# Characterization of a Novel Emaravirus Affecting Ash Species (_Fraximus_ spp.) in Europe
## 2 Materials and Methods
### Determination of the Complete Genome of the Novel Virus  
Preliminary analysis of HTS sequence data identified scaffolds relating to five different genomic RNAs of virus species belonging to the genus _Emaravirus_. In order to determine the complete sequence of the novel virus, full-length RT-PCR was performed, applying the PDAP213 primer [33] using total RNA prepared from _F. excelsior_ leaves (E55270) displaying the shoestring symptom. Full-length PCR products were cloned and sequenced as described in [PERSON] et al. [28] and [PERSON] et al. [19] with at least two identical fragments being required for sequence confirmation. As amplification of the complete RNA1 could not be achieved, eight primer pairs producing overlapping fragments spanning the complete genome segment were constructed based on original HTS scaffolds (Table S1, Supplementary Information). PCR products of the viral RNA1 of sample E55270 were generated by RT-PCR. Following cDNA synthesis from 1-3 ug of total RNA using random hexamer oligonucleotides (Biolegio GmbH, Nijmegen, The Netherlands), RT-PCR was performed in a 50 uL reaction mixture of 37.5 uL MilliQ-H2O, 10 uL 5 \(\times\) HiFi Reaction buffer, 1 uL dNTPs (10 mM each), 0.5 uL forward and reverse primers (50 uM), 1 unit (U)velocity polymerase (Bioline/Meridian Biosciences, Cincinnati, OH, USA) and 1 uL cDNA. Cycling conditions were as follows: initial denaturation at 94 \({}^{\circ}\)C for 2 min, followed by 30 to 35 cycles of denaturation at 94 \({}^{\circ}\)C for 20 s, annealing at 55 \({}^{\circ}\)C for 20 s and elongation at 72 \({}^{\circ}\)C for 15-30 s (adjusted for fragment length according to manufactures instructions). Following their purification using the MSB Spin PCRapace kit (Invitek Molecular, Berlin, Germany), generated products were sequenced from both directions with fragment-specific primers (Macrogen Europe B.V., Amsterdam, The Netherlands). A modified RT-PCR protocol, a tailing PCR, was employed to confirm terminal ends of the RNA1 and 2. Following cDNA synthesis as described above, enzymatic degradation of residual RNA was achieved by adding 3 units (U) of RNase H (Thermo Fisher Scientific, Waltham, MA, USA) to 40 uL of the cDNA and incubating the samples at 37 \({}^{\circ}\)C for 30 min. A poly-A (adenine) tail was added to 3\({}^{\prime}\)cDNA ends with the help of 1 U of terminal transferase (Thermo Fisher Scientific, Waltham, MA, USA). The poly-A cDNA was then purified (MSB Spin PCRapace kit, Invitek Molecular, Berlin, Germany) and used as a template for PCR, utilizing two virus-specific primers in the form of a semi-nested PCR, allowing the amplification of a specific terminal end of a particular genome segment. Primer sets and PCR conditions for the tailing PCR with velocity polymerase are provided in the Supplementary Information (Table S1). PCR products of expected size were cloned in _E. coli_ (XL1 blue, MRF', Thermo Fisher Scientific, Waltham, MA, USA) using the CloneJET PCR cloning kit (Thermo Fisher Scientific, Waltham, MA, USA) and 2-5 clones were sequenced (Macrogen Europe B.V., Amsterdam, The Netherlands) to determine the terminal ends of viral RNA1 and RNA2. All HTS- and RT-PCR-generated sequence data were edited, aligned and translated in BioEdit, version 7.0.5.3 [32]. The single open reading frame (ORF) of each RNA segment was located and translated using the NCBI ORF finder ([[https://www.ncbi.nlm.nih.gov/orfinder](https://www.ncbi.nlm.nih.gov/orfinder)]([https://www.ncbi.nlm.nih.gov/orfinder](https://www.ncbi.nlm.nih.gov/orfinder)) (accessed on 21 March 2021)). A comparison of the RNA1-5 encoded proteins of the novel virus with corresponding proteins of classified and putative members of the genus _Emaravirus_ was performed using the Sequence Identity Matrix function of BioEdit [32]. Phylogenetic analyses were performed using MUSCLE [34] to align the RNA1 and RNA3 encoded amino acid sequences of the novel virus identified in _F. excelsior_ (E55270) and _F.