Figure 1.

HTS and sequence analysis strategy. (A) Primer design for Ion Torrent sequencing of bicyclic peptide libraries (Library A and Library B). (B) Procedure for the analysis of sequencing data applying MatLab scripts. First, reads are separated into several files according to their barcode. Second, low-quality sequences are removed from the dataset, and remaining sequences are translated and sorted by abundance. At this point, two optional steps are performed: distribution of the isolated peptide sequences based on the format (e.g. peptide ring size in the case of bicyclic peptides) and comparison of two different datasets. Then, the sequences of the most abundant peptides (e.g. top 200) are compared and clustered in consensus groups or sub-families of consensus groups, allowing the identification of specific motifs. Finally, the entire pool of sequences is searched for other less abundant sequences sharing such motifs in order to identify promising candidates.