medical Institute / United States
F32 GM108355-01 / GM / NIGMS NIH HHS / United States