Unbiased deep sequencing.

Samples were processed for sequencing in a biosafety level 3 laboratory as described previously (14), with slight modifications. Briefly, for each sample, RNA was isolated from ∼200 μl of plasma by using the Qiagen QIAamp MinElute virus spin kit (Qiagen, Hilden, Germany), omitting carrier RNA. Samples were then treated with DNase, and cDNA synthesis was primed by using random hexamers from a double-stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). Samples were fragmented, and sequencing adaptors were ligated by using the Nextera DNA sample preparation kit (Illumina, San Diego, CA, USA). Deep sequencing was performed on the Illumina MiSeq instrument. Sequence data were processed by using CLC Genomics Workbench 6.5 (CLC Bio, Aarhus, Denmark) and Geneious R5 (Biomatters, Auckland, New Zealand). Low-quality (<Q30, Phred quality score) and short (<100-bp) reads were removed, and coding-complete genome sequences for each virus were acquired by using a combination of mapping and the de novo assembly algorithm in CLC Genomics Workbench version 6.5. Viral genomes were annotated in CLC Genomics Workbench version 6.5, and open reading frames (ORFs) were confirmed by querying the NCBI GenBank database (25).