Genome sequencer flx platform

Fecal genomic DNA extraction, amplification of the V3 region, and pyrosequencing of PCR amplicons were performed as described previously32 (link). Genomic DNA was extracted from fecal samples collected from three groups of mice at age 4, 12 and 24 weeks using bead beating and an InviMag^® DNA kit (Invitek, Berlin, Germany). The V3 region of the 16S rRNA gene was amplified from each sample with the extracted fecal DNA as the template in PCR with 20 cycles. The bacterial universal primer pair for the V3 region consisted of the forward primer 5′-NNNNNNNNCCTACGGGAGGCAGCAG-3′ and the reverse primer 5′-NNNNNNNNATTACCGCGGCTGCT-3′. NNNNNNNN was the sample-unique DNA bar code of eight nucleotide sequences. The PCR amplicons from each sample were quantified with PicoGreen fluorescent dye (Thermo Fisher Scientific, Sunnyvale, USA) by using SpectraMax M5 microplate reader (Molecular Devices, San Francisco, USA), and mixed in equal ratios. The 454 sequencing adapters were ligated to the mixed PCR amplicons by blunt-end ligation. Pyrosequencing was performed on a Genome Sequencer FLX platform (Roche Diagnostics, Mannheim, Germany).

The genome sequence of the PGPR strain B. cereus 905 was already submitted to the National Center for Biotechnology Information (NCBI) database with the accession number of LSTW00000000.1. The B. cereus 905 genome was sequenced using the Genome Sequencer FLX platform (Roche, Mannheim, Germany)¹⁰ . The library for strain 905 was constructed using a 3-kb-long paired-end tag with a GS FLX library preparation kit¹⁰ . The output reads were assembled using the GS De Novo Assembler software program. Gene prediction of this PGPR strain was annotated by the Prokka^{54 (link)} software, which is a rapid prokaryotic genome annotation software. Annotation of the protein-coding sequence was conducted using the Basic Local Alignment Search Tool (BLAST) against the COG, Kyoto Encyclopedia of Genes and Genomes, NCBI nonredundant protein and Interpro databases. The genome sequence of B. cereus ATCC 10987 was set as the reference genome, and the contigs of B. cereus 905 was ordered by Mauve^{55 (link)}. The final annotated chromosome was plotted using CIRCOS^{56 (link)} to show the gene locations, GC skew, and GC content.