Skip to main content

Advertisement

Table 1 Recombination and mutation rates

From: A general method to eliminate laboratory induced recombinants during massive, parallel sequencing of cDNA library

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
  1. 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.