Two data signatures were used to identify well-folded regions: The first is the SHAPE reactivity data generated with the SHAPE-MaP workflow and the ShapeMapper analysis tool (Busan and Weeks, 2018). The second is the Shannon entropy calculated from base-pairing probabilities determined during the SuperFold partition function calculation (Smola et al., 2015b). Two replicate datasets were used, including separate SuperFold predictions.

Local median SHAPE reactivity and Shannon Entropy were calculated in 55nt sliding windows. The global median SHAPE reactivity or Shannon Entropy were subtracted from calculated values to aid in data visualization. Regions with local SHAPE and Shannon Entropy signals 1) below the global median 2) for stretches longer than 40 nucleotides 3) that appear in both replicate datasets were considered well-folded. Disruptions, or regions where local SHAPE or Shannon Entropy rose above the global median, are not considered to disqualify well-folded regions if they extended for less than 40 nucleotides. Arc plots generated from each replicate consensus structure predication were compared for regions that meet sorting criteria described above in order to ensure agreement between secondary structure models generated from each replicate SHAPE-MaP dataset.

Base-pairing distances of well-folded regions were calculated from .ct structure files output from SuperFold consensus predictions, and compared to previously published, publicly available structures for well-folded regions of the HIV genome generated with SHAPE constraints, a max-pairing distance of 500nt, and the SuperFold pipeline (Siegfried et al., 2014).

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