Viral Genomic Sequencing from Clinical Samples

Samples 712 and 808 containing LuJo virus were prepared from human isolates [10 (link)]. RNA was extracted from the cerebrospinal fluid and serum of a liver transplant recipient. After digestion with DNase I to eliminate human chromosomal DNA, RNA preparations were amplified by means of reverse-transcriptase PCR (RT-PCR) with the use of random primers [11 (link), 12 (link)]. Amplification products were pooled and sequenced with the use of the 454 Genome Sequencer FLX platform (Roche, Branford, CT), but DNA fragmentation was omitted. The Zaria bat coronavirus samples 819 and 820 (and the negative control 806) were obtained from the GI tract of bats that tested positive (and negative for the control) for coronavirus by PCR [13 (link)]. Sample 28 containing GBV-D was obtained from bat serum [14 (link)] and prepared as detailed previously. The isolated RNA for both coronavirus and GBV-D samples was converted to cDNA and the library was prepared similarly to the LuJo virus isolates detailed above. The bat parvovirus sample, 1164, was obtained from the spleen of parvovirus PCR-positive bats (like those discovered in [15 (link), 16 (link)]), and DNA was isolated and the prepared libraries were sequenced on the 454 FLX (Roche, Branford, CT). Samples containing MERS-CoV (1500, 1501) [17 ] were prepared as previously described [18 (link)]. Viral cDNA was made using random primer RT-PCR from nasal swabs of camels. Further PCR amplifications were made using overlapping PCR primers spanning 2.0–2.5 kb fragments of MERS-CoV [19 ]. These amplification products were pooled and sequenced on the Ion Torrent PGM platform. The human serum spiked samples containing Y. pestis, F. tularensis, and B. anthracis, ​B. mallei, and B. psuedomallei were prepared for sequencing as described previously [20 (link), 21 (link)] and sequenced on 454 FLX (Roche, Branford, CT), Ion Torrent PGM (Life Technologies, Grand Island, NY), and Illumina MiSeq platforms (Illumina, San Diego, CA). SRA information for each sample analyzed here are available through the NBCI BioProject # PRJNA276557.