Viral RNA Extraction and Sequencing

A fecal suspension (10% w/v) prepared in Tris/HCl/Ca²⁺ buffer was used for nucleic acid extraction by a QIAamp viral RNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s guidelines or using the silica method (Hernandez et al., 2018 (link)). The extracted RNA was quantified by a Qubit 2.0 fluorometer using the Qubit RNA BR assay kit (Thermo Fisher). Then, the samples were subjected to reverse transcription and purified using a cDNA synthesis system kit (Roche, Branford, CT, United States) to obtain pure double-stranded DNA. The DNA was quantified using the Qubit DNA BR assay kit (Thermo Fisher Scientific) and analyzed for fragmentation and quality profile using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States) as recommended by the manufacturer. Subsequently, libraries for sequencing were prepared using the Illumina Nextera XT DNA Library Prep kit and sequenced on an Illumina HiSeq 2500 instrument (Illumina, San Diego, CA, United States) with the high-output V4 2 × 100-bp sequencing kit.