Single-molecule sequencing of RNA to detect epitranscriptomic changes. SMRT sequencing with the Pacific Biosciences RS shows longer times (inter-pulse distances) to incorporate m6A versus standard adenosines. (a) Experimental design for using a DNA primer in a reverse transcription reaction. Sequencing of the unmodified template shows, in a single-molecule sequencing trace, base incorporation via a reverse transcriptase-mediated cDNA synthesis reaction. (b) Shows sequencing as with (a), but using an RNA template with m6A instead of normal adenosines. Incorporation of thymines (T) show significant delay (longer inter-pulse distances). A.U. stands for normalized arbitrary units in fluorescence measurement. (c) Exponential fit of experimentally observed inter-pulse distances (IPDs). (d) Shows the difference between the average IPDs for native As and m6As. The average IPD in each case is the reverse of the exponential decay rate. The error bars indicate the range around each average IPD that includes 83% of the observed IPDs (that is, ±½ of standard deviation of the exponential fit). We used an Ansari-Bradley test in Matlab to confirm that the distribution functions were different (P = 0.0043).