# Rotavirus A Genome Segments Show Distinct Segregation and Codon Usage Patterns
## 2 Materials and Methods  
### Sequence Alignment and Phylogenetic Analysis  
From all complete RVA genomes in NCBI's Virus Variation Resource, 789 complete mammalian rotavirus A genomes isolated between 1974 and 2017 were chosen. To minimize sampling bias, we excluded any isolates where three or more genomes shared the same sequence identity for NSP4. To ensure that the analysis of each RVA segment employed the same set of strains, we filtered out the selected strains from files containing all available genomes for each segment in Python v3.9.2 (xml files available in SI files S2-S12). The pooled sequences of each of the 11 RVA segments were independently aligned using MUSCLE v3.8.31 [75] and visually inspected for obvious sequencing errors or low-quality sequences. Sequences that were unusually long, short, or contained 'N' nucleotides were all removed. We performed phylogenetic analyses using BEAST v1.10.4 [76]. We used tip dating to calibrate molecular clocks and generate time-scaled phylogenies. We note that divergence-date estimating in viruses using tip-date calibration can be especially erroneous if there is a poor model fit to the data [77]. Genetic diversity, a contemporaneous bias of available virus sequences, lineage-specific variation in rates over time, long-term purifying selection, and inappropriate priors can result in substantial errors in the phylogeny and divergence date estimates [78; 79; 80]. We excluded Avian RVA from the phylogenetic analyses to minimize error in divergence-date estimates, which can be exacerbated with the inclusion of deeper nodes. As Mammalian RVA is endemic in the population, and has been for quite some time, this also somewhat minimizes the error associated with sampling bias. The analyses were run under an uncorrelated relaxed clock model using a time-aware Gaussian Markov random field Bayesian Skyride tree prior [81]. Segment alignments were partitioned by coding sequence and untranslated region and run using a GTR + \(\Gamma\) + I substitution model and partitioned by codon position for the coding sequence partition. VP4 was partitioned by the VPS * and VP8 * protein domains. Due to the large insertions in segment 11 (NSP5/6), this segment required three partitions based on insertion locations. Log files in Tracer v1.7.1 [82] were analyzed to confirm sufficient effective sample size (ESS) values. The alignments were run for three chains with a 500,000,000 Markov chain Monte Carlo (MCMC) chain length, analyzed on Tracer v1.7.1, and combined using LogCombiner v1.10.4 [83]. Trees were annotated with a 10% burn-in using TreeAnnotator v1.10.4 [82]. The best trees were visualized using FigTree v1.4.4, with the nodes labeled with posterior probabilities and node bars representing 95% confidence intervals for the divergence dates(annotated tree files available in SI files S13-S23). Bayesian Skyride plots were made in Tracer v1.7.1 to compare each segment's changes in effective population size (used as a proxy for relative diversity) since the root of the tree (-50 years prior to 2017 for most segments). We used the R package 'distory' to calculate the geodesic distances between segments. Geodesic distance uses topology and branch length to visualize the tree space of the 11 segments to determine which segments share a close evolutionary history. To account for phylogenetic uncertainty, 350 post-burn-in, randomly chosen trees were sampled from the BEAST v1.10.4 tree file for each segment. We applied multi-dimensional scaling using the R package, 'tree space' to determine whether the 'time to the most recent common ancestor' (TMRCA) was consistent between segments. The correlation coefficient of TMRCA estimates from all pairwise comparisons of the 11 segment trees was used to estimate tree distance and then the matrix of tree distance was plotted. Variation in branch length between different segment trees was visualized as a cloud of points where the center represents the mean of several hundred trees.