Gs flx sequencing platform

All PCR products were sequenced by standard Sanger sequencing (Macrogen, Inc., Seoul, South Korea). Putative heterozygotes (Sanger sequences containing double peaks) were further analyzed using the Roche GS FLX+ sequencing platform according to manufacturer’s Universal tailed amplicon sequencing design. Original primers were modified by adding a universal tail, which served as a priming site for a second PCR round in which MID sequences were introduced in the PCR product in order to enable post-sequencing individual sequence de-multiplexing. Products were mixed equimolarly and sequenced at Eurofins MWG operon (Ebersberg, Germany). All sequenced reads were sorted according to their MID tags, the MID tags were clipped. Reads corresponding to individual MID tags were further sorted based on the primer sequences using the CD-HIT suite [68 (link)] and a 90% identity cut-off. Primer sequences were trimmed with Cutadapt [69 (link)]. The reads obtained were further clustered by using the CD-HIT suite with the 0.99 sequence identity cut-off and cluster’s consensual sequences were manually compared with the corresponding Sanger sequences in Bioedit [70 ].

RNA was extracted using the High Pure RNA isolation kit (Roche Diagnostics, Germany) according to manufacturer’s instructions. RNA was converted to cDNA using the SuperScript III Reverse Transcriptase kit (Invitrogen, Thermo Fisher, USA) as described previously [24 (link)], and amplified by PCR using primers covering the full viral genome (S1 Table). All 32 PCR fragments from approximately 400–600 nucleotides in length, were sequenced using the 454/Roche GS-FLX sequencing platform. The PCR fragments were pooled in equimolar ratio and purified using the MinElute PCR Purification kit (Qiagen, Germany) according to the manufacturer’s instructions. Rapid Library preparation, Emulsion PCR and Next Generation 454-sequencing were performed according to instructions of the manufacturer (Roche Diagnostics, Germany). Protocols are described in the following manuals: Rapid Library Preparation Method Manual (Roche; May 2010), emPCR Amplification Method Manual–Lib-L (Roche; May 2010) and Sequencing Method Manual (Roche; May 2010). All three samples were sequenced in one run. Samples were pooled using MID adaptors to determine which sequences came from which sample, each sample was assigned two different MID’s. Demultiplexing and basic trimming was done by CLC-bio software to generate raw fastq files (S1 File).