Table 1.

Recombination and mutation rates

a. Recombination rates
RNA (ng)	RT	Virus	PCR Cycles	RR	Lower 95% CI bound	Upper 95% CI bound
160	SSIII	wt + mk mix	27	0.0065	0.0000	0.0132
1600	SSIII	wt + mk mix	27	0.1029	0.0787	0.1391
3990	SSIII	wt + mk mix	27	0.8933	0.3675	1.8378
2000	AMV	wt + mk mix	27	0.0742	0.0640	0.0901
160	SSIII	Heterozygous	27	0.0128	0.0064	0.0248
160	SSIII	wt + mk mix	35	2.9482	2.7699	3.1668
1600	SSIII	wt + mk mix	35	4.6068	4.4601	4.7671
3990	SSIII	wt + mk mix	35	1.061	0.7903	1.4239
b. Mutation Rates
RNA (ng)	RT	Virus	PCR Cycles	MR	Lower 95% CI bound	Upper 95% CI bound
160	SSIII	wt + mk mix	27	0.1198	0.1075	0.1325
1600	SSIII	wt + mk mix	27	0.1551	0.1340	0.1763
3990	SSIII	wt + mk mix	27	0.4549	0.2674	0.6702
2000	AMV	wt + mk mix	27	0.2169	0.2053	0.2288
160	SSIII	Heterozygous	27	0.1658	0.1503	0.1811
160	SSIII	wt + mk mix	35	0.2172	0.1959	0.2396
1600	SSIII	wt + mk mix	35	0.1608	0.1494	0.1717
3990	SSIII	wt + mk mix	35	0.2819	0.2483	0.3166

RNA was extracted from either an equal mix of homozygous WT and MK viruses or from an equivalent amount of heterozygous virus and reverse transcribed using increasing amounts of RNA as template using SSIII reverse transcriptase (RT), engineered to have minimal RNase H activity. In a parallel experiment RNA was also reverse transcribed using AMV with intact RNase H activity. Following first strand synthesis, cDNA was diluted as appropriate and amplified using 27 or 35 PCR cycles. Replicates containing SYBR Green 1 dye were used to ensure the reaction was stopped while in the log linear phase (27 cycles) or at plateau (35 cycles). Replicate samples were pooled and prepared for 454 NGS sequencing. Recombination and mutation rates are expressed as rate per 1000 nucleotides. The 95% confidence interval lower and upper boundaries, as determined by bootstrapping, are shown for each estimate.