Identification, Characterization, and In Vitro Culture of Highly Divergent Arenaviruses from Boa Constrictors and Annulated Tree Boas: Candidate Etiological Agents for Snake Inclusion Body Disease

Stenglein, Mark D.; Sanders, Chris; Kistler, Amy L.; Ruby, J. Graham; Franco, Jessica Y.; Reavill, Drury R.; Dunker, Freeland; DeRisi, Joseph L.

Library preparation and sequencing. Sequencing libraries were prepared essentially as previously described (60) . Two hundred fifty nanograms of RNA was added to 10 l reverse transcription (RT) reaction mixtures containing 50 pmol oligonucleotide MDS-187, 1ϫ reaction buffer, 5 mM dithiothreitol, 1.25 mM (each) deoxynucleoside triphosphates (dNTPs), and 100 U Superscript III (Invitrogen). The sequences of all oligonucleotides are listed in Table S1 in the supplemental material. Reaction mixtures were incubated for 60 min at 42°C and then 15 min at 70°C. Then, 10 U APE1 and 1 U UDG (NEB) diluted in 5 l 1ϫ Sequenase buffer (Affymetrix) were added to reaction mixtures to remove the oligo(dU/dT) RT primer. Reaction mixtures were incubated for 30 min at 37°C and then 94°C for 2 min. To generate end-tagged molecules, primer MDS-4 and 2 U of Sequenase DNA polymerase (Affymetrix) in 5 l of 1ϫ buffer were added to samples, which were ramped from 10°C to 37°C over 8 min and then incubated at 37°C for 8 min. These primer extension reactions were performed twice to generate doubly end-tagged molecules, which were subsequently amplified by PCR. PCR mixtures contained 1ϫ reaction buffer, 2 M primer MDS-189, 0.25 mM dNTPs, 2 U KlenTaq DNA polymerase (Sigma), and 2 l library template. Thermocycling conditions were 95°C for 2 min; 2 cycles of 95°C for 30 s, 40°C for 30 s, and 72°C for 1 min; and then 15 cycles with a 55°C annealing temperature. PCR mixtures were cleaned using the Ampure XP reagent (Agencourt) according to the manufacturer's protocol but using a 1.4:1 ratio of beads to sample. Ten nanograms of library template was then added to PCR mixtures containing 1ϫ reaction buffer, 0.25 mM dNTPs, 0.01 M (each) primers MDS-9 and MDS-10 (bar code), and 12.5 U KlenTaq DNA polymerase. Thermocycling conditions were 95°C for 2 min and 2 cycles of 95°C for 30 s, 40°C for 30 s, and 72°C for 1 min. Then, primers MDS-200 and -201 were added to reaction mixtures to a final concentration of 0.2 M, and 6 more cycles with a 58°C annealing temperature were performed. Reaction mixtures were cleaned again with Ampure XP reagent, and their relative concentrations were quantified in qPCR mixtures containing 1ϫ LC480 Sybr Green master mix (Roche) and 0. Sequence analysis. In addition to the default Illumina quality filtering, low-quality sequences containing any N's were removed from further analysis. Low-complexity sequences with an LZW ratio less than 0.45 (the ratio of the length of the Lempel-Ziv-Welch compressed sequence to the uncompressed length) were additionally removed (60, 61) . The first 6 bases of each sequence, corresponding to the random hexamer used to tag library molecules, were trimmed from sequences. Snake sequences were then filtered using the BLASTN alignment tool (version 2.2.21 [17] ) to query a database composed of a draft (assembly 1) of the boa constrictor genome (16) . Sequences aligning with an expect value of less than 10 Ϫ8 were filtered. Similarly, sequences that aligned with the Illumina adapter sequences (see Table S1 in the supplemental material) or to X174 control sequence were removed. This filtering removed between 90 and 97% of sequences. The remaining sequences were searched against databases of viral protein sequences using the BLASTX algorithm, and sequences matching any viral protein sequence with an expect value of less than 2 were further examined. False positives were eliminated by using BLAST to align putative viral sequenc