Treatments of porcine fecal samples affect high-throughput virome sequencing results

Abstract

The porcine enteric microbiota is currently extensively studied, taking advantage of developments in high-throughput sequencing technologies. However, the viral part of the microbiota, the virome, is being lightly explored, and the impact of the pretreatments used before sequencing the viruses is barely considered. In this study, the impacts of filtration, RNase and DNase treatments on virus reads recovery and diversity after sequencing on a MiSeq platform were assessed on fecal samples individually taken at <3, 5, 12 and 20 weeks from two piglets. None of the four pretreatment series affected the virus read averages or influenced diversity, but the samples with the higher proportion of reads corresponding to an entry in the "nt" database were those receiving the least number of pretreatments. The enzymatic pretreatments affected the detection of the single-stranded RNA viruses of Aichivirus C, porcine astrovirus, Sapovirus and posavirus, which is worrisome, as these viruses can be involved in swine diarrhea. If enzymatic pretreatments are used when sequencing using a high-throughput method, it may impact single-stranded RNA virus recovery, but not the overall virome diversity. Therefore, filtrated samples may be the better option, reducing the amount of bacterial genetic material while preserving the virus reads.

Keywords: High-throughput sequencing; MiSeq; Porcine; Pretreatment; Single-stranded RNA; Viruses.