Ball Python Nidovirus: a Candidate Etiologic Agent for Severe Respiratory Disease in Python regius

Stenglein, Mark D.; Jacobson, Elliott R.; Wozniak, Edward J.; Wellehan, James F. X.; Kincaid, Anne; Gordon, Marcus; Porter, Brian F.; Baumgartner, Wes; Stahl, Scott; Kelley, Karen; Towner, Jonathan S.; DeRisi, Joseph L.

Library preparation and sequencing. Total RNA was extracted from frozen tissues for sequencing library generation and molecular analysis. RNA was extracted as previously described (59) . Two hundred fifty nanograms of RNA was added to 20 l RT reaction mixtures containing 200 pmol oligonucleotide MDS-286 (random hexamer), 1ϫ reaction buffer, 5 mM dithiothreitol, 1.25 mM (each) deoxynucleoside triphosphates (dNTPs), and 100 U SuperScript III (Life Technologies). 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 for 15 min at 70°C. Then, 1 U RNase H (NEB) diluted in 5 l 1ϫ RT buffer with 100 pmol MDS-286 was added to reaction mixtures, which were incubated for 30 min at 37°C and then 94°C for 2 min. Then, primer extension was used to convert single-stranded cDNA to double-stranded DNA (ds-DNA): 2.5 U Klenow DNA polymerase (3= to 5= exo-; NEB) diluted in 5 l 1ϫ RT buffer with 2 mM (each) dNTP was added to reaction mixtures, which were incubated at 37°C for 15 min. DNA was purified using DNA clean and concentrator columns (CC-5; Zymo Research) according to the manufacturer's protocol and using a 3:1 ratio of binding buffer/sample. The DNA concentration was determined fluorometrically, and 10 ng was used as a template in 10 l Nextera transposon reactions, which contained 1ϫ buffer and 0.5 l transposase mix (Illumina), which were incubated at 55°C for 10 min and then placed on ice. Tagmented DNA was cleaned with CC-5 columns (4:1 buffer ratio), eluted in 20 l H 2 O, and used as a template in PCRs that added full-length bar-coded adapters to library molecules. PCR mixtures contained 1ϫ Kapa real-time library amplification PCR mix (Kapa Biosystems), 0.33 M (each) primers MDS-143 and -445 and 0.017 M (each) adapter1 and adapter2 bar-coded primers, and 5-l library template. Thermocycling conditions were 72°C for 3 min, followed by 95°C for 30 s, and then 5 cycles of 98°C for 10 s, 63°C for 30 s, and 72°C for 3 min. Relative concentrations of libraries were then quantified in quantitative PCR (qPCR) reaction mixtures containing 10 mM Tris, pH 8.6, 50 mM KCl, 1.5 mM MgCl2, 5% glycerol, 0.08% IGEPAL CA-630, 0.05% Tween 20, 0.2 mM (each) dNTP, 1ϫ Sybr green (Life Technologies), and 0.5 U Taq polymerase, and 0.1 M (each) primers MDS-143 and -445. Equivalent amounts of each sample were then pooled, and the pool was cleaned with a DNA CC-5 column according to the manufacturer's protocol. The pooled libraries were then size selected (350 to 450 nt) using a LabChip XT device (Caliper), and purified again using a CC-5 column. Size-selected pooled libraries were subjected to a final round of amplification in PCR mixtures containing 1ϫ Kapa real-time library amplification mix, 200 pmol each MDS-143 and -445, and 5 l library template. Reaction conditions were 98°C for 1 min and 16 cycles of 98°C for 15 s, 63°C for 30 s, and 72°C for 3 min. The pooled libraries were finally purified using a DNA CC-5 column and quantified spectroscopically and by using the Illumina library quantification kit (Kapa Biosystems). Sequencing was performed on an Illumina HiSeq 2500 instrument.