# Rotavirus A Genome Segments Show Distinct Segregation and Codon Usage Patterns
## 2 Materials and Methods
### Codon Bias Analysis Comparison  
The relative codon deoptimization index (RCDI) was used to assess if the codon usage of a gene was similar to the codon usage of a reference genome (2953 coding sequences for _Sus scrofa_ (pig), 93,487 coding sequences for _Honto sapiens_ (human), 6017 coding sequences for _Gallus gallus_ (chicken), 13,374 coding sequences from _Bos taurus_ (cow), 1194 coding sequences for _Cantis familiaris_ (dog), and 1115 coding sequences for _Oryctolagus cuniculus_ (rabbit)) [87]. RCDI values range from 0.0 to 1.0 with 1.0 indicating maximum codon usage compatibility with a reference genome. Similarly, the codon adaptation index (CAI) was used as a measure of codon usage adaptation to the most used synonymous codons of a reference genome and was used to predict the expression levels of genes [87]. CAI values range between 0.0 and 1.0, where higher CAI values for a particular reference set indicate higher expression levels. To determine if there were significant differences in CAI/RCDI values between the different segments, we calculated CAI and RCDI values using the CAIcal server ([[http://genomes.urv.es/CAIcal/](http://genomes.urv.es/CAIcal/)]([http://genomes.urv.es/CAIcal/](http://genomes.urv.es/CAIcal/)) (accessed on 15 August 2020)) for each segment using a subset of RVA isolates containing multiple representatives of common mammalian RVA genotypes [88]. Statistical analyses were performed to assess whether RVA was genetically compatible in its codon usage patterns with a set of RVA host reference genomes. To reduce bias that would result from analyzing a small number of genes, the RVA host reference genomes were chosen based on the availability of a large number of genes analyzed for their codon usage tables in the Codon Usage Database ([[http://www.kazusa.orjp/codon/](http://www.kazusa.orjp/codon/)]([http://www.kazusa.orjp/codon/](http://www.kazusa.orjp/codon/)) (accessed on 15 August 2020)). Reference sets for chicken, human, pig, dog, rabbit, and cow genomes were used for the analysis. We performed [PERSON]'s Honest Significant Difference (HSD) test to compare mean CAI and RCDI values between segments. We also used [PERSON]'s HSD tests to compare mean CAI and RCDI values between different host genomes after combining all segments' values.  
To test for neutrality at the third codon position, a neutrality plot was made by comparing the GC contents at the first, second, and third codon positions. GC12 being the average of GC1 and GC2 was plotted against GC3. If GC12 and GC3 are significantly correlated to one another, and the slope of the regression line is close to 1, mutational bias is assumed to be primarily responsible for shaping codon usage patterns rather than translational selection. Selection against mutation bias can lead to larger differences in GC content between positions 1 and 2, and position 3 and little to no correlation between GC12 and GC3 [89].  
RVA strains isolated from specific hosts (pig, human, cow, and avian) were also compared to the pig, human, cow, and chicken genomes to test whether RVA genomes showed evidence of adapting to specific hosts. Seven complete genomes (77 segment genomes) for each host, isolated from either avian, cow, human, or pig hosts. While reassortment and spillover events occur in RVA evolution, there are larger generally bovine/porcine/human/avian clades (Figure 1, SI Figures S1 and S2), so isolates representing their general host strain clades were chosen. If RVA genomes did not appear to differ in CAI and RCDI patterns based on host type, then it would suggest mutational selection was the dominating force over the translational selection, and that selection at the translation level was weak or undetectable.