Skip to main content
. 2019 Apr 8;47(9):4883–4895. doi: 10.1093/nar/gkz216

Figure 5.

Figure 5.

Evaluation of the PARO SELEX by next-generation sequencing. (A) Plot of the RPMs of the Top100. Shown is the minimum and the maximum RPM found in each round (dotted lines) and the median for the Top100 (solid line). The Q1 and Q3 quartile are shown in light grey. The PARO riboswitch precursor P11.2H2 is displayed as a dotted green line. (B) Calculation of the cumulative distribution function (CDF) over all calculated reads per million (RPM) of each sequence within the respective library (for details see ‘Materials and methods’ section and the main text). Increase in AUC and also Gini-index over the course of the experiment can be seen (colour code as inlay). (C) Plot of the information entropy over all sequence frequencies of each selection round for PARO and CFX SELEX. A significantly larger decrease of pool complexity in Capture-SELEX (green line) compared to conventional SELEX (blue line) can be observed from round five on. (D) Density plot of the differences between maximum and minimum folding free energies of each accessible alternative secondary structure for each sequence in the Capture-SELEX and conventional SELEX approach. Comparison of the rounds before enrichment (both times in grey) and the last selection rounds (green for Capture-SELEX, blue for conventional SELEX) for both SELEX strategies. The folding energy span of the PARO riboswitch is displayed as dotted vertical line at 1.8 kcal/mol. (E) Comparison of the PARO Capture-SELEX approach (green line) versus previously published conventional CFX SELEX (blue line). The first 10 000 sequences of the respective last rounds are shown on the x-axis with their corresponding occurrences in reads per million on the y-axis. The precursors of the final riboswitches, namely P11.2H2 (for PARO) and 10A (for CFX) are highlighted. The slopes of the lines show that we obtain a higher degree of enrichment ( = better partitioning during the experiment) with the PARO Capture-SELEX approach. The figure also demonstrates that PARO Capture-SELEX facilitates the finding of the riboswitch precursor (compare P11.2H2 in Top100 to 10A in Top10 000).