Raw Sanger sequencing reads from MSCLV, Cygnus olor fecal virome, sample UTMF1447
Samples were homogenised and filtered through a 0.45 m filter to concentrate virus-like particles (VLPs). Nucleic acids not protected in VLPs were degraded by DNase and RNase incubation at 37C for 1.5 h. Total RNA and DNA were then extracted using a QIAamp Viral RNA Mini Kit (Qiagen).  Reverse transcription was performed using SuperScript IV VILO kit (Invitrogen), cDNAs were purified by a QIAquick PCR Purification Kit (Qiagen)  and complementary strands synthesised by Klenow DNA polymerase I (Promega). Double-stranded DNA was amplified by random PCR amplification  (Q5 Hot Start High-Fidelity kit, New England Biolabs). Samples were barcoded during reverse transcription and PCR steps using homemade 26-nt Dodeca Linkers and PCR  multiplex identifier primers. PCR products were purified using a NucleoSpin gel and PCR clean-up kit (Macherey Nagel). dsDNAs were quantified using a Qubit 1X dsDNA HS assay kit. Libraries were prepared using a NEB NEXT Ultra II DNA Library prep kit, and sequenced on a NovaSeq6000 in 2x150bp paired-end mode (Oxford Genomics, UK). To confirm the genome of the mute swan circo-like virus (MSCLV), DNA was re-extracted from the positive sample using QIAamp fast DNA stool minikit (Qiagen), and amplified by PCR using Promega GoTaq G2 HotStar kit with overlapping primers. Amplification conditions were kept at 95C for 5 min, followed by 35 cycles of denaturation at 95C for 30 s, primer annealing at 56C for 30 s, and elongation at 72C for 2 minutes, followed by a final extension at 72C for 10 min. PCR products were purified using a QIAquick PCR purification kit (Qiagen) and sequenced by Sanger sequencing. Chromatograms were checked for disparities between the forward and reverse reads, and consensus sequence of the MSCLV genome was obtained by amplicons alignment (MAFFT v7.450, E-INS-i algorithm).