Illumina MiSeq 2x150 paired-end Whole Gneome Shotgun Sequencing of E.coli Sakai
The library were prepared with the Nextera DNA Sample Preparation Kit (Illumina, Little Chesterford, United Kingdom) according to the manufacturer’s instructions using 50ng of DNA for each bacterium and adapters P7 with indices for the Sakai strain. The two-step protocol of an AxyPrep™ Mag Fragment Select-I Kit (Serva, Heidelberg, Germany) was used for the size selection of fragments from 400 to 600 bp. These were checked with a High Sensitivity DNA chip on the Agilent 2100 Bioanalyser (Agilent, Waldbronn, Germany) and quantified using Picogreen (Life Technologies). All libraries were diluted to 2 nM in elution buffer EB (Qiagen) and a pool with an equi-molar concentration of all samples was prepared. 10µl of this pool were mixed with 10µl of 0.2 N NaOH and afterwards diluted with hybrization buffer HT1 (Illumina) to a final concentration of 9 pM. The MiSeq run was performed with the MiSeq Reagent Kit (300-cycles PE, Illumina) following the standard protocol for Nextera libraries with 2x151 cycles and 8 additional cycles for the barcode using the MiSeq Control Software v1.2.3. Default settings enabled adapter trimming on the machine. The machine also did base calling and demultiplexing. FASTQ files were created using the MiSeq Reporter Software (RS) v1.3.17 with default settings. Raw sequencing data were reanalyzed on the machine with adapter trimming disabled in order to generate submittable sequencing data of equal read length. Thus, reads may contain adapter contamination at the 5' prime end which have to be removed prior to any down-stream analysis.