Ion Torrent PGM 200bp 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 according to the user guide. 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). The library was diluted to 65 pMol, i.e. 2.5-fold higher input than recommended in the appropriate user guide. The diluted library was used as templates for clonal amplification on Ion Spheres particles with the Ion OneTouch 200 Template Kit v2. Clonal amplification was done with the Ion One Touch System - consisting of OneTouch instrument and Enrichment System (ES) module - according to the instructions of the manufacturer (Life Technologies). The quality of amplification was estimated on the Guava easyCyte 5 system (Millipore, Darmstadt, Germany) as described in the Ion Sphere™ Particles (ISPs) Quality Assessment User Bulletin. For 200bp sequencing a separate Ion 316 chip was loaded and subsequently sequenced using 520 flows according to the Ion PGM 200 Sequencing Kit User Guide. TSS v2.2 software was used for data processing with default settings, except that the more stringent filter was chosen.