Journal: bioRxiv
Article Title: Stochastic variation in surface protein expression diversifies Trypanosoma cruzi infection
doi: 10.1101/2025.04.07.647584
Figure Lengend Snippet: A) Diagram of the YSD screen with sera from Chagas disease patients or healthy individuals. Sera was incubated with the YSD library expressing T. cruzi antigens on the surface and enriched using magnetic-activated cell sorting (MACS). After three rounds of enrichment, the library DNAs were sequenced using Oxford nanopore technology to identify enriched sequences compared to the initial non-enriched library or the library enriched with sera from healthy individuals. B) PCA plot of each sequencing replicate (dots) by each enrichment condition. The experiment was performed in three biological replicates. C) Volcano plot shows YSD screen analysis comparing MGF proteins enriched with ChD patients’ or healthy individuals’ antibodies. ChD or healthy enriched libraries were first compared to non-enriched libraries, and non-significant hits were removed. Only MGF proteins are shown. D) Diagram of T. cruzi TS on top. Not all TSs have FRIP, Asp boxes, or SAPA repeats. All contain VTV motifs. Each motif’s amino acid (aa) sequences are indicated, and x means any aa. The heatmap (bottom graph) displays the location of antibody binding to the TSs identified in the YSD screen, as mapped using nanopore sequencing. TS sequence lengths were normalized from 1-100 for visualization. On the right, sequence comparison of 30 TSs with antibodies reacting at their N-terminus (grey bar). Sequence alignment of aa 30 to 130. The first 30 aa represent predicted signal peptides removed for the analysis. The percentage of identity between all 30 sequences was obtained, and their correlation was analyzed and plotted. E) The heat map displays the locations of antibody binding sites on mucins, MASPs, DGF-1, and GP63, which were identified in the YSD screen and mapped using nanopore sequencing. Protein sequences were normalized to a range of 1-100 for visualization. Experiments were performed in three biological replicates.
Article Snippet: Briefly, Oxford nanopore sequence .fasta files were aligned to the genome using minimap2.
Techniques: Incubation, Expressing, FACS, Sequencing, Binding Assay, Nanopore Sequencing, Comparison