title: mNGS_ZEBOV_metagenomic_Boende_outbreak
design: Metagenomic next-generation sequencing Whole blood samples were subjected to total nucleic acid extraction using the QIAamp Viral RNA kit (Qiagen) at INRB in the DRC. Following extraction, RNA was preserved using RNAStable (Biomatrica, Inc.) and shipped at room temperature to UCSF for metagenomic sequencing and PCR analysis.  Partial degradation of the RNA occurred during shipment, as the RNAStable matrix was inadvertently not fully dried prior to shipment per the manufacturers recommendations. Upon receipt, RNA samples were resuspended in 20 l water. RNA integrity was assessed using the Agilent Bioanalyzer RNA 6000 Pico Kit. For each whole blood sample, 10 l of resuspended extract were treated with 1 unit of Turbo DNase (Ambion) at 37C for 30 min and inactivated with 1.1 l of DNase inactivation reagent for 5 min. RNA was reverse-transcribed with SuperScript III Reverse Transcriptase (Life Technologies) using a random primer attached to a linker adapter (Sol-PrimerA, 5 -GTTTCCCACTGGAGGATA-N9 -3), followed by second-strand DNA synthesis with Sequenase DNA polymerase (Affymetrix), as previously described [10]. Metagenomic next-generation sequencing (mNGS) libraries were constructed from amplified cDNA using the Nextera XT DNA library preparation kit (Illumina). Dual-indexed barcodes were employed to enable pooling of libraries and to assign reads to individual samples after sequencing. Multiplexed barcoded mNGS libraries were sequenced as 150 base pair (bp) paired-end (PE) runs on a HiSeq 2500 instrument (Illumina), with up to 14 sample libraries multiplexed per lane.