Gs flx titanium system

The genome sequence of DOA9 was determined by 454 pyrosequencing analysis using a GS FLX Titanium system (Roche Diagnostics Co., Indianapolis, IN, USA). Genomic DNA (5 μg) was sheared using nebulization to obtain fragments ranging from 300 to 800 bp. Template DNA was prepared according to the supplier’s protocol. The pyrosequencing data were assembled using the MIRA assembler ver. 3 [11 ], and were curated by comparison with the Newbler ver. 2.8 (Roche Diagnostics Co., Indianapolis, IN, USA) assembly or with some close reference strains. Gap closing and resequencing of low-quality regions of the assembled data were performed by PCR and Sanger sequencing. Regions encoding structural RNAs, rRNAs, tRNAs, tmRNAs, noncoding RNAs, and proteins were predicted using the Genaris Annotation System (Genaris, Inc., Kanagawa, Japan).

Bacterial genome sequencing was performed at the DNA Sequencing Service of the University of Seville (Servicio General de Biología, CITIUS, Universidad de Sevilla, Spain). Sample preparation included gDNA isolation by phenolic extraction and ethanol precipitation. Genome sequencing employed Roche 454 FLX + technology on a GS FLX titanium system. Emulsion PCR and 454 pyrosequencing were performed and 1,031,375 reads with an average read length of 810 base pairs, totaling 1000 Mb. Whole genome sequences were analyzed using the Burrow-Wheeler Alignment tool (BWA), specifically the BWA-MEM algorithm^{29 (link),30} . As a reference, the genome sequence of the laboratory stock of S. enterica SL1344 was also analyzed. Genomes were submitted to NCBI GenBank and are available as SUB3834111 “Salmonella typhimurium isolates from Balb/c mice gall bladders (Urdaneta & Casadesus)”. PCR amplification of regions harboring mutations was performed using the primers listed in Table S1. Chromosomal DNA samples obtained by PCR were sequenced by Stab Vida (Caparica, Portugal).

The V1–V3 hypervariable region (about 500 bp) of the 16S rRNA gene was amplified from each DNA sample using fusion primers ArcF-A and ArcR-B (for archaea) or BactF-A and BactR-B (for bacteria) (Table 1). Both primer sets were modified from domain-specific primers that have been commonly used to amplify the V1–V3 region (Nelson, 2011 ) by introducing degenerate bases for maximal inclusiveness (Baker et al., 2003 (link)). One hundred nanogram of each genomic DNA was used in each PCR with the following thermal program: initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturing at 94°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 1 min. The PCR cycles were ended with a final extension at 72°C for 10 min. Quality of the PCR products were examined using agarose gel (1.0%) electrophoresis. The bands of the expected size (approximately 550 bp) were excised, and the DNA amplicons were purified using a Qiaquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). The purified amplicon libraries were pooled at equimolar ratio to a final concentration of 20 ng DNA µl⁻¹ for both bacteria and archaea, and the two pools were then combined at a 9:1 molar ratio and sequenced using a Roche GS FLX Titanium system at the Plant-Microbe Genomics Facility at The Ohio State University.