2.3 Total RNA extraction and NGS sequencing – metatranscriptome RNA
The isolation of total RNA from faecal samples, rRNA depletion, library preparation and sequencing were performed as previously described (Brussel et al., 2020; Chong et al., 2019). Briefly, the RNeasy plus mini kit (Qiagen, Germany) and Zymo-Seq RiboFree Total RNA Library Preparation Kit (Zymo Research, USA) were used for total RNA isolation, rRNA depletion and RNA sequencing library construction (Brussel et al., 2020). RNA libraries were sequenced on the Novaseq6000 platform (Illumina, USA, 150bp paired end) at the Australian Genome Research Facility (AGRF, Melbourne, Australia) (Brussel et al., 2020). Faecal samples from all 24 FPV-cases and 36 healthy controls were processed and RNA libraries sequenced.