100 bp ladder marker

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.

The three polymorphic regions of PvMSP-1 gene, namely F1, F2 and F3 were analyzed according to the previously described method with slight modification (Imwong et al., 2005; Suphakhonchuwong et al., 2018). Total volume of PCR mixture was 20 µl, containing 12.5 µl of 2x PCR mixture (Intron, Singapore), 5 µl of DNA template and 20 pmol of each primer. Primers and PCR condition were as described (Imwong et al., 2005) with slight modification. PCR products were directly analyzed using electrophoreses in agarose gels. Five microliters of PCR product were mixed with 1µl of loading buffer and applied to 2% agarose gel. The fragments of DNA were visualized by UV transillumination after ethidium bromide staining. The size of amplified fragments was compared by size to a 100 bp ladder marker (Invitrogen, 100 bp).