Metagenomic analysis of the RNA fraction of the fecal virome indicates high diversity in pigs infected by porcine endemic diarrhea virus in the United States

Chen, Qi; Wang, Leyi; Zheng, Ying; Zhang, Jianqiang; Guo, Baoqing; Yoon, Kyoung-Jin; Gauger, Phillip C.; Harmon, Karen M.; Main, Rodger G.; Li, Ganwu

Reverse transcription was performed on purified RNA using NEXTflex™ Rapid RNA-Seq Kit (Bioo Scientific Corp, Austin, TX). Double-stranded cDNA was purified with Agencourt® AMPure® XP Beads (Beckman Coulter Life Sciences, Indianapolis, IN) per manufacturer's recommended procedure, and was re-suspended into 9 μl re-suspension buffer. The concentration of cDNA was determined by Qubit® 2.0 Fluorometer and Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA) and adjusted to 0.2 ng/μl for sequencing library preparation. Sequencing library was prepared using Nextera® XT library preparation kit (Illumina, San Diego, CA) with dual-indexing and sequencing was performed on an Illumina MiSeq® instrument (Illumina, San Diego, CA) according to user manual of "MiSeq Sequencing System Guide" for 2 × 150 base pair (bp) reads [9] . Each library composed of 24 pooled samples or fewer without negative control samples for cross contamination assessment.