Roche GS Junior Whole Gneome Shotgun Sequencing of E.coli Sakai
500 ng E. coli Sakai DNA was used to generate a shot gun library following the Roche Rapid Library protocol. DNA fragmentation by nebulization was performed with 3.5 bar nitrogen gas for 1 minute using the nebulization device of the kit. The nebulized DNA was purified with MinElute PCR Purification Kit (Qiagen, Hilden, Germany). This step was followed by fragment end-repair and adaptor ligation. Small fragments were removed with AMPure XP beads (Beckmann Coulter). The library was quantified with RL-Standard (provided in the kit) using the QuantiFluor (Promega, Mannheim, Germany). The library quality was assessed using the High Sensitivity DNA chip on the Agilent 2100 Bioanalyser (Agilent, Waldbronn, Germany). 20 x 107 molecules were used for the emulsion PCR that was carried out on a MasterCycler PCR cycler (Eppendorf, Hamburg, Germany). The following enrichment was performed according to the manufacture’s instructions. Sequencing was performed with the GS Junior Titanium Sequencing Kit under standard conditions. The 454 Sequencing System Software version (v) 2.7 was used with default parameters, i.e., Signal Intensity filter calculation, Primer filter, Valley filter, and Base-call Quality Score filter were all enabled.