O13:16S rRNA pyrosequencing of osteomyelitis of the jaw
For DNA extraction from the samples, the Powersoil DNA Isolation Kit (MO BIO, Carlsbad, CA) was used as follows. The sample (0.2?1.0 wet g) was cut into the pieces less than 5 mm in each of L ? W ? H. All sectioned bones were dipped into buffer provided by the kit and preserved in liquid nitrogen for more than 3 h. The C1 solution and beads from the kit were added into the tube with the tissue and the tissue was homogenized with a Tissue Lyser (Qiagen, Hilden, Germany) at the maximum speed for 10 min. After addition of 250 ?L of phenol/chloroform/isoamlyalcohol (Nacalai Tesque, Kyoto, Japan), homogenization was carried out again. Purification of the crude DNA was carried out according to the manufacturer?s instructions. PCR amplification of the V3?V4 region of the 16S rRNA gene was carried out with bacterial universal primers 338F and 806R, which contained a 454 Life Sciences FLX sequencing primer and a 10-bp barcode sequence for multiplex. Forward: 5?-CCATCTCATCCCTGCGTGTCTCCGACTCAG-10-bp barcode-ACTCCTACGGGAGGCAGCAGT-3?Reverse:5?-CCTATCCCCTGTGTGCCTTGGCAGTC-GGACTACCAGGGTATCTAAT-3?The PCR was carried out in a final volume of 25 ?L including 1?2 ?L template DNA using MightyAmp polymerase (Takara Bio Inc., Otsu, Japan). The amplification was conducted in a 2720 thermal cycler (Applied Biosystems, Foster, CA) under the following conditions: start at 98?C for 2 min, 30 cycles at 98?C for 10 s, 58?C for 15 s, and 68?C for 45 s, and a final extension at 68?C for 2 min. PCR products were purified by gel extraction using a NucleoSpin Gel and PCR Clean-up kit (Takara Bio Inc.). The quality and quantity of the purified amplicon were examined by Agilent Bioanalyzer (Agilent Technologies, Waldbronn, Germany) and quantitative PCR using KAPA library quantification kits (KAPA Biosystems, Woburn, MA).Quantified and equally mixed amplicons were sequenced by using a 454 GS Junior (Roche Diagnostics, Basel, Switzerland) using protocols recommended by the manufacturer.