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An Efficient Strategy Developed for Next-Generation Sequencing of Endosymbiont Genomes Performed Using Crude DNA Isolated from Host Tissues: A Case Study of Blattabacterium cuenoti Inhabiting the Fat Bodies of Cockroaches.

ABSTRACT: Whole-genome sequencing has emerged as one of the most effective means to elucidate the biological roles and molecular features of obligate intracellular symbionts (endosymbionts). However, the de novo assembly of an endosymbiont genome remains a challenge when host and/or mitochondrial DNA sequences are present in a dataset and hinder the assembly of the genome. By focusing on the traits of genome evolution in endosymbionts, we herein developed and investigated a genome-assembly strategy that consisted of two consecutive procedures: the selection of endosymbiont contigs from an output obtained from a de novo assembly performed using a TBLASTX search against a reference genome, named TBLASTX Contig Selection and Filtering (TCSF), and the iterative reassembling of the genome from reads mapped on the selected contigs, named Iterative Mapping and ReAssembling (IMRA), to merge the contigs. In order to validate this approach, we sequenced two strains of the cockroach endosymbiont Blattabacterium cuenoti and applied this strategy to the datasets. TCSF was determined to be highly accurate and sensitive in contig selection even when the genome of a distantly related free-living bacterium was used as a reference genome. Furthermore, the use of IMRA markedly improved sequence assemblies: the genomic sequence of an endosymbiont was almost completed from a dataset containing only 3% of the sequences of the endosymbiont's genome. The efficiency of our strategy may facilitate further studies on endosymbionts.

SUBMITTER: Kinjo Y

PROVIDER: S-EPMC4567559 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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An Efficient Strategy Developed for Next-Generation Sequencing of Endosymbiont Genomes Performed Using Crude DNA Isolated from Host Tissues: A Case Study of Blattabacterium cuenoti Inhabiting the Fat Bodies of Cockroaches.

Kinjo Yukihiro Y Saitoh Seikoh S Tokuda Gaku G

Microbes and environments 20150704 3

Whole-genome sequencing has emerged as one of the most effective means to elucidate the biological roles and molecular features of obligate intracellular symbionts (endosymbionts). However, the de novo assembly of an endosymbiont genome remains a challenge when host and/or mitochondrial DNA sequences are present in a dataset and hinder the assembly of the genome. By focusing on the traits of genome evolution in endosymbionts, we herein developed and investigated a genome-assembly strategy that c ...[more]

PMID: 26156552

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Project description:BackgroundCockroaches are terrestrial insects that strikingly eliminate waste nitrogen as ammonia instead of uric acid. Blattabacterium cuenoti (Mercier 1906) strains Bge and Pam are the obligate primary endosymbionts of the cockroaches Blattella germanica and Periplaneta americana, respectively. The genomes of both bacterial endosymbionts have recently been sequenced, making possible a genome-scale constraint-based reconstruction of their metabolic networks. The mathematical expression of a metabolic network and the subsequent quantitative studies of phenotypic features by Flux Balance Analysis (FBA) represent an efficient functional approach to these uncultivable bacteria.ResultsWe report the metabolic models of Blattabacterium strains Bge (iCG238) and Pam (iCG230), comprising 296 and 289 biochemical reactions, associated with 238 and 230 genes, and 364 and 358 metabolites, respectively. Both models reflect both the striking similarities and the singularities of these microorganisms. FBA was used to analyze the properties, potential and limits of the models, assuming some environmental constraints such as aerobic conditions and the net production of ammonia from these bacterial systems, as has been experimentally observed. In addition, in silico simulations with the iCG238 model have enabled a set of carbon and nitrogen sources to be defined, which would also support a viable phenotype in terms of biomass production in the strain Pam, which lacks the first three steps of the tricarboxylic acid cycle. FBA reveals a metabolic condition that renders these enzymatic steps dispensable, thus offering a possible evolutionary explanation for their elimination. We also confirm, by computational simulations, the fragility of the metabolic networks and their host dependence.ConclusionsThe minimized Blattabacterium metabolic networks are surprisingly similar in strains Bge and Pam, after 140 million years of evolution of these endosymbionts in separate cockroach lineages. FBA performed on the reconstructed networks from the two bacteria helps to refine the functional analysis of the genomes enabling us to postulate how slightly different host metabolic contexts drove their parallel evolution.

Dataset Information

An Efficient Strategy Developed for Next-Generation Sequencing of Endosymbiont Genomes Performed Using Crude DNA Isolated from Host Tissues: A Case Study of Blattabacterium cuenoti Inhabiting the Fat Bodies of Cockroaches.

Publications

An Efficient Strategy Developed for Next-Generation Sequencing of Endosymbiont Genomes Performed Using Crude DNA Isolated from Host Tissues: A Case Study of Blattabacterium cuenoti Inhabiting the Fat Bodies of Cockroaches.

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