Ion Torrent PGM 400bp Whole Gneome Shotgun Sequencing of E.coli Sakai
The Library of the EHEC Sakai strain were generated using 1 μg of the genomic DNA and the Ion Xpress Plus Fragment Library Kit comprising the Ion Shear chemistry with minor modifications to the user guideline: (i) enzymatic shearing for 7 min and (ii) size selection of a 500bp fraction. For size selection E-Gel Size Select 2% Agarose Gels (Life Technologies) were used. All quality control analyses for the libraries were performed with a Caliper Labchip GX using the DNA High Sensitivity Assay (Caliper Life Sciences, Mainz, Germany). Library concentrations were determined with the Qubit dsDNA HS Assay Kit (Life Technologies). A library was diluted to 52 pM (2fold higher input than recommended), pooled equi-molar, and used with the Ion OneTouch 200 Template Kit v2 DL according to the User Guide. For 400bp sequencing of the Sakai library 2 copies per bead (cpb) were used for clonal amplification. The sample sample was sequenced on a 318 chip using 880 nucleotide flows with TSS v3.2 software version with default settings, except that the more stringent filter was chosen the Enable Base Recalibration option was turned off.