Sequencing and Analysis of Ectromelia Virus

Two µg of viral DNA, obtained from semi-purified viral stocks as described [34 (link)], were used for the construction of a library using the GS-FLX Titanium system (454 Life Sciences, Roche, Branford, CT, USA) and sequenced with a FLX Genome Sequencer in the Scientific Park of Madrid, Spain. Reads were de novo assembled and also mapped to ECTV-M or ECTV-N reference genomes with Newbler 2.5.3 (Roche Diagnostics, Branford, CT, USA). For Illumina sequencing, five µg of viral DNA of ECTV-M was used to construct a TruSeq library and reads obtained with a Genome Analyzer IIx in the Scientific Park of Madrid, Spain. These Illumina reads were mapped to ECTV-M genome (AF012825.2) as reference using Bowtie2 with default parameters (http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml; Version 2.1.0) (Table 2). A PCR amplification of the region containing the Direct Repeat III (DRIII) region of ECTV-MK was carried out as described [27 (link)]. The product was analyzed in a 2% agarose gel with a 100 bp ladder marker (Invitrogen) considering the most intense band for the estimation of the number of repeats.