Quality of the reads was assessed using FastQC. For some runs, for which base calling qualities dropped notably toward the end of the reads, read tails were trimmed using Trimmomatic v0.35 (41) with default settings. We ran SeqPrep v1 (https://github.com/jstjohn/SeqPrep) to merge read pairs in case forward and reverse reads overlapped and subsequently remove reads shorter than 70 base pairs (bp). Reads were mapped with bwa mem v0.7.12 with slightly increased accuracy setting (-r 1) (42) to one reference genome consisting of both host (26) and virus (27) reference sequences. We ran Picard (v2.0.1) FixMateInformation and AddOrReplaceReadGroups (http://broadinstitute.github.io/picard), removed alignments with quality 0 with awk, and merged data per time point (across lanes and sequencing runs) using SAMtools v1.3 (43). Resulting .bam files containing aligned reads per time point were sorted, cleaned, and indexed with Picard SortSam, Picard CleanSam, and SAMtools index. For the virus, average genome-wide coverage was >1000× at all time points where virus was present. We used the SAMtools view with the -s parameter to downsample to an average per-position coverage of 1000×; this makes the subsequent analysis less computationally demanding but keeps the empirical coverage distribution intact for later filtering. Table S1 summarizes average coverage of host reads after quality control and alignment for every time point per replicate. If average per-position coverage was below 10×, the time point was completely removed from analysis. Coverage for specific sites was evaluated after .bam files were transformed to .sync format with SAMtools mpileup and Popoolation2 (44) v1.201 mpileup2sync.pl (--min-qual 1). In these .sync files, sites with a coverage outside of the interval (mean ± 3 SDs) or smaller or equal to 10× were set to “not available” with a custom R script. The frequency of the nonreference allele with the highest average frequency across time points was calculated as derived allele frequency.

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