Sequencing of EBOV: typical and atypical cases
Extracted viral RNA samples were treated with ezDNAse. RNA extracts were reverse-transcribed using Superscript III First-Strand Synthesis System. Second strand synthesis was performed at 16C for 16 hours using DNA Polymerase I, E. coli RNase H, E. coli DNA Ligase. Resulting dsDNA was used as template for the TruSeq DNA Nano LP kit (Illumina) following the manufacturers protocol. A total of 316 120-nt custom biotinylated lockdown DNA probes covering the entire EBOV genome in a double tiling approach were designed based on GenBank accession no. KJ660347.2 and purchased from Integrated DNA Technologies. For EBOV-specific library enrichment, equal volumes of up to eight final libraries were pooled with the lock-down probes, per the xGen hybridization capture of DNA libraries protocol Version 2 (Integrated DNA Technologies). Biotinylated probes were captured with streptavidin magnetic beads. Libraries were subjected to twelve cycles of PCR. PCR fragments were purified with Agencourt AMPure XP beads and eluted with Resuspension Buffer provided in the Illumina TruSeq DNA Nano LP kit. The enriched libraries were sequenced without further size selection on a MiSeq instrument using the MiSeq Reagent Kit v3 (600-cycle).