Illumina paired-end sequencing identifies low frequency T54A+R155K double mutant in mock RNA populations.

RNA transcripts were synthesized from WT, T54A, or T54A+R155K plasmid in vitro. Each mock population of RNA transcripts was constructed according to the proportions indicated in the “Expected” column. The proportion of paired-end reads that harbored T54A single mutant, T54A/R155K double mutant, or WT are shown in the “observed” column (%). While the double mutant at a level below 0.1% was detected (Population 3), it was below our experimentally determined threshold for background error rate of ∼0.2% (see Table 2).