Fig. 4: High-throughput sequencing analysis of selected target-specific macrocyclic peptides encoded by yeast.
a DNA extracted from FACS-enriched yeast cell populations was sequenced using both Sanger and next-generation sequencing (NGS) methodologies. DNA samples for high-throughput sequencing analysis were amplified by PCR, indexed using target-specific barcodes, processed using NovaSeq Illumina NGS technology and the obtained FASTQ files analysed using MATLAB scripts. The amino acid sequences are arranged in groups according to sequence similarities. The amino acids are indicated by a one-letter code. Identical or similar amino acids between different peptide sequences are highlighted in colours (C: grey; E and D: red; G: light green; V and A: intense light green; I and L: dark green; S and T: light orange; Y and F: purple; W: violet; H: indigo; P: light brown; Q and N: light blue; R and K: dark blue; M: yellow). Within a single macrocyclic peptide family, the amino acid sequences were listed starting from the clone with the highest abundance (top) to the one with the lowest (bottom). Only sequences with a percentage of abundance >0.1% are reported. b Heat map visualisation of the type of amino acid residues that were enriched or depleted during selection when compared to naïve libraries. The amino acids are indicated by a one-letter code. The colour intensity correlates with the occurrence of each amino acid, with enriched or depleted residues shown in dark blue and dark red, respectively. c Pie chart visualisation of the different macrocyclic peptide topologies enriched during selection against the five different PTs. Macrocyclic peptides with ‘one ring’ and ‘two rings’ are shown in red and blue, respectively. d Pie chart visualisation of the relative abundance of the most frequently selected macrocyclic peptide topologies across different PTs: CX7C (dark red), CX8C (dark salmon), CX9C (light salmon), CX3CX5CX3C (dark blue), CX2CX6CX3C (blue-grey), CCX2CX5C (light blue), CX3CX4CX3C (pink), CCX4CXC (dark grey), CX6CX5CC (dark green), CX2CX5CC (light grey), CX3CX6CX2C (light green), and others (white). Data for b–d are provided as Source data.
