Clc genomics workbench software package

cDNA was prepared from total RNA using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s instructions. Library construction and sequencing were performed by the Max Planck Genome Center Cologne, Germany² using the manufacturers’ protocols. TruSeq libraries were generated from poly-A enriched mRNA. The library was sequenced with an Illumina HiSeq2500 sequencer, and after de-multiplexing 8–11 million 100 bp paired-end reads per sample were obtained. The resulting sequencing data can be freely accessed at doi: 10.17617/3.55. Short 100 bp paired-end Illumina reads were quality-trimmed and filtered for duplicates using default parameters and then assembled de novo using the CLC Genomics Workbench software package (CLC bio Qiagen), using bubble size = 65. Consensus sequences were extracted to give 49,836 contigs.

Genome sequencing of L. monocytogenes strains 10403S, 10403S ΔltaS_sup (ANG2337), and 10403S ΔtagO1-2_sup (ANG2350) was performed by microbesNG (http://microbesng.uk) using an Illumina MiSeq platform and 2× 250-bp paired-end reads. The CLC genomics workbench software package (Qiagen) was used to map the reads to the 10403S reference genome sequence (RefSeq accession no. NC_017544.1), and high-confidence and high-frequency (>85%) changes are listed in Table S3 for the different strains. To determine the conservation of the gtlA gene among other Listeria species, a synteny analysis was performed using the SyntTax web server (http://archaea.u-psud.fr/synttax/) and all available sequenced Listeria genomes (45 (link))

L. monocytogenes genomic DNA was extracted using a bacterial genomic DNA isolation kit (Sigma-Aldrich). Lysis of the bacteria was done using the laboratory produced Ply511 endolysin. Library preparation and sequencing were performed by the Functional Genomics Center Zurich (University of Zurich, Switzerland). The library was prepared using Illumina TruSeq Nano DNA Library reagents, and sequenced at 2 x 250bp paired-end reads for 500 cycles. The CLC genomics workbench software package (Qiagen) was used to map the reads to the WSLC 1042 reference genome sequence, identifying high-confidence changes in the genome of the BIM strains.