Whole genome assembly of a natto production strain Bacillus subtilis natto from very short read data

<p>Abstract</p> <p>Background</p> <p><it>Bacillus subtilis </it>natto is closely related to the laboratory standard strain <it>B. subtilis </it>Marburg 168, and functions as a starter for the production of the traditional Japanese food "natto" made from soybeans. Although re-sequencing whole genomes of several laboratory domesticated <it>B. subtilis </it>168 derivatives has already been attempted using short read sequencing data, the assembly of the whole genome sequence of a closely related strain, <it>B. subtilis </it>natto, from very short read data is more challenging, particularly with our aim to assemble one fully connected scaffold from short reads around 35 bp in length.</p> <p>Results</p> <p>We applied a comparative genome assembly method, which combines <it>de novo </it>assembly and reference guided assembly, to one of the <it>B. subtilis </it>natto strains. We successfully assembled 28 scaffolds and managed to avoid substantial fragmentation. Completion of the assembly through long PCR experiments resulted in one connected scaffold for <it>B. subtilis </it>natto. Based on the assembled genome sequence, our orthologous gene analysis between natto BEST195 and Marburg 168 revealed that 82.4% of 4375 predicted genes in BEST195 are one-to-one orthologous to genes in 168, with two genes in-paralog, 3.2% are deleted in 168, 14.3% are inserted in BEST195, and 5.9% of genes present in 168 are deleted in BEST195. The natto genome contains the same alleles in the promoter region of <it>degQ </it>and the coding region of <it>swrAA </it>as the wild strain, RO-FF-1.</p> <p>These are specific for γ-PGA production ability, which is related to natto production. Further, the <it>B. subtilis </it>natto strain completely lacked a polyketide synthesis operon, disrupted the plipastatin production operon, and possesses previously unidentified transposases.</p> <p>Conclusions</p> <p>The determination of the whole genome sequence of <it>Bacillus subtilis </it>natto provided detailed analyses of a set of genes related to natto production, demonstrating the number and locations of insertion sequences that <it>B. subtilis </it>natto harbors but <it>B. subtilis </it>168 lacks. Multiple genome-level comparisons among five closely related <it>Bacillus </it>species were also carried out. The determined genome sequence of <it>B. subtilis </it>natto and gene annotations are available from the Natto genome browser <url>http://natto-genome.org/</url>.</p

One step assembly of multiple DNA fragments with a designed order and orientation in Bacillus subtilis plasmid

A universal method to reconstitute sets of genes was developed. Owing to the intrinsic nature of the plasmid establishment mechanism in Bacillus subtilis, the assembly of five antibiotic resistance genes with a defined order and orientation was achieved. These five fragments and the plasmid have three-base protruding sequences at both ends. The protruding sequences are designed so that each fragment is ligated once in a row according to the pairing. Ligation by T4 DNA ligase in the presence of 150 mM NaCl and 10% polyethylene glycol at 37°C yielded high molecular tandem repeat linear form DNA. This multimeric form of DNA was preferentially used for plasmid establishment in B.subtilis. The method, referred to as Ordered Gene Assembly in B.subtilis (OGAB), allows for the design of multiple fragments with very high efficiency and great fidelity