Ebola Virus Epidemiology and Evolution in Nigeria

Folarin, Onikepe A.; Ehichioya, Deborah; Schaffner, Stephen F.; Winnicki, Sarah M.; Wohl, Shirlee; Eromon, Philomena; West, Kendra L.; Gladden-Young, Adrianne; Oyejide, Nicholas E.; Matranga, Christian B.; Deme, Awa Bineta; James, Ayorinde; Tomkins-Tinch, Christopher; Onyewurunwa, Kenneth; Ladner, Jason T.; Palacios, Gustavo; Nosamiefan, Iguosadolo; Andersen, Kristian G.; Omilabu, Sunday; Park, Daniel J.; Yozwiak, Nathan L.; Nasidi, Abdusallam; Garry, Robert F.; Tomori, Oyewale; Sabeti, Pardis C.; Happi, Christian T.

To assess sample quality, extracted RNA was quantified using quantitative RT-PCR (qRT-PCR) for both EBOV and human ribosomal RNA (18S). RNA selected for sequencing was quantified using the Power SYBR Green RNA-to-Ct 1-Step qRT-PCR assay (Life Technologies). The Kulesh assay protocol was adapted from a probe-based quantitative PCR (qPCR) assay to a SYBR qPCR assay by omitting the probe [8] . The 10-µL assay mix included 3 µL of RNA, 0.3 µmol/L primer Kulesh forward (TCT GAC ATG GAT TAC CAC AAG ATC), 0.3 µmol/L Kulesh reverse (GGA TGA CTC TTT GCC GAA CAA TC), 5 µL of ×2 Power SYBR Green RT-PCR Mix and 0.08 µL of RT Enzyme Mix (Life Technologies). The cycling conditions were 48°C for 30 minutes and 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 30 seconds with a melt curve of 95°C for 15 seconds, 55°C for 15 seconds, and 95°C for 15 seconds. qRT-PCR was performed on the LightCycler 96 (Roche) instrument at both RUN and the Broad Institute. Synthetic oligonucleotide amplicons were prepared as a standard to quantify the viral copy number in the qRT-PCR assays. These amplicons represent a portion of the EBOV segment within the L gene as a template for PCR. The amplicons were cleaned using AMPure XP beads (Beckman Coulter Genomics) and quantified by the TapeStation system (Ambion). Amplicon concentrations were converted to EBOV copies per microliter for quantification.