Computational protocol: Draft Genome Sequences of Six Rhodobacter capsulatus Strains, YW1, YW2, B6, Y262, R121, and DE442

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Protocol publication

[…] The purple nonsulfur photosynthetic alphaproteobacterium Rhodobacter capsulatus is a model organism for studying a novel mechanism of horizontal gene transfer mediated by gene transfer agents (GTAs), bacteriophage-like entities whose sole function is to mediate gene transfer (). The production of GTAs appears to be regulated in response to environmental signals. The study of the regulation of GTA production has been carried out mainly on the RcGTA of R. capsulatus strain B10 () and its derivative SB1003, the only genome-sequenced strain of R. capsulatus (), and other poorly characterized B10-derived RcGTA overproducer strains, Y262, R121, and DE442, generated by chemical mutagenesis (, ). However, the molecular mechanisms responsible for these RcGTA overproducer strains remain unknown. In addition, some R. capsulatus environmental isolates display altered phenotypes in RcGTA production and/or reception (). To extend our understanding of RcGTA biology, we sequenced the genomes of the R. capsulatus strains YW1, YW2, B6, Y262, R121, and DE442 ().Genomic DNA from each R. capsulatus strain was isolated from a late-log-phase culture grown in the minimal medium RCV (), using the 2012 JGI bacterial DNA isolation CTAB protocol (http://my.jgi.doe.gov/general/protocols/JGI-Bacterial-DNA-isolation-CTAB-Protocol-2012.pdf). DNA concentrations were quantified using the Qubit dsDNA HS Assay kit with the Qubit 2.0 fluorometer (Life Technologies). A total of 2 µg of each DNA sample was used to build indexed PCR-free libraries for each genome using a TruSeq kit (Illumina Hayward, CA). Sequencing was performed on an Illumina MiSeq platform using indexed paired-end 250-nucleotide v2 chemistry. Reads were aligned with the SB1003 reference genome using the Illumina MiSeq Reporter v 2.2 using BWA (), and assemblies were performed using the MiSeq Reporter v2.3.32 running Velvet (, ). The genome sequences of strains Y262, R121, and DE442 were assembled using a k-mer length of 150 with SB1003 as the reference genome (chromosome and plasmid concatenated; NCBI reference sequences NC_014034.1 and NC_014035.1) for contig ordering, whereas the natural isolates YW1, YW2, and B6 were assembled using a k-mer length of 200 without a reference genome. Average k-mer coverages of 158× (YW1), 178× (YW2), 156× (B6), 233× (Y262), 212× (R121), and 198× (DE442) were achieved. Genome annotation used the NCBI Prokaryotic Genome Annotation Pipeline (version 2.0).Our results show that the poorly provenanced R. capsulatus strains Y262, R121, and DE442 are closely related to strain SB1003, because of conserved within-contig syntenies. In addition, 95.2% (Y262), 93.8% (R121), and 94% (DE442) of sequence reads were aligned to the SB1003 genome. Contigs from strains DE442 and R121 lack genes from the SB1003 plasmid pRCB133, confirming results of a DNA microarray study (). The environmental isolates exhibit relatively diverse genome composition, with only 80.5% (YW1), 76.1% (YW2), and 74% (B6) of the reads mapping to the SB1003 genome. Although none of the three environmental isolates appear to produce RcGTA, they all contain RcGTA structural gene clusters. […]

Pipeline specifications

Software tools BWA, Velvet, PGAP
Application Gene expression microarray analysis
Organisms Rhodobacter capsulatus