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.

Metagenomic sequencing and genome assembly. Because consensus PCR approaches did not identify any candidate etiologic agents, we pursued an unbiased metagenomic shotgun sequencing strategy. Suitable frozen tissue was available for 8 of 9 snakes, and total RNA was extracted and converted into sequencing libraries (see Materials and Methods). The libraries were sequenced on an Illumina HiSeq 2500 instrument to an average depth of 2.9 million pairs of 100-nucleotide (nt) sequences per sample (see Table S2 in the supplemental material). We then used a stepwise analysis pipeline to efficiently identify possible pathogen-derived sequences. First, we removed low-quality and low-complexity sequences and trimmed adapter sequences from reads. For each library, identical reads were collapsed to yield sets of unique sequences. Next, we filtered out snake ribosomal sequences and sequences aligning to the Burmese python and boa constrictor genomes (20, 21) . After these operations, approximately 2% of each data set remained.