Department of Chemistry, KU Leuven, Celestijnenlaan 200G, B-3001 Heverlee, Belgium.
Department of Biosystems, KU Leuven, Kasteelpark Arenberg 21, B-3001 Heverlee, Belgium.
Int J Mol Sci. 2021 May 24;22(11):5513. doi: 10.3390/ijms22115513.
The combination of phage display technology with high-throughput sequencing enables in-depth analysis of library diversity and selection-driven dynamics. We applied short-read sequencing of the mutagenized region on focused display libraries of two homologous nucleic acid modification eraser proteins-AlkB and FTO-biopanned against methylated DNA. This revealed enriched genotypes with small indels and concomitant doubtful amino acid motifs within the FTO library. Nanopore sequencing of the entire display vector showed additional enrichment of large deletions overlooked by region-specific sequencing, and further impacted the interpretation of the obtained amino acid motifs. We could attribute enrichment of these corrupted clones to amplification bias due to arduous FTO display slowing down host cell growth as well as phage production. This amplification bias appeared to be stronger than affinity-based target selection. Recommendations are provided for proper sequence analysis of phage display data, which can improve motive discovery in libraries of proteins that are difficult to display.
噬菌体展示技术与高通量测序的结合，使我们能够深入分析文库多样性和选择驱动的动态。我们对经过诱变的两个同源核酸修饰橡皮擦蛋白（AlkB 和 FTO）的聚焦展示文库进行短读测序，该文库针对甲基化 DNA 进行了生物淘选。这揭示了在 FTO 文库中存在丰富的具有小插入缺失和伴随的可疑氨基酸模体的基因型。对整个展示载体的纳米孔测序显示，区域特异性测序忽略了大量缺失的进一步富集，这进一步影响了对获得的氨基酸模体的解释。我们可以将这些受污染克隆的富集归因于扩增偏差，这是由于 FTO 展示的艰难导致宿主细胞生长和噬菌体产生减缓。这种扩增偏差似乎比基于亲和力的目标选择更强。我们为正确分析噬菌体展示数据提供了建议，这可以提高在难以展示的蛋白质文库中发现动机的能力。
