Abstract
BackgroundSequencing of bacterial genomes became an essential approach to study pathogen virulence and the phylogenetic relationship among close related strains. Bacterium Enterococcus faecium emerged as an important nosocomial pathogen that were often associated with resistance to common antibiotics in hospitals. With highly divergent gene contents, it presented a challenge to the next generation sequencing (NGS) technologies featuring high-throughput and shorter read-length. This study was designed to investigate the properties and systematic biases of NGS technologies and evaluate critical parameters influencing the outcomes of hybrid assemblies using combinations of NGS data.ResultsA hospital strain of E. faecium was sequenced using three different NGS platforms: 454 GS-FLX, Illumina GAIIx, and ABI SOLiD4.0, to approximately 28-, 500-, and 400-fold coverage depth. We built a pipeline that merged contigs from each NGS data into hybrid assemblies. The results revealed that each single NGS assembly had a ceiling in continuity that could not be overcome by simply increasing data coverage depth. Each NGS technology displayed some intrinsic properties, i.e. base calling error, systematic bias, etc. The gaps and low coverage regions of each NGS assembly were associated with lower GC contents. In order to optimize the hybrid assembly approach, we tested with varying amount and different combination of NGS data, and obtained optimal conditions for assembly continuity. We also, for the first time, showed that SOLiD data could help make much improved assemblies of E. faecium genome using the hybrid approach when combined with other type of NGS data.ConclusionsThe current study addressed the difficult issue of how to most effectively construct a complete microbial genome using today's state of the art sequencing technologies. We characterized the sequence data and genome assembly from each NGS technologies, tested conditions for hybrid assembly with combinations of NGS data, and obtained optimized parameters for achieving most cost-efficiency assembly. Our study helped form some guidelines to direct genomic work on other microorganisms, thus have important practical implications.
Highlights
Sequencing of bacterial genomes became an essential approach to study pathogen virulence and the phylogenetic relationship among close related strains
Our results revealed that hybrid assembly method greatly improved efficiency in comparison with single next generation sequencing (NGS) technology, which could not be achieved by increasing the coverage depth of a single NGS platform alone
Generating and processing sequence data from NGS platforms In order to investigate the properties of sequencing data from each NGS platform and combine them to achieve the best genome assembly, we sequenced the nosocomial pathogen Enterococcus faecium with the three popular NGS platforms: Roche 454, Illumina GAIIx, and ABI SOLiD 4
Summary
Sequencing of bacterial genomes became an essential approach to study pathogen virulence and the phylogenetic relationship among close related strains. Bacterium Enterococcus faecium emerged as an important nosocomial pathogen that were often associated with resistance to common antibiotics in hospitals. Bacterial isolates of the same species often display surprisingly highly divergent gene contents from vastly different ecological environments Such genome divergence, which is the result of harsh selection and frequent horizontal gene transfer, presents a unique challenge to the modern sequencing technologies featuring high-throughput and short read length, and limits our ability to re-sequence and construct a genome draft of bacterial variant by taking advantage of a “genome reference”. E. faecium emerged as an important nosocomial pathogen from hospital environments and were often associated with the resistance to many common antibiotics. A genome sequence of a vancomycin-resistant E. faecium strain (Aus0004) was completed more recently [4], revealing large segments of repetitive DNA and insertion sequence elements in its genome
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