## 2 Materials and Methods
### Viral Genome Analysis  
To build dual-indexed libraries for Illumina sequencing from the initial virus (EBOV_initial) and from supernatant of MoLu Prim_EBOV 150 dpi (EBOV_150 dpi), we performed DNase treatment using TURBO DNA-free(tm) Kit (Ambion, Austin, TX, USA), cleaned up the reactions with RNA Clean & Concentrator Kit (Zymo Research, Irvine, CA, USA), converted purified RNA to cDNA using the SuperScript(tm) IV First-Strand Synthesis System (Invitrogen, Waltham, MA, USA), and subsequently turned it into dsDNA with NEBNEXT(r) mRNA Second Strand Synthesis Module (New England Biolabs, Ipswich, MA, USA). DNA was purified using MagSi-NGSprep Plus Beads (Steinbrenner Laborsysteme, Wiesenbach, Germany), eluted in TET (Tris-HCl (10 mM), EDTA (1 mM), Tween20 (0.05%)), and fragmented using a Covaris S220 Focused-ultrasonicator with settings to generate 400 bp fragments (intensity = 4, duty cycle = 10%, cycles per burst = 200, treatment time = 55 s, temperature = 7 \({}^{\circ}\)C). Library preparation was performed with NEBNext(r) Ultra(tm) II DNA Library Prep Kit for Illumina(r) (New England Biolabs), and dual-indexes were added using NEBNext(r) Multiplex Oligos for Illumina(r) (New England Biolabs). Dual-indexed libraries were quantified using KAPA Library Quantification Illumina Universal Kit (Roche, Basel, Switzerland).  
To enrich EBOV RNA, we followed the myBaits Hybridization Capture for Targeted NGS protocol (Version 4.01) using custom-made RNA baits (120 nucleotides long, 2-fold tiling; Arbor Biosciences, Ann Arbor, MI, USA) that cover representative genomes of _Zaire ebalavirus_ (KC242801), _Sudan ebalavirus_ (KC242783), _Reston ebalavirus_ (NC_004161), _Tai Forest ebalavirus_ (NC_014372), _Bundibaygo ebalavirus_ (KC545395), and _Marburg marburgvirus_ (FJ750956). Only a fourth of the recommended bait input volume was used. We prepared separate capture reactions for the two EBOV samples, and performed two 24 h long rounds of hybridization capture at a temperature of 65 \({}^{\circ}\)C. After both rounds of capture, capture products were amplified using the KAPA HiFi HotStart ReadyMix and Illumina adapter-specific primers, quantified using the KAPA Library Quantification Illumina Universal Kit, and cleaned up using MinElute PCR Purification Kit. The second-round product was quantified and diluted to 4 nM for sequencing on an Illumina MiSeq platform (EBOV_initial), and to 1 nM for the Illumina iSeq (EBOV_150 dpi).  
EBOV_initial capture product was sequenced on an Illumina MiSeq platform (V3 chemistry, 2 \(\times\) 300 bp reads) for a total of 677,140 unfiltered paired reads. EBOV_150 dpi was sequenced on an Illumina(r) iSeq platform using iSeq 100 i1 Reagents (2 \(\times\) 150-cycle) for a total of 1,585,308 unfiltered paired reads. Sequencing reads were filtered (adapter removal and quality filtering) with Trimmomatic [69] (settings: LEADING:30 TRAILING:30 SLIDINGWINDOW:4:30 MINLEN:40). Read pairs were merged using ClipAndMerge [70], and merged, unmerged, and unpaired reads for each sample were combined into a single file, which was mapped to a _Zaire ebolavirus_ Makona strain (MG572232) using BWA-MEM [71]. For EBOV_initial, 99.55% of 734,731 high quality reads were mapped to the reference, for EBOV_150 dpi 99.84% of 1,896,873 high quality reads were mapped. The mapping files were sorted and duplicates were removed with the tools SortSam and MarkDuplicates from the Picard suite [72], resulting in 41,934 and 64,015 unique mapped reads, respectively. We then used Geneious Prime to assemble consensus genomes, calling bases with a minimum coverage of 20 \(\times\) and a 50% majority. The consensus sequences both contained 18,956 unambiguous positions (out of 18,958 positions in the mapping reference). To compare the genomes of EBOV_initial and EBOV_150 dpi, we aligned the consensus sequences using the MAFFT v7 implemented in Geneious [73] and visually inspected the alignment.