| Design Description | The live ticks were washed with 70% ethanol supplemented with 1% povidone-iodine solution twice and rinsed three times with distilled water to avoid bacterial contaminations on shell surface. Ticks were then ground with 4.8 mm stainless steel beads (TOMY, Tokyo, Japan) using the Micro Smash MS-100R (TOMY) for 2 min at 2,500 rpm. Tick homogenates were treated with a membrane lysis buffer [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM MgCl2, 0.1% IGEPAL CA-630 (Sigma Chemical Co., St. Louis, MO)] for 10 min on ice and centrifuged at 400 ? g for 25 min. The supernatant was centrifuged at 20,000 ? g for 30 min and the pellet containing the bacterial cells was resuspended in a DNAse buffer [25 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 0.8 U/?L DNAse I (Takara, Shiga, Japan)] to remove any contaminating host-cell DNA. After 60 min at 37?C, the reaction was stopped by adding 25 mM EDTA (pH 8.0). The solution was filtered through a 5.0 ?m membrane filter (Millipore, Bedford, MA) by centrifugation at 1,000 ? g. The filtrate was centrifuged at 20,000 ? g for 30 min and the resulting pellet was washed two times with PBS. All the centrifugation was conducted at 4?C. In order to lyse a wide range of bacterial species, this study utilized an Achromopeptidase (ACP) which has been reported to have a potent bacteriolysis activity elsewhere {Ezaki, 1982}. The bacterial pellet was treated with an ACP buffer [10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 1 U/?L ACP (WAKO, Osaka, Japan)] for 60 min at 37?C. Bacterial DNA was then extracted using the NucleoSpin Tissue XS Kit (Macherey-Nagel, D?ren, Gemany) according to the manufacturer's instructions. The genomic DNA was then subjected to a whole-genome amplification using the GenomiPhi? V2 DNA Amplification Kit (GE Healthcare, Chalfont St Giles, UK) according to the manufacturer's instructions. The DNA concentration was measured using the Quant-iT dsDNA BR and the Qubit Fluorometer (Invitrogen, Carlsbad, CA). All the procedures were conducted under sterile conditions in a flow cabinet. Genomic DNA from each tick pool was subjected to pyrosequencing on a Roche/454 Genome Sequencer FLX Titanium (Roch Applied Science/454 Life Science, Brandford, CT) at Hokkaido System Science (Sapporo, Japan). |
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