Figure 4.

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).