Clc bio main workbench

Reads from the three uninoculated samples and the three inoculated samples were mapped to the P. cinnamomi var cinnamomi draft genome downloaded from the Joint Genome Institute (Reeve, 2012, unpublished; JGI Project identity: 1003775). The genome assembly, based on Illumina, 454 and Sanger sequencing, is 77.97 Mbp and the sequence read coverage depth is 69.6x (Grigoriev et al., 2012 (link)). Mapping was performed as described above and assembly of reads and FPKM value were generated using Cufflinks. Any genes or transcripts showing an FPKM value above 100 in the control samples were considered conserved eukaryotic genes and removed from further analysis. Remaining genes or transcripts which showed FPKM values >100 in the three inoculated samples with a coefficient of variation < 0.2 were considered further. Approximately 280 genes satisfied this criteria and were subsequently analyzed using the pathogen host interactions (PHI) database (http://www.phi-base.org/ Winnenburg et al., 2006 (link)) to determine pathogenicity or virulence factors expressed in planta using a local BLASTP search in CLCBio main workbench (version 6.1; Qiagen). The database was downloaded in July 2014 and hits with the lowest e-value were considered. Selected genes were also subjected to Blast2GO® analysis to annotate the gene models.

Multilocus Sequence Typing was performed on 48 isolates, which were selected randomly based on the most common spa-types. Primers [29 (link)] amplifying seven reference genes were used. Amplification was done using the Amplitaq Gold DNA Polymerase kit (Applied Biosystems, CA, USA). Purified PCR products were sequenced (Inqaba Biotech, South Africa). Sequences were assembled using the CLC Bio main workbench (Qiagen, Germany) and analysed using the online database (https://pubmlst.org/saureus/).

Spa-typing was performed on 1467 MRSA isolates. The spa gene was amplified using previously published primers [12 (link)] and the Amplitaq Gold DNA Polymerase kit (Applied Biosystems, CA, USA). Purified PCR products (Qiagen Purification kit; Qiagen, Germany) were sequenced (Inqaba Biotech, South Africa). Sequences were assembled using CLC Bio main workbench (Qiagen, Germany) and analysed using the Ridom StaphType™ software, (Ridom GmbH, Würzburg, Germany).