Comparison of motif-based and whole-unique-sequence-based analyses of phage display library datasets generated by biopanning of anti-Borrelia burgdorferi immune sera
Autoři:
Yurij Ionov aff001; Artem S. Rogovskyy aff002
Působiště autorů:
Department of Cancer Genetics, Roswell Park Comprehensive Cancer Center, Buffalo, New York, United States of America
aff001; Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, United States of America
aff002
Vyšlo v časopise:
PLoS ONE 15(1)
Kategorie:
Research Article
doi:
https://doi.org/10.1371/journal.pone.0226378
Souhrn
Detection of protection-associated epitopes via reverse vaccinology is the first step for development of subunit vaccines against microbial pathogens. Mapping subunit vaccine targets requires high throughput methods, which would allow delineation of epitopes recognized by protective antibodies on a large scale. Phage displayed random peptide library coupled to Next Generation Sequencing (PDRPL/NGS) is the universal platform that enables high-yield identification of peptides that mimic epitopes (mimotopes). Despite being unsurpassed as a tool for discovery of polyclonal serum mimotopes, the PDRPL/NGS is far inferior as a quantitative method of immune response. Difficult-to-control fluctuations in amounts of antibody-bound phages after rounds of selection and amplification diminish the quantitative capacity of the PDRPL/NGS. In an attempt to improve the accuracy of the PDRPL/NGS method, we compared the discriminating capacity of two approaches for PDRPL/NGS data analysis. The whole-unique-sequence-based analysis (WUSA) involved generation of 7-mer peptide profiles and comparison of the numbers of sequencing reads for unique peptide sequences between serum samples. The motif-based analysis (MA) included identification of 4-mer consensus motifs unifying unique 7-mer sequences and comparison of motifs between serum samples. The motif comparison was based not on the numbers of sequencing reads, but on the numbers of distinct 7-mers constituting the motifs. Our PDRPL/NGS datasets generated from biopanning of protective and non-protective anti-Borrelia burgdorferi sera of New Zealand rabbits were used to contrast the two approaches. As a result, the principle component analyses (PCA) showed that the discriminating powers of the WUSA and MA were similar. In contrast, the unsupervised hierarchical clustering obtained via the MA classified the preimmune, non-protective, and protective sera better than the WUSA-based clustering. Also, a total number of discriminating motifs was higher than that of discriminating 7-mers. In sum, our results indicate that MA approach improves the accuracy and quantitative capacity of the PDRPL/NGS method.
Klíčová slova:
Antibodies – Borrelia burgdorferi – Immune response – Phage display – principal component analysis – Rabbits – Sequence analysis – Sequence motif analysis
Zdroje
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